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Year: 2020

Disease characteristics of MCT8 deficiency: an international, retrospective,multicentre cohort study

Groeneweg, Stefan ; van Geest, Ferdy S ; et al ; Enderli, Anina ; Hackenberg, Annette ; Konrad, Daniel; Heinrich, Bianka

Abstract: Background: Disordered thyroid hormone transport, due to mutations in the SLC16A2 geneencoding monocarboxylate transporter 8 (MCT8), is characterised by intellectual and motor disabilityresulting from cerebral hypothyroidism and chronic peripheral thyrotoxicosis. We sought to systematicallyassess the phenotypic characteristics and natural history of patients with MCT8 deficiency. Methods:We did an international, multicentre, cohort study, analysing retrospective data from Jan 1, 2003, to Dec31, 2019, from patients with MCT8 deficiency followed up in 47 hospitals in 22 countries globally. Thekey inclusion criterion was genetically confirmed MCT8 deficiency. There were no exclusion criteria. Ourprimary objective was to analyse the overall survival of patients with MCT8 deficiency and documentcauses of death. We also compared survival between patients who did or did not attain full head controlby age 1·5 years and between patients who were or were not underweight by age 1-3 years (defined as abodyweight-for-age Z score <-2 SDs or <5th percentile according to WHO definition). Other objectiveswere to assess neurocognitive function and outcomes, and clinical parameters including anthropometriccharacteristics, biochemical markers, and neuroimaging findings. Findings: Between Oct 14, 2014, andJan 17, 2020, we enrolled 151 patients with 73 different MCT8 (SLC16A2) mutations. Median age atdiagnosis was 24·0 months (IQR 12·0-60·0, range 0·0-744·0). 32 (21%) of 151 patients died; themain causes of mortality in these patients were pulmonary infection (six [19%]) and sudden death (six[19%]). Median overall survival was 35·0 years (95% CI 8·3-61·7). Individuals who did not attainhead control by age 1·5 years had an increased risk of death compared with patients who did attainhead control (hazard ratio [HR] 3·46, 95% CI 1·76-8·34; log-rank test p=0·0041). Patients who wereunderweight during age 1-3 years had an increased risk for death compared with patients who were ofnormal bodyweight at this age (HR 4·71, 95% CI 1·26-17·58, p=0·021). The few motor and cognitiveabilities of patients did not improve with age, as evidenced by the absence of significant correlationsbetween biological age and scores on the Gross Motor Function Measure-88 and Bayley Scales of InfantDevelopment III. Tri-iodothyronine concentrations were above the age-specific upper limit in 96 (95%) of101 patients and free thyroxine concentrations were below the age-specific lower limit in 94 (89%) of 106patients. 59 (71%) of 83 patients were underweight. 25 (53%) of 47 patients had elevated systolic bloodpressure above the 90th percentile, 34 (76%) of 45 patients had premature atrial contractions, and 20(31%) of 64 had resting tachycardia. The most consistent MRI finding was a global delay in myelination,which occurred in 13 (100%) of 13 patients. Interpretation: Our description of characteristics of MCT8deficiency in a large patient cohort reveals poor survival with a high prevalence of treatable underlyingrisk factors, and provides knowledge that might inform clinical management and future evaluation oftherapies. Funding: Netherlands Organisation for Health Research and Development, and the ShermanFoundation.

DOI: https://doi.org/10.1016/s2213-8587(20)30153-4

Posted at the Zurich Open Repository and Archive, University of ZurichZORA URL: https://doi.org/10.5167/uzh-188600Journal ArticleAccepted Version

Originally published at:Groeneweg, Stefan; van Geest, Ferdy S; et al; Enderli, Anina; Hackenberg, Annette; Konrad, Daniel;Heinrich, Bianka (2020). Disease characteristics of MCT8 deficiency: an international, retrospective,multicentre cohort study. The Lancet. Diabetes Endocrinology, 8(7):594-605.DOI: https://doi.org/10.1016/s2213-8587(20)30153-4

2

The Lancet Diabetes & Endocrinology

Disease characteristics of MCT8 deficiency: an international, retrospective, multicentrecohort study

--Manuscript Draft--

Manuscript Number: THELANCETDE-D-20-00091R2

Article Type: Article (Original Research)

Keywords: MCT8; monocarboxylate transporter 8; MCT8 deficiency; Allan-Herndon-Dudleysyndrome; AHDS; natural history; Survival; life expectancy; prognosis; diseaseoutcomes

Corresponding Author: Stefan GroenewegErasmus Medical CentreRotterdam, NETHERLANDS

First Author: Stefan Groeneweg, MD PhD

Order of Authors: Stefan Groeneweg, MD PhD

Ferdy S van Geest, MD

Ayhan Abacı, Prof. Dr.

Alberto Alcantud, MD

Gautem Ambegaonkar, MD

Christine Armour, MD

Priyanka Bakhtiani, MD

Diana Barca, MD

Enrico S Bertini, Prof. Dr.

Ingrid M van Beynum, MD PhD

Nicola Brunetti-Pierri, Prof. Dr.

Marianna Bugiani, MD

Marco Cappa, Prof. Dr.

Gerarda Cappuccio, MD

Barbara Castellotti, MD

Claudia Castiglioni, MD

Krishna Chatterjee, Prof. Dr.

Irenaeus FM de Coo, MD PhD

Régis Coutant, Prof. Dr.

Dana Craiu, Prof. Dr.

Patricia Crock, DMed

Christian DeGoede, FRCPCH

Korcan Demir, Assoc. Prof. Dr.

Alice Dica, MD

Paul Dimitri, Prof. BSc, MBChB, FRCPCH, PhD

Anna Dolcetta Capuzzo, MD

Marjolein HG Dremmen, MD

Rachana Dubey, MD

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Anina Enderli, MD

Jan Fairchild, MD PhD

Jonathan Gallichan, MBChB

Belinda George, MD, DM

Evelien F Gevers, MD

Annette Hackenberg, MD

Zita Halász, MD PhD

Bianka Heinrich

Tony Huynh, MBBS PhD FRACP RCPA

Anna Kłosowska, MD

Marjo S van der Knaap, Prof. Dr.

Marieke M van der Knoop, MSc

Daniel Konrad, Prof. Dr.

David A Koolen, MD PhD

Heiko Krude, Prof. Dr.

Amy Lawson-Yuen, MD PhD

Jan Lebl, Prof. Dr.

Michaela Linder-Lucht, MD

Cláudia F Lorea, MD

Charles M Lourenço, MD PhD

Roelineke J Lunsing, MD PhD

Greta Lyons

Jana Malikova, MD

Edna E Mancilla, MD

Anne McGowan, MD

Veronica Mericq, Prof. Dr.

Felipe Monti Lora, MD

Carla Moran, MD PhD

Katalin E Müller, MD PhD

Isabelle Oliver-Petit, MD

Laura Paone, MD

Praveen G Paul, MD

Michel Polak, Prof. Dr.

Francesco Porta, MD

Fabiano O Poswar, MD

Christina Reinauer, MD

Klara Rozenkova, MD

Tuba Seven Menevse, MD

Peter Simm, MD

Anna Simon, Prof. Dr.

Yogen Singh, MDPowered by Editor ial Manager® and ProduXion Manager® from Aries System s Corporat ion

Marco Spada, MD PhD

Jet van der Spek, MD

Milou AM Stals, MD

Athanasia Stoupa, MD

Gopinath M Subramanian, MD

Davide Tonduti, MD PhD

Serap Turan, Prof. Dr.

Corstiaan A den Uil, MD PhD

Joel Vanderniet, MBBS

Adri van der Walt, MD

Jean-Louis Wémeau, Prof. Dr.

Jolante Wierzba, MD

Marie-Claire Y de Wit, MD PhD

Nicole I Wolf, MD PhD

Michael Wurm, MD

Federica Zibordi, MD

Amnon Zung, MD

Nitash Zwaveling-Soonawala, MD

W Edward Visser, MD PhD

Manuscript Region of Origin: NETHERLANDS

Abstract: Background: Disordered thyroid hormone transport, due to mutations inmonocarboxylate transporter 8 (MCT8; gene: SLC16A2 ), is characterized byintellectual and motor disability due to cerebral hypothyroidism and chronic peripheralthyrotoxicosis. Phenotypic characteristics and natural history of MCT8 deficiency havenot been systematically evaluated.Methods: In this international, multicentre, study, retrospective data (2003 to 2019)from patients with MCT8 deficiency followed in 47 centres, was analysed. Our primaryobjectives were to determine neurocognitive outcomes and overall survival. We alsoassessed clinical parameters, including anthropometric characteristics, biochemicalmarkers and neuroimaging findings.Results: 151 subjects with 73 different MCT8 ( SLC16A2 ) mutations were included.21·2% (32/151) of patients died, with main causes of mortality in these patients beingpulmonary infection (18·8%) and sudden death (18·8%). The median overall survivalwas 35·0 (95%CI 8·3-61·7) years. Survival differed significantly between individualswho attained head control by the age of 1·5 years or not (log-rank test: p=0·0041;hazard ratio 3·46 95%CI 1·76-8·34). Patients who were underweight during earlychildhood (1-3 years of age) had an increased risk for death compared with patientswho were not underweight at this age (HR 4·71, 95% CI 1·26-17·58, p=0·021).  Thelimited motor and cognitive abilities of patients did not improve with age. T3concentrations were elevated in 95·1% (96/101) and total T4 concentrations werereduced in 89·5% (94/105) of patients. 71·1% (59/83) patients were underweight (<-2SD). Cardiovascular abnormalities were frequent, with 53·2% (25/47) of patientsexhibiting elevated systolic blood pressure, and 75·6% (34/45) of patients havingpremature atrial contractions and 31·3% (20/60) having resting tachycardia.Interpretation Our description of characteristics of MCT8 deficiency in a large patientcohort reveals poor survival with a high prevalence of treatable underlying risk factorsand provides knowledge which informs clinical management and future evaluation oftherapies.  Funding Our study was funded by the Netherlands Organisation for Health Researchand Development (project number 113303005; to WEV), and the Sherman Foundation(to WEV).

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1

Disease characteristics of MCT8 deficiency: an international, retrospective, multicentre

cohort study

Stefan Groeneweg MD1, Ferdy S van Geest MD1, Prof Ayhan Abacı MD2, Alberto Alcantud MD3,

Gautem P Ambegaonkar MRCPCH4, Christine M Armour MD5, Priyanka Bakhtiani MD6, Diana

Barca MD7, Prof Enrico S Bertini MD8, Ingrid M van Beynum MD9, Prof Nicola Brunetti-Pierri

MD10,11, Marianna Bugiani MD12,13, Marco Cappa MD14, Gerarda Cappuccio MD10,11, Barbara

Castellotti MD15, Claudia Castiglioni MD16, Prof Krishna Chatterjee FRCP17, Irenaeus FM de Coo

MD18, Prof Régis Coutant MD19, Prof Dana Craiu MD7, Patricia Crock DMed20, Christian

DeGoede FRCPCH21, Korcan Demir MD2, Alice Dica MD7, Prof Paul Dimitri PhD22, Anna Dolcetta

Capuzzo MD1,23, Marjolein HG Dremmen MD24, Rachana Dubey DM25, Anina Enderli MD26, Jan

Fairchild FRACP27, Jonathan Gallichan MBChB28, Belinda George DM29, Evelien F Gevers PhD30,

Annette Hackenberg MD26, Zita Halász MD31, Bianka Heinrich26, Tony Huynh PhD32,33,34, Anna

Kłosowska MD35, Prof Marjo S van der Knaap MD12, Marieke M van der Knoop MSc18, Prof

Daniel Konrad MD36, David A Koolen MD37, Prof Heiko Krude MD38, Amy Lawson–Yuen MD39,

Prof Jan Lebl MD40, Michaela Linder-Lucht MD41, Cláudia F Lorea MD42, Charles M Lourenço

MD43, Roelineke J Lunsing MD44, Greta Lyons RGN17, Jana Malikova MD40, Edna E Mancilla

MD45, Anne McGowan MD17, Prof Veronica Mericq MD46,47, Felipe Monti Lora MD48, Carla

Moran MB17, Katalin E Müller MD49, Isabelle Oliver-Petit MD50, Laura Paone MD14, Praveen G

Paul MD51, Prof Michel Polak MD52, Francesco Porta MD53, Fabiano O Poswar MD54, Christina

Reinauer MD55, Klara Rozenkova MD40, Tuba Seven Menevse MD56, Peter Simm MBBS57, Anna

Simon MD51, Yogen Singh MD58, Marco Spada MD53, Jet van der Spek MD37, Milou AM Stals

MD1, Athanasia Stoupa MD52, Gopinath M Subramanian FRACP20, Davide Tonduti MD59, Prof

Serap Turan MD56, Corstiaan A den Uil MD60, Joel Vanderniet MBBS20, Adri van der Walt MD61,

Prof Jean-Louis Wémeau MD62, Jolante Wierzba MD35, Marie-Claire Y de Wit MD18, Nicole I

Wolf MD12, Michael Wurm MD63,64, Federica Zibordi MD59, Prof Amnon Zung MD65,66, Nitash

Zwaveling-Soonawala MD67, W Edward Visser MD1

1 Academic Center for Thyroid Diseases, Erasmus Medical Centre, Rotterdam, Netherlands

2 Division of Pediatric Endocrinology, Faculty of Medicine, Dokuz Eylul University, İzmir, Turkey

3 Pediatric neurology section, Hospital Francesc de Borja de Gandia, Spain

4 Department of Paediatric Neurology, Addenbrooke's Hospital, Cambridge University

Hospitals NHS Foundation Trust, Cambridge, UK

5 Regional Genetics Program, Children's Hospital of Eastern Ontario, and Children's Hospital

of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Canada

6 University of Louisville, Kentucky, USA

7 Paediatric Neurology Clinic, Alexandru Obregia Hospital, Bucharest, Romania; Department

of Neurosciences, Paediatric Neurology Discipline II, Carol Davila University of Medicine,

Bucharest, Romania

8 Unit of Neuromuscular and Neurodegenerative Disorders, Bambino Gesu' Children's

Research Hospital IRCCS, Rome, Italy

Manuscript

2

9 Sophia Children's Hospital, Division of Paediatric Cardiology, Erasmus Medical Centre,

Rotterdam, Netherlands

10 Department of Translational Medicine, Federico II University, 80131 Naples, Italy

11 Telethon Institute of Genetics and Medicine, Pozzuoli, 80078 Naples, Italy

12 Department of Child Neurology, Center for Childhood White Matter Diseases, Emma

Children's Hospital, Amsterdam University Medical Centers, Vrije Universiteit Amsterdam,

and Amsterdam Neuroscience, Amsterdam, The Netherlands.

