supplementary figure 1 sequencing traces of hist1h4c dna ... · supplementary figure 6 rna...

Supplementary Figure 1

Sequencing traces of HIST1H4C DNA from patients.

For all patients, the causative substitutions are indicated (black arrowhead). Patient 1 carries the rs61735681 SNP (asterisk); Patients 2

and 3 carry the rs198852 SNP (hash).

Nature Genetics: doi:10.1038/ng.3956


Sequence reads of the HIST1H4C gene indicate low-level mosaicism in the father of patients 2 and 3.

Integrated Genomics Viewer (IGV) screenshots from the mutated position of patient 2 (left), the mother (middle) and the father (right).

The mother is homozygous for SNP rs198852. The mutation in the child only occurs on reference reads, indicating that the mutation

occurred in the paternal germ line. The reads of the father (right) show 2 mutated reads and 47 reference reads. The two mutated reads

are present on a duplicate read, indicating that these originated from the same molecule.



Protein sequence alignment of HIST1H4C homologs.

The amino acid affected in the patients (K91) is indicated by a black arrow.



Relative expression of histone H4 genes in adult human tissues.

Histograms were drawn using data retrieved from the EMBL-EBI expression atlas (https://www.ebi.ac.uk/gxa/home) and previously

published in the Genotype-Tissue Expression (GTEx) project (Nat. Genet. 45, 580–585, 2013). Gene IDs for the reported genes are as

follows: ENSG00000197061, HIST1H4C; ENSG00000158406, HIST1H4H; ENSG00000197238, HIST1H4J; ENSG00000197837,

HIST4H4; ENSG00000270276, HIST2H4B; ENSG00000270882, HIST2H4A; ENSG00000273542, HIST1H4K; ENSG00000274618,

HIST1H4F; ENSG00000275126, HIST1H4L; ENSG00000275663, HIST1H4G; ENSG00000276180, HIST1H4I; ENSG00000276966,

HIST1H4E; ENSG00000277157, HIST1H4D; ENSG00000278637, HIST1H4A; ENSG00000278705, HIST1H4B.


https://www.ebi.ac.uk/gxa/home


Histone H4 K91Q and K91R expression induces convergence-extension defects in zebrafish embryos.

(a) mRNA in situ hybridization for krox20 (*; hindbrain rhombomeres 3 and 5) and myoD (+; muscle cell precursors) at the ten-somite

stage in embryos microinjected with mRNA encoding K91Q and K91R HIST1H4C. Note that, in comparison to the normal situation

(class A), embryos microinjected with the K91Q or K91R mutant mRNA often display mildly broader and relatively shorter signals for

both krox20 and myoD (class B) to stronger convergence-extension defects (class C). (b) Quantification of the classes defined in a per

treatment. Scale bar, 100 m.



RNA sequencing data for HIST1H4C mutant carriers and control fibroblast cell lines.

RNA sequence traces as seen in IGV and pie charts showing the normalized relative amounts of sequence reads for HIST1H4 gene

family members are shown for a control cell line and the two described HIST1H4C-mutant cell lines. (a) RNA sequencing results for the

control fibroblast cell line. (b) RNA sequencing results for the HIST1H4C p.Lys91Gln variant; the numbers of reference and variant

sequence reads are shown in parentheses. SNP rs61735681 is found in the variant haplotype. (c) RNA sequencing reads for the

p.Lys91Arg variant; the numbers of reference and variant sequence reads are shown in parentheses. Just upstream of this variant, a

very common SNP (rs198852) is present. As can be seen in the pie charts for all three variants, the relative amount of HIST1H4C

sequence reads ranges from 14 to 17% of all HIST1H4 family member reads, of which approximately half correspond to the mutant

allele.



Mass spectrometry analysis shows the presence of mutant histones H4 in patient-derived fibroblasts.

(a) Fragmentation spectra from a single experiment on fibroblasts form patient 3 showing the existence of both WT (TVTAMDVVYALK)

and mutant K91R (TVTAMDVVYALR) peptide species. (b) Also included are the data of C-terminal dibasic missed cleavage species of

both peptides, again showing the existence of the mutant histone H4. (c) To estimate the amount of the K91R H4 mutant in vivo, we

digested the chromatin fraction of patient fibroblasts (patients 2 and 3) with trypsin and used high-resolution LC–MS/MS to detect

peptides derived exclusively from the mutant protein as well as wild-type H4. Using the precursor area detector node of Proteome

Discoverer 2.1, we determined the summed intensity ratio between mutant (K91R) and wild-type peptides. Plotted are the ratios of

three replicates. The black horizontal bar indicates the mean value for each condition.



Apoptosis in K91Q- and K91R-expressing zebrafish larvae.

(a) Expression of the K91Q and K91R HIST1H4C variants induces increased apoptosis as shown by acridine orange (AcrOr) staining in

live embryos at 28 h.p.f. Representative magnifications of the head and tail (corresponding to the whole-embryo pictures shown) show

increased occurrence of cellular apoptosis in these regions. The experiment was replicated twice on embryos originating from group

matings of adult zebrafish. For each condition and replicate, a sample consisted of a minimum of 25 embryos. (b) Increased numbers of

apoptotic cells in the head and tail of zebrafish larvae are also shown by DAPI staining of fragmented nuclei visualized as bright

fluorescent circular spots in K91Q- and K91R-expressing larvae at 28 h.p.f. Scale bars, 100 m in a and 50 m in b. Imaging was

carried out on three different embryos per condition. One representative image is shown.



Exogenous HAT4 and DTX3L retain their histone H4 acetyltransferase and ubiquitin ligase activity, respectively, in zebrafish embryos.

(a) H4K91 acetylation in control zebrafish embryos and zebrafish embryos injected with HAT4 mRNA as detected with anti-H4K91ac

antibody. Detection with anti-H4 antibody was used as a loading control. Normalized ratios of H4K91ac and H4 are shown below the

panels. HAT4 mRNA injection results in increased levels of H4K91ac. (b) Co-injection of DTX3L mRNA with H4-FLAG mRNA results in

an increased fraction of monoubiquitinated H4. Detection of H4-FLAG and Ubi-H4-FLAG was carried out with anti-FLAG antibody. One

representative immunoblot of a minimum of two fully independent experiments is shown.



Coexpression of the histone acetyltransferase HAT4 does not rescue the occurrence of -H2AX signals in

embryos expressing K91Q or K91R HIST1H4.

Expression of the K91Q and K91R HIST1H4C variants results in significant accumulation of DSBs. Coexpression of HAT4 does not

reduce the occurrence of -H2AX-positive cells in K91Q- or K91R-expressing embryos. For all conditions, n = 20. For all graphs,

significance was determined by two-tailed Mann–Whitney U test: ns, not significant; **P < 0.01; ***P < 0.001. The black horizontal bar

indicates the mean value for each condition. Data were collected on one technical replicate. All embryos analyzed originated from

group matings of adult zebrafish.



Inactivated DTX3L-M2 does not rescue the occurrence of -H2AX signals in embryos expressing K91Q or K91R HIST1H4.

Expression of the K91Q and K91R HIST1H4C variants results in significant accumulation of DSBs. DTX3L was inactivated by

introducing four mutations affecting its RING domain (Online Methods). The inactive form of DTX3L was named DTX3L-M2,

abbreviated here as M2. Coexpression of M2 does not reduce the occurrence of -H2AX-positive cells in K91Q- or K91R-expressing

embryos. For all graphs, significance was determined by two-tailed Mann–Whitney U test: ns, not significant; the black horizontal bar

indicates the mean value for each condition. Data were collected on two technical replicates. All embryos analyzed originated from

group matings of adult zebrafish.



FACS analysis of zFUCCI embryos expressing H4 mutants.

The FACS profiles displayed show Hoechst 34580 staining (405-nm channel; x axis) in vitro of zebrafish FUCCI live cell suspensions

either in G1 phase or S/G2/M phases as determined by their fluorescence (mKO2 for G1; mAG for S/G2/M). The ratio displayed is

calculated based on the mKO2 (G1) and mAG (S/G2/M) fluorescence detected by the FACS analyzer.



p53 levels are increased in embryos expressing H4 mutants.

Western blot analysis and quantification display upregulation of p53 in embryos expressing H4 mutants. One representative immunoblot of two biological and technical replicates is shown.