13 Department of Pathology, Amsterdam Neuroscience, Amsterdam University Medical

Centers, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

14 Division of Endocrinology, Bambino Gesu' Children's Research Hospital IRCCS, Rome, Italy

15 Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo

Besta, Milan

16 Departamento de Neurologia Pediatrica, Clinica Las Condes, Santiago, Chile

17 Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of

Cambridge, Cambridge, UK

18 Sophia Children's Hospital, Department of Paediatric Neurology, Erasmus Medical Centre,

Rotterdam, Netherlands

19 Department of Pediatric Endocrinology and Diabetology, University Hospital, Angers,

France

20 John Hunter Children’s Hospital and University of Newcastle, Newcastle, NSW Australia

21 Lancashire Teaching Hospitals NHS Trust

22 Sheffield Children’s NHS Foundation Trust, Sheffield Hallam University and University of

Sheffield

23 Università Vita-Salute San Raffaele, Milan, Italy

24 Sophia Children's Hospital, Division of Paediatric Radiology, Erasmus Medical Centre,

Rotterdam, Netherlands

25 Medanta Superspeciality Hospital, Indore, India

26 Department of Neuropediatrics, University Children’s Hospital Zurich, Steinwiesstrasse 75, 8032 Zürich, Switzerland

27 Department of Diabetes and Endocrinology, Women’s and Children’s Hospital , North Adelaide 5066 South Australia

28 Plymouth Hospitals NHS Trust, Plymouth, PL6 8DH, UK

29 Department of Endocrinology, St. John’s Medical College Hospital, Bengaluru

30 Centre for Endocrinology, William Harvey Research institute, Queen Mary University

London and Dept of Paediatric Endocrinology, Barts Health NHS Trust

31 Department of Paediatrics, Semmelweis University, Budapest

32 Department of Endocrinology & Diabetes, Queensland Children’s Hospital, South Brisbane Queensland, Australia

33 Department of Chemical Pathology, Mater Pathology, South Brisbane, Queensland,

Australia

34 Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia

35 Medical University of Gdańsk, Department of Paediatrics, Haemathology & Oncology, Department of General Nursery, Gdańsk, Poland

36 Division of Pediatric Endocrinology and Diabetology and Children’s Research Center, University Children's Hospital, CH-8032 Zurich, Switzerland

37 Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour,

Radboud university medical center (Radboudumc), Nijmegen, The Netherlands

3

38 Department of Paediatric Endocrinology and Diabetology, Charité-Universitätsmedizin

Berlin, Berlin, Germany

39 Genomics Institute Mary Bridge Children's Hospital, MultiCare Health System Tacoma

Washington

40 Department of Paediatrics, Second Faculty of Medicine, Charles University, University

Hospital Motol, Prague, Czech Republic

41 Division of Neuropediatrics and Muscular Disorders, Department of Pediatrics and

Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany

42 Teaching Hospital of Universidade Federal de Pelotas, Brazil

43 Faculdade de Medicina - Centro Universitario Estácio de Ribeirão Preto, Brazil

44 Department of Child Neurology, University Medical Center Groningen, University of

Groningen, Groningen, Netherlands

45 Division of Endocrinology and Diabetes, Children's Hospital of Philadelphia, Perelman

School of Medicine, University of Pennsylvania

46 Institute of Maternal and child Research University of Chile

47 Department of Pediatrics, Clinica Las Condes, Santiago Chile

48 Pediatric Endocrinology Group, Santa Catarina Hospital, São Paulo, Brazil

49 University of Debrecen, Pediatric Institute, Debrecen, Hungary

50 Department of Paediatric Endocrinology and Genetics, Children's Hospital, Toulouse

University Hospital, Toulouse, France

51 Department of Paediatrics, Christian Medical College, Vellore, South India

52 Paediatric Endocrinology, Diabetology and Gynaecology Department, Necker Children's

University Hospital, Imagine Institute, Paris, France

53 Department of Paediatrics, AOU Città della Salute e della Scienza di Torino, University of

Torino, Italy

54 Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

55 Department of General Pediatrics, Neonatology and Pediatric Cardiology, University

Children's Hospital, Medical Faculty, Duesseldorf, Germany

56 Marmara University School of Medicine Department of Pediatric Endocrinology, Istanbul,

Turkey

57 Royal Children's Hospital, Parkville, Australia

58 Department of Paediatric Cardiology, Addenbrooke's Hospital, Cambridge University

Hospitals NHS Foundation Trust, Cambridge, UK

59 Child Neurology Unit, Fondazione IRCCS, Istituto Neurologico Carlo Besta, Milan, Italy

60 Department of Cardiology and Intensive Care Medicine, Erasmus Medical Centre,

Rotterdam, Netherlands

61 Private practice Dr A van der Walt, Durbanville, South Africa

62 University of Lille, France

63 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine, University of

Freiburg, Germany

64 KUNO Children’s University Hospital, Campus St. Hedwig, University of Regensburg, Germany

65 Paediatric Endocrinology Unit, Kaplan Medical Center

66 Hebrew University of Jerusalem, Israel

67 Emma Children’s Hospital, Department of Paediatric Endocrinology, Amsterdam UMC,

University of Amsterdam, Amsterdam, The Netherlands

4

Corresponding author

Dr. W. Edward Visser, Department of Internal Medicine, Erasmus Medical Centre Rotterdam,

Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands (w.e.visser@erasmusmc.nl;

telephone 0031107040570).

5

Abstract 1

Background: Disordered thyroid hormone transport, due to mutations in monocarboxylate 2

transporter 8 (MCT8; gene: SLC16A2), is characterized by intellectual and motor disability due 3

to cerebral hypothyroidism and chronic peripheral thyrotoxicosis. Phenotypic characteristics 4

and natural history of MCT8 deficiency have not been systematically evaluated. 5

Methods: In this international, multicentre, study, retrospective data (2003 to 2019) from 6

patients with MCT8 deficiency followed in 47 centres, was analysed. Our primary objectives 7

were to determine neurocognitive outcomes and overall survival. We also assessed clinical 8

parameters, including anthropometric characteristics, biochemical markers and neuroimaging 9

findings. 10

Results: 151 subjects with 73 different MCT8 (SLC16A2) mutations were included. 21·2% 11

(32/151) of patients died, with main causes of mortality in these patients being pulmonary 12

infection (18·8%) and sudden death (18·8%). The median overall survival was 35·0 (95%CI 8·3-13

61·7) years. Survival differed significantly between individuals who attained head control by 14

the age of 1·5 years or not (log-rank test: p=0·0041; hazard ratio 3·46 95%CI 1·76-8·34). 15

Patients who were underweight during early childhood (1-3 years of age) had an increased 16

risk for death compared with patients who were not underweight at this age (HR 4·71, 95% CI 17

1·26-17·58, p=0·021). The limited motor and cognitive abilities of patients did not improve 18

with age. T3 concentrations were elevated in 95·1% (96/101) and total T4 concentrations were 19

reduced in 89·5% (94/105) of patients. 71·1% (59/83) patients were underweight (<-2SD). 20

Cardiovascular abnormalities were frequent, with 53·2% (25/47) of patients exhibiting 21

elevated systolic blood pressure, and 75·6% (34/45) of patients having premature atrial 22

contractions and 31·3% (20/60) having resting tachycardia. 23

6

Interpretation Our description of characteristics of MCT8 deficiency in a large patient cohort 24

reveals poor survival with a high prevalence of treatable underlying risk factors and provides 25

knowledge which informs clinical management and future evaluation of therapies. 26

Funding Our study was funded by the Netherlands Organisation for Health Research and 27

Development (project number 113303005; to WEV), and the Sherman Foundation (to WEV). 28

29

7

Research in context 30

31

Evidence before this study 32

Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disorder with devastating 33

consequences including intellectual and motor disability due to cerebral hypothyroidism and 34

severe clinical sequelae secondary to chronic peripheral thyrotoxicosis. We searched Pubmed 35

for studies published in English to January 1, 2020, using the search terms “MCT8 deficiency”, 36

“Allan-Herndon-Dudley Syndrome”, “AHDS”, “natural history” and “life expectancy”. Prior to 37

this study, given the rarity of the disorder, knowledge on the phenotypic characteristics, 38

natural history and life expectancy of monocarboxylate transporter 8 (MCT8) deficiency was 39

limited. Previous studies consisted of case reports, had small patient cohorts (<25 patients), 40

and neglected the peripheral features of the disorder. Comprehensive and structured in-depth 41

characterisation of the phenotype of MCT8 deficiency is urgently needed to accelerate early 42

diagnosis and inform management, including the use of a new disease-modifying therapy. 43

44

Added value of this study 45

151 patients from 47 centres across the world were included in the largest study on MCT8 46

deficiency, to our knowledge. This is the first multicentre, international study to provide in-47

depth quantitative data on the natural history and life expectancy of patients with MCT8 48

deficiency. Our data report poor survival in this disorder, with 30% of patients dying in 49

childhood. Having identified pulmonary infection and sudden death (our data suggests cardiac 50

arrhythmia as underlying basis) as the major causes of mortality, timely intervention with Triac 51

8

therapy may ameliorate the poor prognosis in this disease. Also, the identification of 52

underweight being strongly linked to survival provides a direct target for clinical management. 53

Our detailed description of key clinical features together with biochemical and radiological 54

correlates constitutes a signature for the disorder which may facilitate its early diagnosis and 55

discrimination of this entity from other developmental disorders. Our data will be used as 56

natural history control data for an ongoing trial of with Triac in young children with MCT8 57

deficiency (NCT02396459). These data will also be important for future clinical trials 58

investigating treatment options for MCT8 deficiency, such as gene therapy. 59

60

Implications of all the available evidence 61

Systemic in-depth description of international natural history data will inform clinical 62

management of patients with MCT8 deficiency. Our findings underscore the need for a 63

multidisciplinary approach in the management and follow-up of patients with MCT8 64

deficiency. The current data indicate a unique combination of clinical presentation, 65

biochemical markers and brain imaging features that will enhance early diagnosis. The low T4 66

concentrations measured in the neonatal screening indicates that current neonatal screening 67

strategy holds potential to detect MCT8 deficiency. These observations hinting at the 68

possibility of early diagnosis are particularly relevant in the context of Triac therapy recently 69

reported, which has the potential to ameliorate the devastating course of the disorder if left 70

untreated. In addition, robust natural history data can be used as controls in clinical trials for 71

rare diseases in which accrual of placebo controls in group might not be feasible. 72

73

9

Introduction 74

Thyroid hormones are crucial for normal physiological processes, particularly 75

neurodevelopment, and regulation of basal metabolic rate, throughout life (1, 2). Intracellular 76

bioavailability of thyroid hormones is governed by membrane transporter proteins that 77

facilitate their cellular entry (3). Monocarboxylate transporter 8 (MCT8) is a specific thyroid 78

hormone transporter that is crucial for transport of triiodothyronine (T3) and thyroxine (T4) 79

in several tissues, including the brain (4-8). Mutations in the gene encoding MCT8 (SLC16A2 80

on chromosome Xq13.2) cause MCT8 deficiency, also known as Allan-Herndon-Dudley 81

syndrome (AHDS), a debilitating disorder with an estimated prevalence of 1 in 70 000 male 82

individuals (9-11). 83

MCT8 deficiency is characterized by profound neurodevelopmental delay and a wide 84

range of severe clinical sequelae secondary to chronic peripheral thyrotoxicosis which cannot 85

be effectively treated with conventional (anti)thyroid drugs (3, 10, 11). In 2019, a clinical trial 86

showed that treatment with triiodothyroacetic acid (Triac) ameliorates key features of 87

peripheral thyrotoxicosis and might improve neurocognitive outcomes if treatment is 88

commenced early in life (12). 89

Robust, comprehensive data regarding the phenotypic characteristics and natural 90

history of patients with MCT8 deficiency are lacking, as the phenotype has only been recorded 91

in single case reports or small case series with related patients [e.g. (13, 14)]. Furthermore, 92

these reports used differing clinical methods precluding consistent assessments, and merely 93

focused on the neurological phenotype, neglecting the peripheral clinical features of the 94

disorder (3, 14). Data on survival and neurodevelopmental outcomes in this disorder are not 95

known. The lack of consistent quantitative knowledge of the natural history and the 96

10

phenotypic spectrum of MCT8 deficiency hampers early diagnosis and uniform clinical 97

management including the evaluation of a disease-modifying therapy. 98

Given the paucity of recorded data and with access to a large patient cohort via an 99

international collaborative network for this rare disorder, we have sought to provide 100

comprehensive and uniform phenotypic characterization of MCT8 deficiency using clinical, 101

radiological, and biochemical data. 102

Methods 103

Study design and participants 104

This international study was initiated on 14 October 2014 by founding a consortium of 105

centres where patients with MCT8 deficiency were followed before and after this date. 106

The key inclusion criterion was genetically confirmed MCT8 deficiency. Additionally, 107

data on first-degree and second-degree male relatives with clinical MCT8 deficiency (when 108

genetic testing was not available at that time) were included. There were no exclusion criteria. 109

Our cohort consisted of patients, evaluated with a standardized protocol, who had been 110

enrolled in the international, multicentre Triac Trial [NCT02060474, (12)] and patients who 111

participated in the named patient program for Triac treatment and historical cases for whom 112

Erasmus MC fulfilled a consultancy role following the first reports of MCT8 deficiency in 2004 113

(10, 11) (figure s1). The group of historical cases therefore contain patients who were alive 114

and patients who were already deceased at time of enrollment. A subgroup of participants 115

has been reported before with available individual case descriptions (n=47), or has been 116

reported on aggregated level (n=46, (12)) (figure s2). For such patients, updated and 117

exhaustive data were collected. For analysis of serum thyroid function tests, only patients 118

11

whose measurements were performed in the central laboratory of the Erasmus MC were 119

considered to avoid inter-assay variation. For in-depth clinical and biochemical phenotyping 120

only those patients were enrolled that either participated in the Triac Trial (12) or in the 121

named patient program to ensure data had been captured by trained personnel and according 122

to standard operating procedures. 123

Ethical considerations 124

This study conforms to the Declaration of Helsinki, Good Clinical Practice guidelines 125

and was evaluated and approved by the appropriate local institutional review boards or ethics 126

committees. However, for the retrospective analysis of existing datasets of patients in routine 127

clinical care, the majority of centres did not require additional specific institutional review 128

board approval. For other centres, studies were either ethically approved or the ethics 129

committee provided a waiver for approval. Informed consent was obtained from the parents 130

or legal representatives of all enrolled patients, unless the relevant institutional review board 131

and/or local regulations had authorized the use of anonymised patient data without additional 132

consent. 133

Procedures 134

An overview of study assessments and investigations is provided in figure s1 and in the 135

Supplementary Methods. 136

Outcomes 137

Our primary objective was to analyse the overall survival of patients with MCT8 138

deficiency and document causes of death. We also compared survival between patients who 139

12

did or did not attain full head control by the age of 1·5 years and between patients who were 140

or were not already underweight by early childhood (between 1-3 years of age). 141

Other key objectives were to document neurocognitive function using uniform criteria 142

and assess their relationship to biological age as a measure of disease progression and 143

developmental outcome and to describe the occurrence of extra-neurological features. 144

Statistical analysis 145

We summarised continuous variables as mean and standard deviation (SD), or median 146

and range. We established overall survival and compared patients with and without full head 147

control and cases who were or were not underweight during early childhood with log-rank 148

analysis. Survival was defined as the age at last date known alive. Hazard ratios were 149

calculated using Cox regression models. Correlations between biological age and scores on 150

different neuropsychological assessments were explored using linear regression. For these 151

analyses, we excluded patients with a less severe neurocognitive phenotype, defined as 152

individuals that attained at least two of the following developmental milestones: talking in 153

simple words, achieving head control, sitting independently, and/or walking with assistance. 154

Higher developmental attainment in these patients is more likely to be due to the milder 155

impact of the underlying MCT8 mutation than to the effect of aging. Assumptions for linear 156

regression analyses were met. All statistical tests were two-sided, and p values of less than 157

0·05 were considered statistically significant. Statistical analyses were performed using 158

GraphPad Prism, version 6 (GraphPad, La Jolla, CA, USA). 159

Role of funding source 160

13

The funder of the study had no role in study design, data collection, data analysis, data 161

interpretation, or writing of the report. The corresponding author had full access to all the 162

data in the study and final responsibility for the decision to submit for publication. 163

Results 164

In 47 centres, 151 patients of 22 different nationalities (8 ethnicities) were enrolled 165

between October 14th 2014 and January 17th 2020 (figure s1), thereby including 50 percent of 166

families reported thus far (figure s2). In 106 cases serum thyroid function tests had been 167

measured in the central laboratory, and 86 had been checked according to standardised 168

protocols for in-depth phenotyping at a median age of 4·8 years (interquartile range [IQR] 1·9-169

9·8, range 0·4-66·8) (figure s1). 170

The demographics and characteristics of the enrolled patients are summarised in table 171

s1. In the 151 enrolled cases, 73 different underlying MCT8 mutations were identified, of 172

which 36 had not been reported before (figure s3). A total of 17 mutations were identified in 173

at least two unrelated families. All 35 missense mutations were located in the transmembrane 174

helices (figure s4). The median age at diagnosis was 24·0 months (range: 0·0-744·0) (figure 175

1A), but the median age at onset of first symptoms was 4·0 months (range: 0·0-13·0) (figure 176

1A). Consequently, the median time to diagnosis was 18·0 months (IQR 7·8-63·0, range 0·0-177

738·0). The most frequently reported initial concerns that prompted medical evaluation were 178

gross developmental delay (78·6%), hypotonia (39·8%), feeding problems (8·2%), and poor 179

weight gain (7·1%) (figure 1B). 180

32 (21·2%) patients had died and the median age of their demise was 10·5 years (IQR 181

5·3-18·8, range 1·6-71·0). The main causes of death reported for these patients were 182

14

pulmonary infections (18·8%), sudden death (18·8%), and aspiration pneumonia (9·4%) (figure 183

1C). In 15 (46·9 %) of 32 deceased subjects the cause of death was unclear and postmortem 184

examinations had not been performed. The median overall survival was 35·0 years (95% CI 185

8·3-61·7; figure 1D). The 10-, 18-, and 60-year survival probabilities were 85% (95% CI 78·0-186

92·0), 69·8% (58·2-80·3), and 34·8% (10·2-59·3), respectively. Patients not attaining full head 187

control by the age of 1·5 years had an increased risk for death compared with patients who 188

did attain head control (HR 3·46, 95% CI 1·76-8·34, p=0·0041; figure 1E). Patients who were 189

underweight during early childhood (1-3 years of age) had an increased risk for death 190

compared with patients who had a normal body weight by this age (HR 4·71, 95% CI 1·26-191

17·58, p=0·021, figure 1F). 192

The prevalence of specific neurological features in patients included in the in-depth 193

phenotyped cohort (N=86, median age 4·8 years, IQR 1·9-9·8, range 0·4-66·8) is reported in 194

table 1 and figure 2, and neurological sequelae are summarized in figure s5 and figure s6. All 195

patients had moderate-to-severe intellectual disability with a severe delay in motor and 196

language development (table s2). Only 6 (7·7%) patients achieved independent sitting and 197

were less severely affected than the other patients (figure 2A). The median score on the Gross 198

Motor Function Measure (GMFM)-88 (15) did not exceed 10% of the total score that should 199

be obtained by healthy 4-year old children (figure 2B, table s2). Among 28 subjects that had 200

been evaluated at a median age of 6·4 years (range 0·4-44·6) with the Bayley Scales of Infant 201

Development (BSID)-III (16), the median developmental age was well-below 12 months on all 202

tested sub-domains (figure 2C-G, figure s7B-F, table s2). Similar findings were obtained with 203

the Vineland Adaptive Behavior Scale (VABS)-II (figure s8, table s2). The scores in any of the 204

developmental domains did not correlate positively with age (e.g. motor skills: GMFM-88 B=-205

0·10 (95% CI, -0·29-0·09; p=0.29), BSID-III fine motor skills B= -0·11 (-0·23 - 0·01; p=0.072), and 206

15

BSID-III gross motor skills B= -0·04(-0·11- 0·02; p=0.17); figure 2C-G) and scores of patients 207

with different age categories were not different (table s2)). 208

Pregnancy and delivery were unremarkable in the majority of cases, with most infants 209

having normal birth weight and head circumference (table 1). At first presentation, most 210

patients had global hypotonia with a pronounced head-lag on vertical suspension and upper 211

trunk slipping through. Typically, by the end of the first year, dystonic posturing of the limbs 212

and neck were noted. Exaggerated deep tendon reflexes were present in 80·3% (57/71) of 213

cases, and 90·5% (67/74) of patients developed hypertonia in wrists, knees or heels with age 214

attributed to dystonia and spasticity. Primitive reflexes remained present in 91·1% (51/56) 215

cases, with a positive tonic neck reflex (81·0%) and glabellar sign (80·0%) being most prevalent, 216

irrespective of patient age. Electro-encephalogram (EEG)-confirmed seizures were observed 217

in 15 (23·1%) of 65 patients, and mostly involved generalized, absence-like episodes without 218

a clear motor component. 219

MRI scans of the brain were available in 13 patients, performed at a median age of 8·0 220

months (range: 5·0-187·0), with 8 patients having at least one follow-up scan available (table 221

1, figure s5, table s3). The most consistent finding was a global delay in myelination, evidenced 222

by diffuse residual hyperintense white matter in specific brain regions on T2-weighted images. 223

Myelination improved with age, but had not fully normalized in the oldest patient (15 years) 224

with available data. The neuroradiological findings were supported by postmortem findings 225

(see supplementary results). 226

Serum thyroid function tests were available in 106 treatment-naïve patients at a 227

median age of 5·3 years (IQR 2·1-11·0, range 0·4-66·8). Serum TSH concentrations were within 228

the normal range in 93 (88·6 %) of 105 patients (figure 3A). Serum free and total T4 229

16

concentrations were below the age-specific lower limit in 94 (88·7 %) of 106 and 94 (89·5%) 230

of 105 patients, respectively (figure 3B and figure s9A). Mean serum T3 concentrations 231

exceeded the age-specific upper limit in 96 (95·1%) of 101 patients (figure 3C), which resulted 232

in a pronounced increase in the T3/T4 ratio (figure s9B). Reverse T3 (rT3) concentrations were 233

decreased in 76 (90·5%) of 84 patients (figure s9C), with a concomitant increase in the T3/rT3 234

ratio (figure s9D). This endocrine signature was present regardless of age (figure s9E-H). In 3 235

out of 7 subjects TRH-stimulation tests showed an inadequate TSH response. In 7 (87·5%) out 236

of 8 subjects in whom T4-based neonatal screening results were available, total T4 237

concentrations were below the 20th percentile (figure 3D), and in 5 out of 8 (60%) below the 238

10th percentile. By contrast, neonatal TSH concentrations were <15 mU/L in 8/8 (100%) of 239

patients with available data (figure s9I). Serum total T4 concentrations were significantly less 240

reduced in patients with less severe versus those with a severe neurocognitive phenotype 241

(1·05±0·22 vs 0·71±0·18 times the age-specific lower limit of normal, p<0·0001) (figure s10A). 242

Serum T3 concentrations were not significantly different between these groups (1·46±0·23 vs 243

1·51±0·44 times the age-specific upper limit of normal, p=0·76)(figure s10B). Consequently, 244

the T3/T4 ratio, a marker of thyroid hormone metabolism in peripheral tissues, was 245

significantly lower in patients with a less severe phenotype (1·44±0·40 vs 2·27±0·91 times the 246

age-specific upper limit of normal, p=0·019) (figure s10C). 247

The main findings of in-depth phenotyping of peripheral clinical features (n=86) are 248

summarised in table 2 and table s4. Body weight for age showed progressive deterioration 249

over time, with 59 (71·1%) of 83 patients being severely underweight (<-2SD) (figure 3E, table 250

2). 27 (35·5%) of 76 patients were tube fed, although impaired swallowing function was 251

reported in 55 (71·4%) of 77 patients. Body height for age also deteriorated with age (figure 252

3F). Delayed sexual maturation was present in 5 (26·3%) patients (figure s11A-D). Among 253

17

patients over 8 years of age, bone mineral density (BMD) was below the 5th percentile, but 254

bone turnover markers were generally within the low-normal range (figure s12A-D). 255

The mean resting heart rate was 110 (±20) beats per minute, with 20 (31·3%) of 64 256

patients exceeding the 90th percentile for age (figure 3G) (17). Systolic blood pressure 257

exceeded the 90th percentile in 25 (53·2%) of 47 patients whereas the diastolic blood pressure 258

exceeded the 90th percentile in 17 (36·2%) of 47 patients (figure 3H) (18, 19). Detailed 259

cardiovascular assessment was available in 50 patients. At the time of evaluation, 47 (94%) of 260

50 patients reportedly had no cardiovascular abnormalities and were not receiving any 261

treatment. 3 (6·0%) of 50 patients had second-degree atrioventricular block (Mobitz I: 1; 262

Mobitz II: 2) and 6 (12·0%) of 50 patients had (incomplete) right bundle branch block. In 263

addition, corrected QT interval (QTc) was above the 98th percentile in three out of 39 (7·7%) 264

patients (table 2, figure s13A). Even though most patients were completely immobile, 24h 265

ambulatory cardiac monitoring showed a high resting heart rate (103±13 beats per minute) 266

with frequent episodes of tachycardia and premature atrial or ventricular contractions (figure 267