Supplementary Table 1: Overview of genes encoding histone H4 in the human genome

Supplementary Table 2 (Differentially expressed genes). MS Excel file.

Supplementary Table 3 (GO term analysis). MS Excel file.


Supplementary Note Contributing members of the DDD consortium Central DDD Team Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK & The Ethox Centre, Nuffield Department of Population Health, University of Oxford, Old Road Campus, Oxford, OX3 7LF, UK DDD Management Team (* Principal Investigator) Jeffrey C. Barrett, Nigel P. Carter, Helen V. Firth, David R. FitzPatrick , Matthew E. Hurles*, Michael Parker, Caroline F. Wright DDD Laboratory Team Kirsty Ambridge, Daniel M. Barrett, Tanya Bayzetinova, Susan Gribble, Netravathi Krishnappa, Laura E. Mason, Elena Prigmore, Diana Rajan DDD Model Organisms Eve L. Coomber, Sebastian S. Gerety DDD Informatics Team Stephen Clayton, Tomas W. Fitzgerald, Philip Jones, Ray Miller, Adrian R. Tivey DDD Analysis Team Nadia Akawi, Saeed Al-Turki, Jeffrey C. Barrett, Tomas W. Fitzgerald, Matthew E. Hurles, Wendy D. Jones, Daniel King, Margriet van Kogelenberg, Jeremy McRae, Katherine I. Morley, Vijaya Parthiban, Alejandro Sifrim DDD Ethics, Social Science and Policy Team Anna Middleton, Michael Parker, Caroline F. Wright Wellcome Trust Sanger Institute Staff Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK DECIPHER Team A. Paul Bevan, Eugene Bragin, G. Jawahar Swaminathan WTSI Pipelines Staff (sample QC, genotyping, pulldown, sequencing, informatics) Rob Andrews, John Burton, Suzannah J. Bumpstead, Sarah Edkins, Peter Ellis, Emma Gray, David Jones, Carol Scott, Douglas Simpkin, Danielle Walker, Sara Widaa WTSI FISH Team Ruby Banerjee, Beiyuan Fu, Sandra Louzada Gomes Pereira, Fentang Yang, Nature Genetics: doi:10.1038/ng.3410 UK NHS Regional Genetic Services (* local Principal Investigator) Aberdeen (North of Scotland Regional Genetics Service, NHS Grampian, Department of Medical Genetics Medical School, Foresterhill, Aberdeen, AB25 2ZD,


UK) Recruiting Consultant Clinical Geneticists: John Dean*, Ruth McGowan, Alison Ross Research Nurse/ Genetic Counsellors: Mariella D’Alessandro Diagnostic Laboratory scientists: Paul Batstone, Shalaka Samant Belfast (Northern Ireland Regional Genetics Centre, Belfast Health and Social Care Trust, Belfast City Hospital, Lisburn Road, Belfast, BT9 7AB, UK) Recruiting Consultant Clinical Geneticists: Tabib Dabir, Deirdre Donnelly, Alex Magee, Vivienne McConnell, Shane McKee*, Fiona Stewart, Research Nurse/ Genetic Counsellors: Claire Kirk Diagnostic Laboratory scientists: Mervyn Humphreys, Susan McNerlan Birmingham (West Midlands Regional Genetics Service, Birmingham Women's NHS Foundation Trust, Birmingham Women's Hospital, Edgbaston, Birmingham, B15 2TG, UK) Recruiting Consultant Clinical Geneticists: Louise Brueton, Trevor Cole*, Nicola Cooper, Helen Cox, Joanna Jarvis, Derek Lim , Jenny Morton, Andrew Norman, Chirag Patel, Nicola Ragge, Saba Sharif, Mark Tein , Julie Vogt , Denise Williams Research Nurse/ Genetic Counsellors: Gail Kirby Diagnostic Laboratory scientists: David Bohanna, Kirsten McKay, Dominic J McMullan Bristol (Bristol Genetics Service (Avon, Somerset, Gloucs and West Wilts), University Hospitals Bristol NHS Foundation Trust, St Michael's Hospital, St Michael's Hill, Bristol, BS2 8DT, UK) Recruiting Consultant Clinical Geneticists: Ruth Newbury-Ecob*, Sarah Smithson Research Nurse/ Genetic Counsellors: Rose Hawkins Diagnostic Laboratory scientists: Eileen Roberts, Christopher Wragg Cambridge (East Anglian Medical Genetics Service, Box 134, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge ,CB2 0QQ, UK) Recruiting Consultant Clinical Geneticists: Ruth Armstrong, Helen Firth*, Simon Holden, Sarju Mehta, Soo-Mi Park, Joan Paterson, Lucy Raymond, Richard Sandford, Geoff Woods Research Nurse/ Genetic Counsellors: Jonathan Roberts, Sarah Wilcox Diagnostic Laboratory scientists: Ingrid Simonic, Becky Treacy Cardiff (Institute Of Medical Genetics, University Hospital Of Wales, Heath Park, Cardiff, CF14 4XW, UK and Department of Clinical Genetics, Block 12, Glan Clwyd Hospital, Rhyl, Denbighshire, LL18 5UJ, UK) Recruiting Consultant Clinical Geneticists: Hayley Archer, Sally Davies, Dhavendra Kumar, Emma McCann*, Daniela T. Pilz*, Annie Procter Research Nurse/ Genetic Counsellors: Karenza Evans Diagnostic Laboratory scientists: Sian Morgan, Hood Mugalaasi Nature Genetics: doi:10.1038/ng.3410 Dublin (National Centre for Medical Genetics, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland) Recruiting Consultant Clinical Geneticists: Sally Ann Lynch*