3I, table s4). One childhood patient had an episode of atrial fibrillation and another had non-268

sustained ventricular tachycardia (table 2). Cardiac echocardiography studies performed in 26 269

patients revealed dilated aortic root (>+2SD for age, range 2·0-3·4 SD) in 7 (26·9%) patients, 270

and relatively small left ventricular wall dimensions (figure s13B). 271

Serum concentrations of sex hormone binding globulin were elevated in 69 (88·5%) of 272

78 patients (figure 3J, figure s14). Serum alanine aminotransferase, aspartate 273

aminotransferase, and gamma-glutamyl transferase concentrations were mildly elevated in a 274

substantial proportion of patients (table 2, figure s15D-F), and two patients reportedly had an 275

episode of hepatic dysfunction following a (viral) infection. 276

18

43 (84·3%) of 51 patients had low muscle mass. Creatinine concentrations in serum 277

were within the low-normal range for age (figure 3K, s14B). Serum creatine kinase 278

concentrations were mostly low-normal (figure 3L, s14C), with some exceptions in patients 279

with recent seizures or severe dystonic episodes. Other biochemical tests are shown in table 280

s4 and figure s15A-O. 281

Gastroesophageal reflux disease was present in 79·2% (38/48) of patients and this 282

often required pharmacological intervention. Spontaneous gastrointestinal bleeding was 283

reported in 2 patients and was the cause of death in one of them. Constipation was present 284

in 62·3% (37/63) of patients. 29 (69·1%) of 42 patients had recurrent (pulmonary) infections 285

(table 2). 286

Discussion 287

To our knowledge, this international, multicentre, retrospective study reports the 288

quantitative evaluation of the disease characteristics of MCT8 deficiency, in the largest cohort 289

of patients with this disorder. We have documented key clinical features together with 290

biochemical and radiological correlates as well as outcomes in this rare but potentially 291

treatable condition. Our findings will facilitate accurate diagnosis, guide management, and 292

inform conduct of future therapeutic intervention trials. 293

A principal finding is that overall survival of patients with MCT8 deficiency is greatly 294

diminished, with an overall median life expectancy of 35 years. Stratification of analyses 295

revealed that patients who attain full head control are more likely to survive longer than those 296

who do not. Accordingly, attaining full head control, as a marker of improved 297

19

neurodevelopment, could be a relevant endpoint for future therapeutic trials in MCT8 298

deficiency. 299

The most common cause of death was pneumonia, caused either by aspiration or by 300

infections. Aspiration, due to impaired swallowing function, is frequently observed in patients 301

with MCT8 deficiency, and could be mitigated by tube feeding. However, a substantial number 302

of patients that exhibited swallowing problems were not tube fed and thus remained at risk 303

for aspiration. With our study suggesting that being underweight is strongly linked to reduced 304

survival, tube feeding can prevent adverse clinical sequelae and potentially improve life 305

expectancy (20). The second major cause of mortality was sudden death. Although its 306

aetiology remains unclear, available data may suggest a cardiac cause, with the high 307

prevalence of premature atrial and ventricular contractions, which are uncommon in healthy 308

individuals especially in childhood (21-25). We also observed non-sustained ventricular 309

tachycardia and QTc prolongation in some patients, with both considered risk factors for 310

sudden cardiac death. Moreover, a substantial proportion of patients exhibited systolic 311

hypertension and/or tachycardia and had several echocardiographic and electrophysiological 312

cardiac changes that have been linked to these traits. As the vast majority (94·0%) of patients 313

reportedly had no history of cardiac problems, these cardiovascular abnormalities likely 314

remain clinically undiagnosed in this population. This observation calls for inclusion of 315

cardiovascular assessment in the management of this disorder. With loss of body weight and 316

many cardiovascular abnormalities being attributable to chronic thyrotoxicosis, reduction in 317

circulating T3 concentrations in patients could represent effective treatment for these aspects 318

of the disorder. Indeed, in a recent clinical trial, treatment with the thyroid hormone analogue 319

Triac efficiently reduced serum T3 concentrations and improved key clinical features such as 320

20

loss of body weight and reversal of abnormal cardiovascular parameters in MCT8 deficiency 321

(12). 322

The current study also identified several other clinical features that require treatment 323

or close follow-up, of which gastro-esophageal reflux disease, scoliosis, hip luxation, and 324

constipation have the highest prevalence. The presence of mildly elevated aminotransferases 325

and the occurrence of transient hepatic failure in at least three reported cases following a viral 326

infection [this report and (11)], suggests that drugs with hepatotoxic side effects (e.g. anti-327

epileptic drugs as frequently used in this population) should be used with extra caution. 328

Our comprehensive documentation of neurological sequelae in patients with MCT8 329

deficiency revealed that the combination of global hypotonia, hypertonia due to dystonia and 330

spasticity and persistence of primitive reflexes was present in up to 90% of patients. Delayed 331

myelination on MRI was consistent with other studies (14, 26, 27). Taken together, these 332

clinical and neuroimaging characteristics may facilitate early diagnosis of MCT8 deficiency and 333

in discriminating this entity from other neurodevelopmental disorders. 334

Our study highlights major delay in diagnosis of this disorder, with a minority of cases 335

being identified in the first year of life. This is mainly attributable to the non-specific initial 336

clinical features with lack of awareness of the specific characteristics of this disorder among 337

clinicians. Having documented that circulating T3 concentrations are elevated in patients 338

below one year of age, the combination of clinical and radiological features with measurement 339

of serum T3 concentrations may constitute a key clue for early diagnosis. The low T4 340

concentrations measured in patients with MCT8 deficiency in the neonatal screening indicates 341

the potential to diagnose MCT8 deficiency in newborns. This may engender debate on 342

whether modification of the current neonatal screening strategy is warranted. The importance 343

21

of early diagnosis is supported by preclinical studies in which Triac completely prevented 344

abnormal neurological development in animal models of MCT8 deficiency when administrated 345

at birth (28). A future phase 2 clinical trial will investigate the effects of Triac on 346

neurodevelopment, with treatment being commenced at a very young age (NCT02396459). 347

This study has limitations inherent to its retrospective design. In general, such study 348

design is prone to collection of an incomplete dataset, possibly resulting in selection bias. 349

Indeed, most assessments could not be carried out in all patients, resulting in missing data. 350

The cause of this mostly reflected disease characteristics, such as a poor clinical condition of 351

patients, their inability to follow instructions and dystonic posturing that hampered 352

investigations for which patients needed proper positioning. Therefore, some conclusions are 353

based on a limited number of observations. Yet, calculated prevalence rates for many clinical 354

features were similar to those observed in smaller cohort studies and through analysis of 355

existing literature (14). It was also not possible to obtain uniform long-term follow-up data. 356

However, MCT8 deficiency is a rare disorder with surviving patients being located throughout 357

the world such that retrospective analysis of available clinical data was the most suitable way 358

of increasing our understanding of this disorder. In the majority of cases data had been 359

collected uniformly during baseline assessment of patients whether participating in the Triac 360

Trial or in named patient treatment programs, providing an unique opportunity for systematic 361

cross-sectional evaluation of key clinical outcomes. Should MCT8 deficiency result in an 362

increased rate of miscarriage this will likely remain unascertained, resulting in survivor bias. 363

Although selection bias cannot be excluded, probably because not all newly diagnosed cases 364

are brought to our attention and patients who die before the diagnosis has been established 365

are being missed, our study included a substantial proportion of currently diagnosed patients. 366

22

With advent of Triac as possible disease-modifying therapy, a future, prospective cohort study 367

of the natural history of the disorder is unlikely to be possible. 368

In summary, this study provides a comprehensive and structured in-depth 369

characterisation of the phenotype of MCT8 deficiency. The current study first reports poor 370

survival in this disorder, with 30% of patients dying in childhood. Having identified pulmonary 371

infection and sudden death (our data suggests cardiac arrhythmia as underlying basis) as the 372

major causes of mortality, timely intervention with Triac therapy may ameliorate the poor 373

prognosis in this disease. Furthermore, our finding that survival is particularly poor in patients 374

with impaired neurological development (head control) or who are underweight, provides a 375

basis for therapeutic intervention targeted at this subgroup. Our findings underscore the need 376

for a multidisciplinary approach in the management and follow-up of patients with MCT8 377

deficiency. In addition, our observations represent an unique, quantitative dataset of the 378

characteristics of this disorder which may serve as a historical control for future interventional 379

studies in this rare disorder, for which a biological control group is often deemed not feasible. 380

Accordingly, we suggest that this study enhances our understanding of the clinical sequelae 381

and longterm outcome of MCT8 deficiency and also facilitates the diagnosis and management 382

of this disorder. 383

Contributors 384

SG, FSvG, WEV, IFMdC, and MD designed the study, acquired and analysed the results and 385

drafted and approved the manuscript. All other authors contributed to the acquisition, 386

analysis, and interpretation of data, and approved the manuscript. 387

388

23

Declaration of Interest 389

WEV reports grants from Netherlands Organisation for Health Research and Development 390

and from Sherman Foundation during the conduct of the study. WEV, SG, FSvG report other 391

from Rare Thyroid Therapeutics, outside the submitted work. DC reports grants from 392

BioMarine, grants from UCB, grants from A&D pharma, outside the submitted work. 393

MCYdW reports other from Hoffmann-La Roche Ltd, other from Ionis, outside the submitted 394

work.. All other authors declare no competing interests. 395

Acknowledgements 396

We thank the patients for contributing to this study and their families for the ongoing support. 397

Our study was funded by the Netherlands Organisation for Health Research and Development 398

(project number 113303005; to WEV), and the Sherman Foundation (to WEV). The centres in 399

Rotterdam, Berlin, Paris, Prague, Angers and Toulouse are part of the European Reference 400

Network on rare endocrine conditions (Endo-ERN). The centre in Rome is part of the European 401

Reference Network for Rare Neurological Disorders (ERN RND). The centre in Cambridge is 402

supported by the Wellcome Trust and NIHR Biomedical Research Centre. 403

Data sharing 404

Because of the rarity of MCT8 deficiency, individual participant data beyond that reported 405

here will not be shared, to safeguard patient privacy. 406

407

408

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Legends to the Figures 508

Figure 1 – Panel A graphically presents the mean ± SEM (black lines) age at onset of symptoms 509

and the age at time of diagnosis. Grey dots represent measurements in individual patients. 510

Panel B shows the most commonly reported features that prompted parents to seek medical 511

attention. Panel C shows the causes of death in patient with MCT8 deficiency based on the 512

available information derived from the 32 patients in our cohort who died at a median age of 513

10·5 years (IQR 5·3-18·8, range 1·6-71·0). HR: hazard ratio, calculated using Cox regression 514

models. Panel D shows the overall survival based on age at last follow-up (Kaplan-Meier 515

estimates). Panel E shows the Kaplan-Meier estimates of MCT8-specific survival in patients 516

who attained head control (red line) by the age of 1·5 years versus those who did not (blue 517

line) and panel F those in patients with underweight (blue line) versus normal body weight 518

(red line) in early childhood (1-3 years of age). Underweight was defined as a body weight for 519

age z score <-2SDs (or below the fifth percentile), following the definition of the World Health 520

Organization. Please note, since potential confounding factors could not be accounted for, a 521

causal relationship on survival cannot be inferred from the applied stratification variables. 522

Figure 2 – Panel A shows the prevalence of clinical, radiological and developmental key 523

features in MCT8 deficiency. Bars indicate the proportion of patients presenting the indicated 524

feature at first presentation. Panel B represents the gross motor function development in 525

patients with MCT8 deficiency measured by the Gross Motor Function Measure (GMFM)-88 526

(15). A 100% score indicates the level of development that is achieved by a healthy 4-year old 527

child. Panel C shows cognition, panel D receptive language, panel E expressive language, panel 528

F fine motor skills and panel G gross motor skills, measured by the respective sub-domains of 529

the Bayley Scales of Infant Development (BSID)-III (16). Scores are expressed as developmental 530

age in months. In panels B-G, the left figures indicate measurements in individual patients 531

27

(blue dots) and black lines indicate the median score and IQR from all patients with available 532

data. The right figures show the relation between the neurodevelopmental scores versus the 533

chronological age using linear regression. A few, predominantly older patients , with an 534

unexpectedly less severe neurocognitive phenotype (defined as having at least 2 of the 535

following abilities: talk in simple words, attain head control, independent sitting, and/or 536

walking with assistance) were excluded from the regression analysis (n=4 for GMFM-88 in 537

panel B, and n=1 for BSID-III in panel C-G), which focused on individuals with classical sequelae 538

of severe MCT8 deficiency (indicated with grey dashed lines in the left figure of each panel; 539

n=40 for GMFM-88 in panel A, and n=27 for BSID-III in panel C-G). Linear regression was used 540

to plot the trend (blue solid lines) and the 95% confidence intervals (blue dotted lines). Besides 541

age, no other factors were considered in the models. Patients harboring the same genetic 542

mutation are displayed in the same color: p.F230del (green), c.651-652+20del (blue), G564R 543

(purple), p.V566X (pink), and R271H (orange). Unique mutations are colored in grey. 544

Figure 3 – Mean ± SEM (black lines) serum concentrations of thyroid stimulating hormone 545

(TSH) (n=106) (panel A) and free T4 (n=106) (panel B). Blue dots represent measurements in 546

individual patients and grey areas the normal range. Panel C presents the serum total T3 547

concentrations versus age (n=101). Panel D shows the available results on total T4 548

measurements during neonatal screening expressed in SDs (n=8). See figure s9I for TSH 549

measurements during neonatal screening. Panel E shows the natural course of bodyweight 550

change in patients with MCT8 deficiency. Blue dots represent available historical bodyweight 551

measurements (n=300) in 86 untreated patients. Non-linear (third order) polynomial 552

regression was used to plot the trend with its 95% error band. Similarly, panel F shows the 553

natural course of body height. Accurate measurement of body height can be hampered by 554

muscle contractions and involuntary movements. Panel G shows the resting heart rate by age 555

28

(n=64). Normal range in healthy children is derived from (17). Panel H shows the mean ± SEM 556

diastolic and systolic blood pressure (n=47). The orange line represents the threshold for 557

classification as elevated blood pressure and the red line the threshold of hypertension, as 558

defined by the guidelines from the American Academy of Pediatrics (18) and the American 559

College of Cardiology and American Heart Association (19). Panel I shows the mean ± SEM 560

(black lines) occurrence of indicated features during 24h cardiac monitoring in 45 individuals. 561

Serum concentration of sex hormone binding globulin (SHBG) (n=78) (panel J), creatinine 562

(n=79) (panel K), and creatine kinase (n=79) (panel L) are expressed relative to the age-specific 563

lower (panel K) or upper (panel J and L) limit of the normal range. Abbreviations: TSH, thyroid 564

stimulating hormone; T4, thyroxine; T3, triiodothyronine; PACs, premature atrial contractions; 565

PVCs premature ventricular contractions; CK, creatine kinase; SHBG, sex hormone binding 566

globulin; LL, lower limit; UL, upper limit. The absolute mean values of all parameters are 567

summarized in table s4. 568

569

570

1

Disease characteristics of MCT8 deficiency: an international, retrospective, multicentre

cohort study

Stefan Groeneweg MD1, Ferdy S van Geest MD1, Prof Ayhan Abacı MD2, Alberto Alcantud MD3,

Gautem P Ambegaonkar MRCPCH4, Christine M Armour MD5, Priyanka Bakhtiani MD6, Diana

Barca MD7, Prof Enrico S Bertini MD8, Ingrid M van Beynum MD9, Prof Nicola Brunetti-Pierri

MD10,11, Marianna Bugiani MD12,13, Marco Cappa MD14, Gerarda Cappuccio MD10,11, Barbara

Castellotti MD15, Claudia Castiglioni MD16, Prof Krishna Chatterjee FRCP17, Irenaeus FM de Coo

MD18, Prof Régis Coutant MD19, Prof Dana Craiu MD7, Patricia Crock DMed20, Christian

DeGoede FRCPCH21, Korcan Demir MD2, Alice Dica MD7, Prof Paul Dimitri PhD22, Anna Dolcetta

Capuzzo MD1,23, Marjolein HG Dremmen MD24, Rachana Dubey DM25, Anina Enderli MD26, Jan

Fairchild FRACP27, Jonathan Gallichan MBChB28, Belinda George DM29, Evelien F Gevers PhD30,

Annette Hackenberg MD26, Zita Halász MD31, Bianka Heinrich26, Tony Huynh PhD32,33,34, Anna

Kłosowska MD35, Prof Marjo S van der Knaap MD12, Marieke M van der Knoop MSc18, Prof

Daniel Konrad MD36, David A Koolen MD37, Prof Heiko Krude MD38, Amy Lawson–Yuen MD39,

Prof Jan Lebl MD40, Michaela Linder-Lucht MD41, Cláudia F Lorea MD42, Charles M Lourenço

MD43, Roelineke J Lunsing MD44, Greta Lyons RGN17, Jana Malikova MD40, Edna E Mancilla

MD45, Anne McGowan MD17, Prof Veronica Mericq MD46,47, Felipe Monti Lora MD48, Carla

Moran MB17, Katalin E Müller MD49, Isabelle Oliver-Petit MD50, Laura Paone MD14, Praveen G

Paul MD51, Prof Michel Polak MD52, Francesco Porta MD53, Fabiano O Poswar MD54, Christina

Reinauer MD55, Klara Rozenkova MD40, Tuba Seven Menevse MD56, Peter Simm MBBS57, Anna

Simon MD51, Yogen Singh MD58, Marco Spada MD53, Jet van der Spek MD37, Milou AM Stals

MD1, Athanasia Stoupa MD52, Gopinath M Subramanian FRACP20, Davide Tonduti MD59, Prof

Serap Turan MD56, Corstiaan A den Uil MD60, Joel Vanderniet MBBS20, Adri van der Walt MD61,

Prof Jean-Louis Wémeau MD62, Jolante Wierzba MD35, Marie-Claire Y de Wit MD18, Nicole I

Wolf MD12, Michael Wurm MD63,64, Federica Zibordi MD59, Prof Amnon Zung MD65,66, Nitash

Zwaveling-Soonawala MD67, W Edward Visser MD1

1 Academic Center for Thyroid Diseases, Erasmus Medical Centre, Rotterdam, Netherlands

2 Division of Pediatric Endocrinology, Faculty of Medicine, Dokuz Eylul University, İzmir, Turkey

3 Pediatric neurology section, Hospital Francesc de Borja de Gandia, Spain

4 Department of Paediatric Neurology, Addenbrooke's Hospital, Cambridge University

Hospitals NHS Foundation Trust, Cambridge, UK

5 Regional Genetics Program, Children's Hospital of Eastern Ontario, and Children's Hospital

of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Canada

6 University of Louisville, Kentucky, USA

7 Paediatric Neurology Clinic, Alexandru Obregia Hospital, Bucharest, Romania; Department

of Neurosciences, Paediatric Neurology Discipline II, Carol Davila University of Medicine,

Bucharest, Romania

8 Unit of Neuromuscular and Neurodegenerative Disorders, Bambino Gesu' Children's

Research Hospital IRCCS, Rome, Italy

Manuscript with revisions highlighted

2

9 Sophia Children's Hospital, Division of Paediatric Cardiology, Erasmus Medical Centre,

Rotterdam, Netherlands

10 Department of Translational Medicine, Federico II University, 80131 Naples, Italy

11 Telethon Institute of Genetics and Medicine, Pozzuoli, 80078 Naples, Italy

12 Department of Child Neurology, Center for Childhood White Matter Diseases, Emma

Children's Hospital, Amsterdam University Medical Centers, Vrije Universiteit Amsterdam,

and Amsterdam Neuroscience, Amsterdam, The Netherlands.

13 Department of Pathology, Amsterdam Neuroscience, Amsterdam University Medical

Centers, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

14 Division of Endocrinology, Bambino Gesu' Children's Research Hospital IRCCS, Rome, Italy

15 Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo

Besta, Milan

16 Departamento de Neurologia Pediatrica, Clinica Las Condes, Santiago, Chile

17 Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of

Cambridge, Cambridge, UK

18 Sophia Children's Hospital, Department of Paediatric Neurology, Erasmus Medical Centre,

Rotterdam, Netherlands

19 Department of Pediatric Endocrinology and Diabetology, University Hospital, Angers,

France

20 John Hunter Children’s Hospital and University of Newcastle, Newcastle, NSW Australia

21 Lancashire Teaching Hospitals NHS Trust

22 Sheffield Children’s NHS Foundation Trust, Sheffield Hallam University and University of

Sheffield

23 Università Vita-Salute San Raffaele, Milan, Italy

24 Sophia Children's Hospital, Division of Paediatric Radiology, Erasmus Medical Centre,

Rotterdam, Netherlands

25 Medanta Superspeciality Hospital, Indore, India

26 Department of Neuropediatrics, University Children’s Hospital Zurich, Steinwiesstrasse 75, 8032 Zürich, Switzerland

27 Department of Diabetes and Endocrinology, Women’s and Children’s Hospital , North Adelaide 5066 South Australia

28 Plymouth Hospitals NHS Trust, Plymouth, PL6 8DH, UK

29 Department of Endocrinology, St. John’s Medical College Hospital, Bengaluru

30 Centre for Endocrinology, William Harvey Research institute, Queen Mary University

London and Dept of Paediatric Endocrinology, Barts Health NHS Trust

31 Department of Paediatrics, Semmelweis University, Budapest

32 Department of Endocrinology & Diabetes, Queensland Children’s Hospital, South Brisbane Queensland, Australia

33 Department of Chemical Pathology, Mater Pathology, South Brisbane, Queensland,

Australia

34 Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia

35 Medical University of Gdańsk, Department of Paediatrics, Haemathology & Oncology, Department of General Nursery, Gdańsk, Poland

36 Division of Pediatric Endocrinology and Diabetology and Children’s Research Center, University Children's Hospital, CH-8032 Zurich, Switzerland

37 Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour,

Radboud university medical center (Radboudumc), Nijmegen, The Netherlands

3

38 Department of Paediatric Endocrinology and Diabetology, Charité-Universitätsmedizin

Berlin, Berlin, Germany

39 Genomics Institute Mary Bridge Children's Hospital, MultiCare Health System Tacoma

Washington

40 Department of Paediatrics, Second Faculty of Medicine, Charles University, University

Hospital Motol, Prague, Czech Republic

41 Division of Neuropediatrics and Muscular Disorders, Department of Pediatrics and

Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany

42 Teaching Hospital of Universidade Federal de Pelotas, Brazil

43 Faculdade de Medicina - Centro Universitario Estácio de Ribeirão Preto, Brazil

44 Department of Child Neurology, University Medical Center Groningen, University of

Groningen, Groningen, Netherlands

45 Division of Endocrinology and Diabetes, Children's Hospital of Philadelphia, Perelman

School of Medicine, University of Pennsylvania

46 Institute of Maternal and child Research University of Chile

47 Department of Pediatrics, Clinica Las Condes, Santiago Chile

48 Pediatric Endocrinology Group, Santa Catarina Hospital, São Paulo, Brazil

49 University of Debrecen, Pediatric Institute, Debrecen, Hungary

50 Department of Paediatric Endocrinology and Genetics, Children's Hospital, Toulouse

University Hospital, Toulouse, France

51 Department of Paediatrics, Christian Medical College, Vellore, South India

52 Paediatric Endocrinology, Diabetology and Gynaecology Department, Necker Children's

University Hospital, Imagine Institute, Paris, France

53 Department of Paediatrics, AOU Città della Salute e della Scienza di Torino, University of

Torino, Italy

54 Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

55 Department of General Pediatrics, Neonatology and Pediatric Cardiology, University

Children's Hospital, Medical Faculty, Duesseldorf, Germany

56 Marmara University School of Medicine Department of Pediatric Endocrinology, Istanbul,

Turkey

57 Royal Children's Hospital, Parkville, Australia

58 Department of Paediatric Cardiology, Addenbrooke's Hospital, Cambridge University

Hospitals NHS Foundation Trust, Cambridge, UK

59 Child Neurology Unit, Fondazione IRCCS, Istituto Neurologico Carlo Besta, Milan, Italy

60 Department of Cardiology and Intensive Care Medicine, Erasmus Medical Centre,

Rotterdam, Netherlands

61 Private practice Dr A van der Walt, Durbanville, South Africa

62 University of Lille, France

63 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine, University of

Freiburg, Germany

64 KUNO Children’s University Hospital, Campus St. Hedwig, University of Regensburg, Germany

65 Paediatric Endocrinology Unit, Kaplan Medical Center

66 Hebrew University of Jerusalem, Israel

67 Emma Children’s Hospital, Department of Paediatric Endocrinology, Amsterdam UMC,

University of Amsterdam, Amsterdam, The Netherlands

4

Corresponding author

Dr. W. Edward Visser, Department of Internal Medicine, Erasmus Medical Centre Rotterdam,

Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands (w.e.visser@erasmusmc.nl;

telephone 0031107040570).