Research Nurse/ Genetic Counsellors: Rosie O’Shea Dundee (East of Scotland Regional Genetics Service, Human Genetics Unit, Pathology Department, NHS Tayside, Ninewells Hospital, Dundee, DD1 9SY, UK) Recruiting Consultant Clinical Geneticists: Jonathan Berg*, David Goudie, Susann Schweiger Research Nurse/ Genetic Counsellors: Debbie Rice Diagnostic Laboratory scientists: David Baty, Norman Pratt Edinburgh (MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK) Recruiting Consultant Clinical Geneticists: David R. FitzPatrick*, Wayne Lam, Anne Lampe Research Nurse/ Genetic Counsellors: Philip Greene Diagnostic Laboratory scientists: Eddy Maher, David Moore Exeter (Peninsula Clinical Genetics Service, Royal Devon and Exeter NHS Foundation Trust, Clinical Genetics Department, Royal Devon & Exeter Hospital (Heavitree), Gladstone Road, Exeter, EX1 2ED, UK) Recruiting Consultant Clinical Geneticists: Carole Brewer, Bruce Castle, Emma Kivuva*, Julia Rankin, Charles Shaw-Smith, Claire Turner, Peter Turnpenny Research Nurse/ Genetic Counsellors: Gemma Devlin, Sarah Everest Diagnostic Laboratory scientists: Sian Ellard, Carolyn Tysoe Glasgow (West of Scotland Regional Genetics Service, NHS Greater Glasgow and Clyde, Institute Of Medical Genetics, Yorkhill Hospital, Glasgow, G3 8SJ, UK) Recruiting Consultant Clinical Geneticists: Rosemarie Davidson, Carol Gardiner, Shelagh Joss, Esther Kinning, Victoria Murday, John Tolmie*, Margo Whiteford Research Nurse/ Genetic Counsellors: Alexis Duncan Diagnostic Laboratory scientists: Gordon Lowther, Nicola Williams Leeds (Yorkshire Regional Genetics Service, Leeds Teaching Hospitals NHS Trust, Department of Clinical Genetics, Chapel Allerton Hospital, Chapeltown Road, Leeds, LS7 4SA, UK) Recruiting Consultant Clinical Geneticists: Chris Bennett, Moira Blyth*, Emma Hobson, Alison Kraus, Katrina Prescott*, Audrey Smith, Jenny Thomson Research Nurse/ Genetic Counsellors: Miranda Squires Diagnostic Laboratory scientists: Andrea Coates, Sarah Hewitt, Paul Roberts Leicester (Leicestershire Genetics Centre, University Hospitals of Leicester NHS Trust, Leicester Royal Infirmary (NHS Trust), Leicester, LE1 5WW, UK) Recruiting Consultant Clinical Geneticists: Pradeep Vasudevan* Research Nurse/ Genetic Counsellors: Beckie Kaemba, Sandra Kazembe Diagnostic Laboratory scientists: Lara Cresswell Nature Genetics: doi:10.1038/ng.3410 Liverpool (Merseyside and Cheshire Genetics Service, Liverpool Women's NHS Foundation Trust, Department of Clinical Genetics, Royal Liverpool Children's Hospital Alder Hey, Eaton Road, Liverpool, L12 2AP, UK) Recruiting Consultant Clinical Geneticists: Astrid Weber*, Alan Fryer, Lynn Greenhalgh, Elizabeth Sweeney