5

Abstract 1

Background: Disordered thyroid hormone transport, due to mutations in monocarboxylate 2

transporter 8 (MCT8; gene: SLC16A2), is characterized by intellectual and motor disability due 3

to cerebral hypothyroidism and chronic peripheral thyrotoxicosis. Phenotypic characteristics 4

and natural history of MCT8 deficiency have not been systematically evaluated. 5

Methods: In this international, multicentre, study, retrospective data (2003 to 2019) from 6

patients with MCT8 deficiency followed in 47 centres, was analysed. Our primary objectives 7

were to determine neurocognitive outcomes and overall survival. We also assessed clinical 8

parameters, including anthropometric characteristics, biochemical markers and neuroimaging 9

findings. 10

Results: 151 subjects with 73 different MCT8 (SLC16A2) mutations were included. 21·2% 11

(32/151) of patients died, with main causes of mortality in these patients being pulmonary 12

infection (18·8%) and sudden death (18·8%). The median overall survival was 35·0 (95%CI 8·3-13

61·7) years. Survival differed significantly between individuals who attained head control by 14

the age of 1·5 years or not (log-rank test: p=0·0041; hazard ratio 3·46 95%CI 1·76-8·34). 15

Patients who were underweight during early childhood (1-3 years of age) had an increased 16

risk for death compared with patients who were not underweight at this age (HR 4·71, 95% CI 17

1·26-17·58, p=0·021). The limited motor and cognitive abilities of patients did not improve 18

with age. T3 concentrations were elevated in 95·1% (96/101) and total T4 concentrations were 19

reduced in 89·5% (94/105) of patients. 71·1% (59/83) patients were underweight (<-2SD). 20

Cardiovascular abnormalities were frequent, with 53·2% (25/47) of patients exhibiting 21

elevated systolic blood pressure, and 75·6% (34/45) of patients having premature atrial 22

contractions and 31·3% (20/60) having resting tachycardia. 23

6

Interpretation Our description of characteristics of MCT8 deficiency in a large patient cohort 24

reveals poor survival with a high prevalence of treatable underlying risk factors and provides 25

knowledge which informs clinical management and future evaluation of therapies. 26

Funding Our study was funded by the Netherlands Organisation for Health Research and 27

Development (project number 113303005; to WEV), and the Sherman Foundation (to WEV). 28

29

7

Research in context 30

31

Evidence before this study 32

Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disorder with devastating 33

consequences including intellectual and motor disability due to cerebral hypothyroidism and 34

severe clinical sequelae secondary to chronic peripheral thyrotoxicosis. We searched Pubmed 35

for studies published in English to January 1, 2020, using the search terms “MCT8 deficiency”, 36

“Allan-Herndon-Dudley Syndrome”, “AHDS”, “natural history” and “life expectancy”. Prior to 37

this study, given the rarity of the disorder, knowledge on the phenotypic characteristics, 38

natural history and life expectancy of monocarboxylate transporter 8 (MCT8) deficiency was 39

limited. Previous studies consisted of case reports, had small patient cohorts (<25 patients), 40

and neglected the peripheral features of the disorder. Comprehensive and structured in-depth 41

characterisation of the phenotype of MCT8 deficiency is urgently needed to accelerate early 42

diagnosis and inform management, including the use of a new disease-modifying therapy. 43

44

Added value of this study 45

151 patients from 47 centres across the world were included in the largest study on MCT8 46

deficiency, to our knowledge. This is the first multicentre, international study to provide in-47

depth quantitative data on the natural history and life expectancy of patients with MCT8 48

deficiency. Our data report poor survival in this disorder, with 30% of patients dying in 49

childhood. Having identified pulmonary infection and sudden death (our data suggests cardiac 50

arrhythmia as underlying basis) as the major causes of mortality, timely intervention with Triac 51

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8

therapy may ameliorate the poor prognosis in this disease. Also, the identification of 52

underweight being strongly linked to survival provides a direct target for clinical management. 53

Our detailed description of key clinical features together with biochemical and radiological 54

correlates constitutes a signature for the disorder which may facilitate its early diagnosis and 55

discrimination of this entity from other developmental disorders. Our data will be used as 56

natural history control data for an ongoing trial of with Triac in young children with MCT8 57

deficiency (NCT02396459). These data will also be important for future clinical trials 58

investigating treatment options for MCT8 deficiency, such as gene therapy. 59

60

Implications of all the available evidence 61

Systemic in-depth description of international natural history data will inform clinical 62

management of patients with MCT8 deficiency. Our findings underscore the need for a 63

multidisciplinary approach in the management and follow-up of patients with MCT8 64

deficiency. The current data indicate a unique combination of clinical presentation, 65

biochemical markers and brain imaging features that will enhance early diagnosis. The low T4 66

concentrations measured in the neonatal screening indicates that current neonatal screening 67

strategy holds potential to detect MCT8 deficiency. These observations hinting at the 68

possibility of early diagnosis are particularly relevant in the context of Triac therapy recently 69

reported, which has the potential to ameliorate the devastating course of the disorder if left 70

untreated. In addition, robust natural history data can be used as controls in clinical trials for 71

rare diseases in which accrual of placebo controls in group might not be feasible. 72

73

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9

Introduction 74

Thyroid hormones are crucial for normal physiological processes, particularly 75

neurodevelopment, and regulation of basal metabolic rate, throughout life (1, 2). Intracellular 76

bioavailability of thyroid hormones is governed by membrane transporter proteins that 77

facilitate their cellular entry (3). Monocarboxylate transporter 8 (MCT8) is a specific thyroid 78

hormone transporter that is crucial for transport of triiodothyronine (T3) and thyroxine (T4) 79

in several tissues, including the brain (4-8). Mutations in the gene encoding MCT8 (SLC16A2 80

on chromosome Xq13.2) cause MCT8 deficiency, also known as Allan-Herndon-Dudley 81

syndrome (AHDS), a debilitating disorder with an estimated prevalence of 1 in 70 000 male 82

individuals (9-11). 83

MCT8 deficiency is characterized by profound neurodevelopmental delay and a wide 84

range of severe clinical sequelae secondary to chronic peripheral thyrotoxicosis which cannot 85

be effectively treated with conventional (anti)thyroid drugs (3, 10, 11). In 2019, a clinical trial 86

showed that treatment with triiodothyroacetic acid (Triac) ameliorates key features of 87

peripheral thyrotoxicosis and might improve neurocognitive outcomes if treatment is 88

commenced early in life (12). 89

Robust, comprehensive data regarding the phenotypic characteristics and natural 90

history of patients with MCT8 deficiency are lacking, as the phenotype has only been recorded 91

in single case reports or small case series with related patients [e.g. (13, 14)]. Furthermore, 92

these reports used differing clinical methods precluding consistent assessments, and merely 93

focused on the neurological phenotype, neglecting the peripheral clinical features of the 94

disorder (3, 14). Data on survival and neurodevelopmental outcomes in this disorder are not 95

known. The lack of consistent quantitative knowledge of the natural history and the 96

10

phenotypic spectrum of MCT8 deficiency hampers early diagnosis and uniform clinical 97

management including the evaluation of a disease-modifying therapy. 98

Given the paucity of recorded data and with access to a large patient cohort via an 99

international collaborative network for this rare disorder, we have sought to provide 100

comprehensive and uniform phenotypic characterization of MCT8 deficiency using clinical, 101

radiological, and biochemical data. 102

Methods 103

Study design and participants 104

This international study was initiated on 14 October 2014 by founding a consortium of 105

centres where patients with MCT8 deficiency were followed before and after this date. 106

The key inclusion criterion was genetically confirmed MCT8 deficiency. Additionally, 107

data on first-degree and second-degree male relatives with clinical MCT8 deficiency (when 108

genetic testing was not available at that time) were included. There were no exclusion criteria. 109

Our cohort consisted of patients, evaluated with a standardized protocol, who had been 110

enrolled in the international, multicentre Triac Trial [NCT02060474, (12)] and patients who 111

participated in the named patient program for Triac treatment and historical cases for whom 112

Erasmus MC fulfilled a consultancy role following the first reports of MCT8 deficiency in 2004 113

(10, 11) (figure s1). The group of historical cases therefore contain patients who were alive 114

and patients who were already deceased at time of enrollment. A subgroup of participants 115

has been reported before with available individual case descriptions (n=47), or has been 116

reported on aggregated level (n=46, (12)) (figure s2). For such patients, updated and 117

exhaustive data were collected. For analysis of serum thyroid function tests, only patients 118

11

whose measurements were performed in the central laboratory of the Erasmus MC were 119

considered to avoid inter-assay variation. For in-depth clinical and biochemical phenotyping 120

only those patients were enrolled that either participated in the Triac Trial (12) or in the 121

named patient program to ensure data had been captured by trained personnel and according 122

to standard operating procedures. 123

Ethical considerations 124

This study conforms to the Declaration of Helsinki, Good Clinical Practice guidelines 125

and was evaluated and approved by the appropriate local institutional review boards or ethics 126

committees. However, for the retrospective analysis of existing datasets of patients in routine 127

clinical care, the majority of centres did not require additional specific institutional review 128

board approval. For other centres, studies were either ethically approved or the ethics 129

committee provided a waiver for approval. Informed consent was obtained from the parents 130

or legal representatives of all enrolled patients, unless the relevant institutional review board 131

and/or local regulations had authorized the use of anonymised patient data without additional 132

consent. 133

Procedures 134

An overview of study assessments and investigations is provided in figure s1 and in the 135

Supplementary Methods. 136

Outcomes 137

Our primary objective was to analyse the overall survival of patients with MCT8 138

deficiency and document causes of death. We also compared survival between patients who 139

12

did or did not attain full head control by the age of 1·5 years and between patients who were 140

or were not already underweight by early childhood (between 1-3 years of age). 141

Other key objectives were to document neurocognitive function using uniform criteria 142

and assess their relationship to biological age as a measure of disease progression and 143

developmental outcome and to describe the occurrence of extra-neurological features. 144

Statistical analysis 145

We summarised continuous variables as mean and standard deviation (SD), or median 146

and range. We established overall survival and compared patients with and without full head 147

control and cases who were or were not underweight during early childhood with log-rank 148

analysis. Survival was defined as the age at last date known alive. Hazard ratios were 149

calculated using Cox regression models. Correlations between biological age and scores on 150

different neuropsychological assessments were explored using linear regression. For these 151

analyses, we excluded patients with a n unexpectedly less severe neurocognitive phenotype, 152

defined as individuals that attained at least two of the following developmental milestones: 153

talking in simple words, achieving head control, sitting independently, and/or walking with 154

assistance. Higher developmental attainment in these patients is more likely to be due to the 155

milder impact of the underlying MCT8 mutation than to the effect of aging. Assumptions for 156

linear regression analyses were met. All statistical tests were two-sided, and p values of less 157

than 0·05 were considered statistically significant. Statistical analyses were performed using 158

GraphPad Prism, version 6 (GraphPad, La Jolla, CA, USA). 159

Role of funding source 160

13

The funder of the study had no role in study design, data collection, data analysis, data 161

interpretation, or writing of the report. The corresponding author had full access to all the 162

data in the study and had final responsibility for the decision to submit for publication. 163

Results 164

In 47 centres, 151 patients of 22 different nationalities (8 ethnicities) were enrolled 165

between October 14th 2014 and January 17th 2020 (figure s1), thereby including 50 percent of 166

families reported thus far (figure s2). In 106 cases serum thyroid function tests had been 167

measured in the central laboratory, and 86 had been checked according to standardised 168

protocols for in-depth phenotyping at a median age of 4·8 years (interquartile range [IQR] 1·9-169

9·8, range 0·4-66·8) (figure s1). 170

The demographics and characteristics of the enrolled patients are summarised in table 171

s1. In the 151 enrolled cases, 73 different underlying MCT8 mutations were identified, of 172

which 36 had not been reported before (figure s3). A total of 17 mutations were identified in 173

at least two unrelated families. All 35 missense mutations were located in the transmembrane 174

helices (figure s4). The median age at diagnosis was 24·0 months (range: 0·0-744·0) (figure 175

1A), but the median age at onset of first symptoms was 4·0 months (range: 0·0-13·0) (figure 176

1A). Consequently, the median time to diagnosis was 18·0 months (IQR 7·8-63·0, range 0·0-177

738·0). The most frequently reported initial concerns that prompted medical evaluation were 178

gross developmental delay (78·6%), hypotonia (39·8%), feeding problems (8·2%), and poor 179

weight gain (7·1%) (figure 1B). 180

32 (21·2%) patients had died and the median age of their demise was 10·5 years (IQR 181

5·3-18·8, range 1·6-71·0). The main causes of death reported for these patients were 182

14

pulmonary infections (18·8%), sudden death (18·8%), and aspiration pneumonia (9·4%) (figure 183

1C). In 15 (46·9 %) of 32 deceased subjects the cause of death was unclear and postmortem 184

examinations had not been performed. The median overall survival was 35·0 years (95% CI 185

8·3-61·7; figure 1D). The 10-, 18-, and 60-year survival probabilities were 85% (95% CI 78·0-186

92·0), 69·8% (58·2-80·3), and 34·8% (10·2-59·3), respectively. Patients not attaining full head 187

control by the age of 1·5 years had an increased risk for death compared with patients who 188

did attain head control (HR 3·46, 95% CI 1·76-8·34, p=0·0041; figure 1E). Patients who were 189

underweight during early childhood (1-3 years of age) had an increased risk for death 190

compared with patients who were not underweighthad a normal body weight by this age (HR 191

4·71, 95% CI 1·26-17·58, p=0·021, figure 1F). 192

The prevalence of specific neurological features in patients included in the in-depth 193

phenotyped cohort (N=86, median age 4·8 years, IQR 1·9-9·8, range 0·4-66·8) is reported in 194

table 1 and figure 2, and neurological sequelae are summarized in figure s5 and figure s6. All 195

patients had moderate-to-severe intellectual disability with a severe delay in motor and 196

language development (table s2). Only 6 (7·7%) patients achieved independent sitting and 197

were less severely affected than the other patients (figure 2A). The median score on the Gross 198

Motor Function Measure (GMFM)-88 (15) did not exceed 10% of the total score that should 199

be obtained by healthy 4-year old children (figure 2B, table s2). Among 28 subjects that had 200

been evaluated at a median age of 6·4 years (range 0·4-44·6) with the Bayley Scales of Infant 201

Development (BSID)-III (16), the median developmental age was well-below 12 months on all 202

tested sub-domains (figure 2C-G, figure s7B-F, table s2). Similar findings were obtained with 203

the Vineland Adaptive Behavior Scale (VABS)-II (figure s8, table s2). The scores in any of the 204

developmental domains did not correlate positively with age (e.g. motor skills: GMFM-88 B=-205

0·10 (95% CI, -0·29-0·09; p=0.29), BSID-III fine motor skills B= -0·11 (-0·23 - 0·01; p=0.072), and 206

15

BSID-III gross motor skills B= -0·04(-0·11- 0·02; p=0.17); figure 2C-G) and scores of patients 207

with different age categories were not different (table s2)). 208

Pregnancy and delivery were unremarkable in the majority of cases, with most infants 209

having good Apgar scores, normal birth weight at term and head circumference (table 1). At 210

first presentation, most patients had global hypotonia with a pronounced head-lag on vertical 211

suspension and upper trunk slipping through. Typically, by the end of the first year, dystonic 212

posturing of the limbs and neck were noted. Exaggerated deep tendon reflexes were present 213

in 80·3% (57/71) of cases, and 90·5% (67/74) of patients developed hypertonia in wrists, knees 214

or heels with age attributed to dystonia and spasticity. Primitive reflexes remained present in 215

91·1% (51/56) cases, with a positive tonic neck reflex (81·0%) and glabellar sign (80·0%) being 216

most prevalent, irrespective of patient age. Electro-encephalogram (EEG)-confirmed seizures 217

were observed in 15 (23·1%) of 65 patients, and mostly involved generalized, absence-like 218

episodes without a clear motor component. 219

MRI scans of the brain were available in 13 patients, performed at a median age of 8·0 220

months (range: 5·0-187·0), with 8 patients having at least one follow-up scan available (table 221

1, figure s5, table s3). The most consistent finding was a global delay in myelination, evidenced 222

by diffuse residual hyperintense white matter in specific brain regions on T2-weighted images. 223

Myelination improved with age, but had not fully normalized in the oldest patient (15 years) 224

with available data. Most cases showed diffuse cortical and subcortical atrophy with dilatation 225

of the ventricles, widening of the subarachnoid spaces demonstrated by prominence of the 226

supra- and infra-tentorial sulci. These neuroradiological findings were supported by 227

postmortem findings (see supplementary results). 228

16

Serum thyroid function tests were available in 106 treatment-naïve patients at a 229

median age of 5·3 years (IQR 2·1-11·0, range 0·4-66·8). Serum TSH concentrations were within 230

the normal range in 93 (88·6 %) of 105 patients (figure 3A). Serum free and total T4 231

concentrations were below the age-specific lower limit in 94 (88·7 %) of 106 and 94 (89·5%) 232

of 105 patients, respectively (figure 3B and figure s9A). Mean serum T3 concentrations 233

exceeded the age-specific upper limit in 96 (95·1%) of 101 patients (figure 3C), which resulted 234

in a pronounced increase in the T3/T4 ratio (figure s9B). Reverse T3 (rT3) concentrations were 235

decreased in 76 (90·5%) of 84 patients (figure s9C), with a concomitant increase in the T3/rT3 236

ratio (figure s9D). This endocrine signature was present regardless of age (figure s9E-H). In 3 237

out of 7 subjects TRH-stimulation tests showed an inadequate TSH response. In 7 (87·5%) out 238

of 8 subjects in whom T4-based neonatal screening results were available, total T4 239

concentrations were below the 20th percentile (figure 3D), and in 5 out of 8 (60%) below the 240

10th percentile. By contrast, neonatal TSH concentrations were <15 mU/L in 8/8 (100%) of 241

patients with available data (figure s9I). Serum total T4 concentrations were significantly less 242

reduced in patients with less severe versus those with a severe neurocognitive phenotype 243

(1·05±0·22 vs 0·71±0·18 times the age-specific lower limit of normal, p<0·0001) (figure s10A). 244

Serum T3 concentrations were not significantly different between these groups (1·46±0·23 vs 245

1·51±0·44 times the age-specific upper limit of normal, p=0·76)(figure s10B). Consequently, 246

the T3/T4 ratio, a marker of thyroid hormone metabolism in peripheral tissues, was 247

significantly lower in patients with a less severe phenotype (1·44±0·40 vs 2·27±0·91 times the 248

age-specific upper limit of normal, p=0·019) (figure s10C). 249

The main findings of in-depth phenotyping of peripheral clinical features (n=86) are 250

summarised in table 2 and table s4. Body weight for age showed progressive deterioration 251

over time, with 59 (71·1%) of 83 patients being severely underweight (<-2SD) (figure 3E, table 252

17

2). 27 (35·5%) of 76 patients were tube fed, although impaired swallowing function was 253

reported in 55 (71·4%) of 77 patients. Body height for age also deteriorated with age (figure 254

3F). Delayed sexual maturation was present in 5 (26·3%) patients (figure s11A-D). Among 255

patients over 8 years of age, bone mineral density (BMD) was below the 5th percentile, but 256

bone turnover markers were generally within the low-normal range (figure s12A-D). 257

The mean resting heart rate was 110 (±20) beats per minute, with 20 (31·3%) of 64 258

patients exceeding the 90th percentile for age (figure 3G) (17). Systolic blood pressure 259

exceeded the 90th percentile in 25 (53·2%) of 47 patients whereas the diastolic blood pressure 260

exceeded the 90th percentile in 17 (36·2%) of 47 patients (figure 3H) (18, 19). Detailed 261

cardiovascular assessment was available in 50 patients. At the time of evaluation, 47 (94%) of 262

50 patients reportedly had no cardiovascular abnormalities and were not receiving any 263

treatment. 3 (6·0%) of 50 patients had second-degree atrioventricular block (Mobitz I: 1; 264

Mobitz II: 2) and 6 (12·0%) of 50 patients had (incomplete) right bundle branch block. In 265

addition, corrected QT interval (QTc) was above the 98th percentile in three out of 39 (7·7%) 266

patients (table 2, figure s13A). Even though most patients were completely immobile, 24h 267

ambulatory cardiac monitoring showed a high resting heart rate (103±13 beats per minute) 268

with frequent episodes of tachycardia and premature atrial or ventricular contractions (figure 269

3I, table s4). One childhood patient had an episode of atrial fibrillation and another had non-270

sustained ventricular tachycardia (table 2). Cardiac echocardiography studies performed in 26 271

patients revealed dilated aortic root (>+2SD for age, range 2·0-3·4 SD) in 7 (26·9%) patients, 272

and relatively small left ventricular wall dimensions (figure s13B). 273

Serum concentrations of sex hormone binding globulin were elevated in 69 (88·5%) of 274

78 patients (figure 3J, figure s14). Serum alanine aminotransferase, aspartate 275

18

aminotransferase, and gamma-glutamyl transferase concentrations were mildly elevated in a 276

substantial proportion of patients (table 2, figure s15D-F), and two patients reportedly had an 277

episode of hepatic dysfunction following a (viral) infection. 278

43 (84·3%) of 51 patients had low muscle mass. Creatinine concentrations in serum 279

were within the low-normal range for age (figure 3K, s14B). Serum creatine kinase 280

concentrations were mostly low-normal (figure 3L, s14C), with some exceptions in patients 281

with recent seizures or severe dystonic episodes. Other biochemical tests are shown in table 282

s4 and figure s15A-O. 283

Gastroesophageal reflux disease was present in 79·2% (38/48) of patients and this 284

often required pharmacological intervention. Spontaneous gastrointestinal bleeding was 285

reported in 2 patients and was the cause of death in one of them. Constipation was present 286

in 62·3% (37/63) of patients. 29 (69·1%) of 42 patients had recurrent (pulmonary) infections 287

(table 2). 288

Discussion 289

To our knowledge, this international, multicentre, retrospective study reports the 290

quantitative evaluation of the disease characteristics of MCT8 deficiency, in the largest cohort 291

of patients with this disorder. We have documented key clinical features together with 292

biochemical and radiological correlates as well as outcomes in this rare but potentially 293

treatable condition. Our findings will facilitate accurate diagnosis, guide management, and 294

inform conduct of future therapeutic intervention trials. 295

A principal finding is that overall survival of patients with MCT8 deficiency is greatly 296

diminished, with an overall median life expectancy of 35 years. Stratification of analyses 297

19

revealed that patients who attain full head control are more likely to survive longer than those 298

who do not. Accordingly, attaining full head control, as a marker of improved 299

neurodevelopment, could be a relevant endpoint for future therapeutic trials in MCT8 300

deficiency. 301

The most common cause of death was pneumonia, caused either by aspiration or by 302

infections. Aspiration, due to impaired swallowing function, is frequently observed in patients 303

with MCT8 deficiency, and could be mitigated by tube feeding. However, a substantial number 304

of patients that exhibited swallowing problems were not tube fed and thus remained at risk 305

for aspiration. With our study suggesting that being underweight is strongly linked to reduced 306

survival, tube feeding can prevent adverse clinical sequelae and potentially improve life 307

expectancy (20). The second major cause of mortality was sudden death. Although its 308

aetiology remains unclear, available data may suggest a cardiac cause, with the high 309

prevalence of premature atrial and ventricular contractions, which are uncommon in healthy 310

individuals especially in childhood (21-25). We also observed non-sustained ventricular 311

tachycardia and QTc prolongation in some patients, with both considered risk factors for 312

sudden cardiac death. Moreover, a substantial proportion of patients exhibited systolic 313

hypertension and/or tachycardia and had several echocardiographic and electrophysiological 314

cardiac changes that have been linked to these traits. As the vast majority (94·0%) of patients 315

reportedly had no history of cardiac problems, these cardiovascular abnormalities likely 316

remain clinically undiagnosed in this population. This observation calls for inclusion of 317

cardiovascular assessment in the management of this disorder. With loss of body weight and 318

many cardiovascular abnormalities being attributable to chronic thyrotoxicosis, reduction in 319

circulating T3 concentrations in patients could represent effective treatment for these aspects 320

of the disorder. Indeed, in a recent clinical trial, treatment with the thyroid hormone analogue 321

20

Triac efficiently reduced serum T3 concentrations and improved key clinical features such as 322

loss of body weight and reversal of abnormal cardiovascular parameters in MCT8 deficiency 323

(12). 324

The current study also identified several other clinical features that require treatment 325

or close follow-up, of which gastro-esophageal reflux disease, scoliosis, hip luxation, and 326

constipation have the highest prevalence. The presence of mildly elevated aminotransferases 327

and the occurrence of transient hepatic failure in at least three reported cases following a viral 328

infection [this report and (11)], suggests that drugs with hepatotoxic side effects (e.g. anti-329

epileptic drugs as frequently used in this population) should be used with extra caution. 330

Our comprehensive documentation of neurological sequelae in patients with MCT8 331

deficiency revealed that the combination of global hypotonia, hypertonia due to dystonia and 332

spasticity and persistence of primitive reflexes was present in up to 90% of patients. Delayed 333

myelination on MRI was consistent with other studies (14, 26, 27). Taken together, these 334

clinical and neuroimaging characteristics may facilitate early diagnosis of MCT8 deficiency and 335

in discriminating this entity from other neurodevelopmental disorders. 336

Our study highlights major delay in diagnosis of this disorder, with a minority of cases 337

being identified in the first year of life. This is mainly attributable to the non-specific initial 338

clinical features with lack of awareness of the specific characteristics of this disorder among 339

clinicians. Having documented that circulating T3 concentrations are elevated in patients 340

below one year of age, the combination of clinical and radiological features with measurement 341

of serum T3 concentrations may constitute a key clue for early diagnosis. The low T4 342

concentrations measured in patients with MCT8 deficiency in the neonatal screening indicates 343

the potential to diagnose MCT8 deficiency in newborns. This may engender debate on 344

21

whether modification of the current neonatal screening strategy is warranted. The importance 345

of early diagnosis is supported by preclinical studies in which Triac completely prevented 346

abnormal neurological development in animal models of MCT8 deficiency when administrated 347

at birth (28). A future phase 2 clinical trial will investigate the effects of Triac on 348

neurodevelopment, with treatment being commenced at a very young age (NCT02396459). 349

This study has limitations inherent to its retrospective design. In general, such study 350

design is prone to collection of an incomplete dataset, possibly resulting in selection bias. 351

Indeed, most assessments could not be carried out in all patients, resulting in missing data. 352

The cause of this mostly reflected disease characteristics, such as a poor clinical condition of 353

patients, their inability to follow instructions and dystonic posturing that hampered 354

investigations for which patients needed proper positioning. Therefore, some conclusions are 355

based on a limited number of observations. Yet, calculated prevalence rates for many clinical 356

features were similar to those observed in smaller cohort studies and through analysis of 357

existing literature (14). It was also not possible to obtain uniform long-term follow-up data. 358

However, MCT8 deficiency is a rare disorder with surviving patients being located throughout 359

the world such that retrospective analysis of available clinical data was the most suitable way 360

of increasing our understanding of this disorder. In the majority of cases data had been 361

collected uniformly during baseline assessment of patients whether participating in the Triac 362

Trial or in named patient treatment programs, providing an unique opportunity for systematic 363

cross-sectional evaluation of key clinical outcomes. Should MCT8 deficiency result in an 364

increased rate of miscarriage this will likely remain unascertained, resulting in survivor bias. 365

Although selection bias cannot be excluded, probably because not all newly diagnosed cases 366

are brought to our attention and patients who die before the diagnosis has been established 367

are being missed, our study included a substantial proportion of currently diagnosed patients. 368

22

With advent of Triac as possible disease-modifying therapy, a future, prospective cohort study 369

of the natural history of the disorder is unlikely to be possible. 370

In summary, this study provides a comprehensive and structured in-depth 371

characterisation of the phenotype of MCT8 deficiency. The current study first reports poor 372

survival in this disorder, with 30% of patients dying in childhood. Having identified pulmonary 373

infection and sudden death (our data suggests cardiac arrhythmia as underlying basis) as the 374

major causes of mortality, timely intervention with Triac therapy may ameliorate the poor 375

prognosis in this disease. Furthermore, our finding that survival is particularly poor in patients 376

with impaired neurological development (head control) or who are underweight, provides a 377

basis for therapeutic intervention targeted at this subgroup. Our findings underscore the need 378

for a multidisciplinary approach in the management and follow-up of patients with MCT8 379

deficiency. In addition, our observations represent an unique, quantitative dataset of the 380

characteristics of this disorder which may serve as a historical control for future interventional 381

studies in this rare disorder, for which a biological control group is often deemed not feasible. 382