Research Nurse/ Genetic Counsellors: Gillian Roberts, Vivienne Sutton Diagnostic Laboratory scientists: Angela Douglas, Una Maye London - North West Thames (North West Thames Regional Genetics Centre, North West London Hospitals NHS Trust, The Kennedy Galton Centre, Northwick Park And St Mark's NHS Trust Watford Road, Harrow, HA1 3UJ, UK) Recruiting Consultant Clinical Geneticists: Birgitta Bernhard, Angela Brady, Natalie Canham*, Neeti Ghali, Susan Holder, Anthony Vandersteen Emma Wakeling Research Nurse/ Genetic Counsellors: Cheryl Sequeira, Roldan Singzon Diagnostic Laboratory scientists: Louise Bourdon, Stewart Payne London - Great Ormond Street (North East Thames Regional Genetics Service, Great Ormond Street Hospital for Children NHS Foundation Trust, Great Ormond Street Hospital, Great Ormond Street, London, WC1N 3JH, UK) Recruiting Consultant Clinical Geneticists: Jane Hurst*, Melissa Lees , Elisabeth Rosser, Richard Scott Research Nurse/ Genetic Counsellors: Kate Brunstrom, Georgina Hollingsworth Diagnostic Laboratory scientists: Lucy Jenkins, Jonathon Waters London - Guy's (South East Thames Regional Genetics Centre, Guy's and St Thomas' NHS Foundation Trust, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK) Recruiting Consultant Clinical Geneticists: Fiona Connell, Charu Deshpande, Frances Flinter, Melita Irving, Dragana Josifova, Shehla Mohammed*, Leema Robert Research Nurse/ Genetic Counsellors: Tina Fendick, Caroline Langman Diagnostic Laboratory scientists: Caroline Ogilvie, Michael Yau London - St George's (South West Thames Regional Genetics Centre, St George's Healthcare NHS Trust, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK) Recruiting Consultant Clinical Geneticists: Frances Elmslie, Tessa Homfray, Sahar Mansour*, Meriel McEntagart, Anand Saggar, Kate Tatton-Brown Research Nurse/ Genetic Counsellors: Uruj Anjum Diagnostic Laboratory scientists: Karen Marks, Rohan Taylor Manchester (Manchester Centre for Genomic Medicine, St Mary’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester M13 9WL) Recruiting Consultant Clinical Geneticists: Kate Chandler, Jill Clayton-Smith*, Yanick Crow, Elizabeth Jones, Bronwyn Kerr, Kay Metcalfe Research Nurse/ Genetic Counsellors: Carina Donnelly, Zara Skitt Diagnostic Laboratory scientists: Lorraine Gaunt, Emma Miles Nature Genetics: doi:10.1038/ng.3410 Newcastle (Northern Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK) Recruiting Consultant Clinical Geneticists: John Burn, Richard Fisher, Judith Goodship , Alex Henderson, Tara Montgomery, Miranda Splitt*, Michael Wright Research Nurse/ Genetic Counsellors: Linda Sneddon Diagnostic Laboratory scientists: David Bourn, Stephen Hellens