Accordingly, we suggest that this study enhances our understanding of the clinical sequelae 383

and longterm outcome of MCT8 deficiency and also facilitates the diagnosis and management 384

of this disorder. 385

Contributors 386

SG, FSvG, WEV, IFMdC, and MD designed the study, acquired and analysed the results and 387

drafted and approved the manuscript. All other authors contributed to the acquisition, 388

analysis, and interpretation of data, and approved the manuscript. 389

390

23

Declaration of Interest 391

Dr. VisserWEV reports grants from Netherlands Organisation for Health Research and 392

Development and from Sherman Foundation during the conduct of the study. Drs 393

VisserWEV, GroenewegSG, Van GeestFSvG report other from Rare Thyroid Therapeutics, 394

outside the submitted work. Dr. CraiuDC reports grants from BioMarine, grants from UCB, 395

grants from A&D pharma, outside the submitted work. Dr. de WitMCYdW reports other 396

from Hoffmann-La Roche Ltd, other from Ionis, outside the submitted work.The Erasmus 397

Medical Centre (Rotterdam, Netherlands), which employs SG, FSvG, IMvB, MD, MMvdK, 398

CAU, MCYdW, and WEV, might receive royalties from Rare Thyroid Therapeutics (the 399

manufacturer of Triac) in the future, dependent on any future commercialisation. None of 400

the authors will benefit personally from any royalties. Rare Thyroid Therapeutics had no 401

influence on the conduct or analysis of this study. All other authors declare no competing 402

interests. 403

Acknowledgements 404

We thank the patients for contributing to this study and their families for the ongoing support. 405

Our study was funded by the Netherlands Organisation for Health Research and Development 406

(project number 113303005; to WEV), and the Sherman Foundation (to WEV). The centres in 407

Rotterdam, Berlin, Paris, Prague, Angers and Toulouse are part of the European Reference 408

Network on rare endocrine conditions (Endo-ERN). The centre in Rome is part of the European 409

Reference Network for Rare Neurological Disorders (ERN RND). The centre in Cambridge is 410

supported by the Wellcome Trust and NIHR Biomedical Research Centre. 411

Data sharing 412

24

Because of the rarity of MCT8 deficiency, individual participant data beyond that reported 413

here will not be shared, to safeguard patient privacy. 414

415

416

25

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Legends to the Figures 516

Figure 1 – Panel A graphically presents the mean ± SEM (black lines) age at onset of symptoms 517

and the age at time of diagnosis. Grey dots represent measurements in individual patients. 518

Panel B shows the most commonly reported features that prompted parents to seek medical 519

attention. Panel C shows the causes of death in patient with MCT8 deficiency based on the 520

available information derived from the 32 patients in our cohort who died at a median age of 521

10·5 years (IQR 5·3-18·8, range 1·6-71·0). HR: hazard ratio, calculated using Cox regression 522

models. Panel D shows the overall survival based on age at last follow-up (Kaplan-Meier 523

estimates). Panel E shows the Kaplan-Meier estimates of MCT8-specific survival in patients 524

who attained head control (red line) by the age of 1·5 years versus those who did not (blue 525

line) and panel F those in patients with underweight (blue line) versus without normal body 526

weight (red line) underweight in early childhood (1-3 years of age). Underweight was defined 527

as a body weight for age z score <-2SDs (or below the fifth percentile), following the definition 528

of the World Health Organization. Please note, since potential confounding factors could not 529

be accounted for, a causal relationship on survival cannot be inferred from the applied 530

stratification variables. 531

Figure 2 – Panel A shows the prevalence of clinical, radiological and developmental key 532

features in MCT8 deficiency. Bars indicate the proportion of patients presenting the indicated 533

feature at first presentation. Panel B represents the gross motor function development in 534

patients with MCT8 deficiency measured by the Gross Motor Function Measure (GMFM)-88 535

(15). A 100% score indicates the level of development that is achieved by a healthy 4-year old 536

child. Panel C shows cognition, panel D receptive language, panel E expressive language, panel 537

F fine motor skills and panel G gross motor skills, measured by the respective sub-domains of 538

the Bayley Scales of Infant Development (BSID)-III (16). Scores are expressed as developmental 539

28

age in months. In panels B-G, the left figures indicate measurements in individual patients 540

(blue dots) and black lines indicate the median score and IQR from all patients with available 541

data. The right figures show the relation between the neurodevelopmental scores versus the 542

chronological age using linear regression. A few, predominantly older patients , with an 543

unexpectedly less severe neurocognitive phenotype (defined as having at least 2 of the 544

following abilities: talk in simple words, attain head control, independent sitting, and/or 545

walking with assistance) were excluded from the regression analysis (n=4 for GMFM-88 in 546

panel B, and n=1 for BSID-III in panel C-G), which focused on individuals with classical sequelae 547

of severe MCT8 deficiency (indicated with grey dashed lines in the left figure of each panel ; 548

n=40 for GMFM-88 in panel A, and n=27 for BSID-III in panel C-G) Linear regression was used 549

to plot the trend (blue solid lines) and the 95% confidence intervals (blue dotted lines). Besides 550

age, no other factors were considered in the models. Patients harboring the same genetic 551

mutation are displayed in the same color: p.F230del (green), c.651-652+20del (blue), G564R 552

(purple), p.A565fs566X V566X (pink), and R271H (orange). Unique mutations are colored in 553

grey. 554

Figure 3 – Mean ± SEM (black lines) serum concentrations of thyroid stimulating hormone 555

(TSH) (n=106) (panel A) and free T4 (n=106) (panel B). Blue dots represent measurements in 556

individual patients and grey areas the normal range. Panel C presents the serum total T3 557

concentrations versus age (n=101). Panel D shows the available results on total T4 558

measurements during neonatal screening expressed in SDs (n=8). See figure s9I for TSH 559

measurements during neonatal screening. Panel E shows the natural course of bodyweight 560

change in patients with MCT8 deficiency. Blue dots represent available historical bodyweight 561

measurements (n=300) in 86 untreated patients. Non-linear (third order) polynomial 562

regression was used to plot the trend with its 95% error band. Similarly, panel F shows the 563

29

natural course of body height. Accurate measurement of body height can be hampered by 564

muscle contractions and involuntary movements. Panel G shows the resting heart rate by age 565

(n=64). Normal range in healthy children is derived from (17). Panel H shows the mean ± SEM 566

diastolic and systolic blood pressure (n=47). The orange line represents the threshold for 567

classification as elevated blood pressure and the red line the threshold of hypertension, as 568

defined by the guidelines from the American Academy of Pediatrics (18) and the American 569

College of Cardiology and American Heart Association (19). Panel I shows the mean ± SEM 570

(black lines) occurrence of indicated features during 24h cardiac monitoring in 45 individuals. 571

Serum concentration of sex hormone binding globulin (SHBG) (n=78) (panel J), creatinine 572

(n=79) (panel K), and creatine kinase (n=79) (panel L) are expressed relative to the age-specific 573

lower (panel K) or upper (panel J and L) limit of the normal range. Abbreviations: TSH, thyroid 574

stimulating hormone; T4, thyroxine; T3, triiodothyronine; PACs, premature atrial contractions; 575

PVCs premature ventricular contractions; CK, creatine kinase; SHBG, sex hormone binding 576

globulin; LL, lower limit; UL, upper limit. The absolute mean values of all parameters are 577

summarized in table s4. 578

579

580 Formatted: Normal

1

Table 1. In depth phenotyping of neurodevelopmental features

N=86

Age at assessment (years) 4·8 (0·44-66·8)

Perinatal features

Pregnancy duration (weeks) 40·0 (32·0-42·3)

Apgar scores >8 after 5 min (n=16) 15 (93·8)

Term birth weight (grams) 3584 (±517)

Microcephaly (<3th centile) at birth (n=11) 2 (18·2)

Neurological examination

Hypotonia (n=72) 72 (100%)

Primitive reflexes (>1 present) (n=56) 51 (91·1%)

Tonic neck reflex (n=21) 17 (81·0%)

Glabellar sign (n=55) 44 (80·0%)

Startle response (n=25) 17 (68·0%)

Scoliosis (>8 years) (n=17) 15 (88·2%)

Muscle hypoplasia (n=51) 43 (84·3%)

Dystonia (n=69) 57 (82·6%)

Spasticity (n=71) 57 (80·3%)

Urinary / faecal incontinence (>4 years) (n=41) 33 (80·5%)

Feeding problems (n=77) 55 (71·4%)

Hip dislocation (>8 years, n=15) 10 (66·7%)

Plantar extension response (Babinski sign, n=57) 38 (66·7%)

Delayed evoked potentials (<6 months, n=6)* 3 (50·0%)

Sleep problems (n=51) 20 (39·2%)

Tube feeding (n=76) 27 (35·5%)

Strabismus (n=54) 19 (35·2%)

Microcephaly (<3th centile) (n=59) 19 (32·2%)

Nystagmus (n=49) 13 (26·5%)

Extrapyramidal signs (other) (n=28) 7 (25·0%)

Seizures (EEG proven) (n=65) 15 (23·1%)

Apneusis (n=32) 7 (21·9%)

Abnormal hearing (n=44) 1 (2·3%)

Delayed evoked potentials (>1 year, n=3) 0 (0·0%)

Development

Head control (n=77) 19 (24·7%)

Speech (at least 1 word) (n=76) 5 (6·6%)

Independent sitting (n=78) 6 (7·7%)

Independent walking (n=77) 4 (5·2%)

MRI/MRS characteristics*

Normal global anatomy (n=13) 13 (100%)

Delayed myelination (n=13) 13 (100%)

Reduced cerebral white matter volume (n=13) 13 (100%)

Periventricular white matter lesions (n=10) 10 (100%)

Prominent supratentorial ventricular system (n=13) 13 (100%)

Prominent peripheral liquor spaces (n=13) 13 (100%)

Low NAA peak (n=7) 6 (85·7%)

High choline peak (n=7) 6 (85·7%)

Data are median (range), n (%), or mean (±SD). Systematic deep

phenotyping of neurological phenotype in 86 eligible patients. Median age

at last available MRI scan: 18·0 months, range 5·0-187·0); MRS was

available in 7 cases. Details are provided in table s2.

* In particular the brainstem-evoked response audiometry was abnormal

in children < 1 year of age and showed prolongation of the I-V interval.

1

Tables

2

2

Table 2. In depth phenotyping of peripheral features

Characteristic N=106

Serum thyroid function tests

Age at measurement (years) 5·3 (0·4-66·8)

Elevated T3 concentrations (n=101) 96 (95·1%)

Reduced free T4 concentrations (n=106) 94 (88·7%)

Deep phenotyping N=86

Age at assessment (years) 4·8 (0·44-66·8)

Biochemical measurements *

Elevated sex hormone binding globulin (n=78) 69 (88·5%)

Elevated alanine aminotransferase (n=65) 30 (46·2%)

Reduced creatinine (n=79) 22 (27·8%)

Elevated lactate (n=11) 3 (27·3%)

Reduced total cholesterol (n=65) 12 (18·5%)

Elevated aspartate aminotransferase (n=56) 11 (19·6%)

Elevated creatine kinase (n=79) 3 (3·8%)

Clinical features

Low bone mineral density (>8 years, n=5) 5 (100%)

Hypotrophic musculature (n=51) 43 (84·3%)

Gastro-esophageal reflux disease (n=48) 38 (79·2%)

Premature atrial complexes (n=45) 34 (75·6%)

Recurrent (pulmonary) infections (n=42) 29 (69·0%)

Underweight (<-2 SD, n=83) 59 (71·1%)

Constipation (n=63) 37 (58·7%)

Elevated systolic blood pressure ¶ (n=47) 25 (53·2%)

Increased perspiration (n=60) 29 (48·3%)

Short stature (<-2 SD, n=67) 27 (40·3%)

Premature ventricular complexes (n=45) 19 (42·2%)

Tachycardia in rest † (n=64) 20 (31·3%)

Aortic root dilatation (n=26) 7 (26·9%)

Elevated diastolic blood pressure ¶ (n=47) 17 (36·2%)

Delayed sexual maturation (>8 years, n=19) 5 (26·3%)

Cardiac conduction abnormalities ‡ (n=50) 9 (18·0%)

Cryptorchidism (n=49) 9 (18·4%)

Prolonged QTc interval (39) 3 (7·7%)

Supraventricular tachycardia (n=48) 2 (4·2%)

(Non-sustained) ventricular tachycardia (n=48) 2 (4·2%)

Atrial fibrillation (n=48) 1 (2·1%)

Data are median (range), or n (%). Systematic deep phenotyping of the

peripheral phenotype. Please note that most parameters have not been

captured in all patients. All absolute and relative values are provided in

table s4.

* Reduced and elevated indicated concentrations below or above the

normal range (2.5-97.5 centile in the healthy population). † Tachycardia was defined as a resting heart rate above the 90th

percentile for the corresponding age, with cut-offs described by

Fleming and colleagues (17).

‡Three out of 50 patients (5·5%) had a second degree atrioventricular

block (Mobitz I:1; Mobitz II:2) and 6 out of 50 patients (12·0%) had an

(incomplete) right bundle branch block and 1 patient (2·0%) had a left

posterior hemiblock. ¶ Elevated systolic and diastolic blood pressure were defined using the

guidelines from the American Academy of Pediatrics (18) and the

American College of Cardiology and American Heart Association (19).

3

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