Nottingham (Nottingham Regional Genetics Service, City Hospital Campus, Nottingham University Hospitals NHS Trust, The Gables, Hucknall Road, Nottingham NG5 1PB, UK) Recruiting Consultant Clinical Geneticists: Abhijit Dixit, Jacqueline Eason*, Ajoy Sarkar, Nora Shannon, Mohnish Suri Research Nurse/ Genetic Counsellors: Ann Selby Diagnostic Laboratory scientists: Gareth Cross, Katherine Martin Oxford (Oxford Regional Genetics Service, Oxford Radcliffe Hospitals NHS Trust, The Churchill Old Road, Oxford, OX3 7LJ, UK) Recruiting Consultant Clinical Geneticists: Edward Blair, Richard Gibbons, Usha Kini*, Sue Price, Debbie Shears, Helen Stewart Research Nurse/ Genetic Counsellors: Julie Phipps, Abigail Pridham, Hellen Purnell Diagnostic Laboratory scientists: Susan Clasper, Anneke Seller Sheffield (Sheffield Regional Genetics Services, Sheffield Children's NHS Trust, Western Bank, Sheffield, S10 2TH, UK) Recruiting Consultant Clinical Geneticists: Meena Balasubramanian, Diana Johnson, Michael Parker* Research Nurse/ Genetic Counsellors: Louise Nevitt, Stuart Ingram, Cat Taylor Diagnostic Laboratory scientists: Emma Shearing, Kath Smith Southampton/Wessex (Wessex Clinical Genetics Service, University Hospital Southampton, Princess Anne Hospital, Coxford Road, Southampton, SO16 5YA, UK and Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury District Hospital, Odstock Road, Salisbury, Wiltshire, SP2 8BJ, UK and Faculty of Medicine, University of Southampton) Recruiting Consultant Clinical Geneticists: Munaza Ahmed, Diana Baralle, Amanda Collins, Nicola Foulds, Katherine Lachlan, I. Karen Temple*, Diana Wellesley Research Nurse/ Genetic Counsellors: Lucy Harrison, Audrey Torokwa Diagnostic Laboratory scientists: David J. Bunyan , Morag N. Collinson


Experimental Sample Size

Figure 2d:

Conditions:

No inj: n=44

WT n=44

K91Q n=44

K91R n=44

Figure 2f:

Conditions:

No inj: n=27

No inj+DTX3L n=26

WT n=34

WT+DTX3L n=31

K91Q n=32

K91Q+DTX3L n=32

K91R n=32

K91R+DTX3L n=34

Supplementary Figure 11:

Conditions:

No inj: n=34

No inj+M2 n=28

WT n=32

WT+M2 n=35

K91Q n=36

K91Q+M2 n=32

K91R n=40

K91R+M2 n=40


Patient Reports

Patient 1

Child is currently 9 years and 5 months. Both her parents are European British with no family

history of note. She has a sibling with no developmental problems.

She was previously reported as girl with distinctive features, but no diagnosis1, to try to find

other similar children.

age weight length ofc

birth 2400g (2nd centile) 46.2cm (2nd centile) 34cm (50th centile)

12 months 6.14 kg (0.4th centile 7.15kg)

66cm (0.4th centile 68cm)

41.9cm (0.4th centile 43.2cm)

7 years 9 months 15.7 kg (0.4th centile 17.5kg)

109.4 cm (0.4th centile 112cm)

46.5 (0.4th centile 49.5cm)

9 years 17.25 kg (0.4th centile 20 kg)

115.2 cm (0.4th centile 117 cm)

Her distinctive facial features were noted from birth: hypertelorism (inner canthal distance 97th

centile and outer canthal distance 1cm> 97th centile at 3 years), ptosis, exorbitism, up-slanting

palpebral fissures, a bifid nasal tip, pseudo-cleft of the upper lip wide mouth, a skin tag in the

external auditory meatus and pre-auricular skin tags. Her hair is fair and was slow growing in

infancy.

She has a left unilateral post-axial oligodactyly with an absent 5th ray and a hypoplastic 5th toe

on the right. In her hands she has a gap between the middle and ring fingers and 5th finger

clinodactyly.

Internally echocardiogram confirmed a secundum atrial septal defect (surgical repair age 2

years). She has small kidneys with lack of cortico-medullary differentiation and simple cysts. Her

renal function is normal at 9 years. To maintain weight gain and nutrition and help with reflux,

she was PEG fed but is now taking increasing volumes of oral feeds. She has no interest in

food.

She also had tonsillectomy for sleep apnoe and grommets for glue ear. Her hearing is otherwise

normal.

Ophthalmological examination confirmed a squint but ERG evaluation was normal. She has

myopia with a -5 dioptre lens. Her eyes are sensitive to the sun. She has never been sunburnt.

Growth has been below expected range from birth and her head circumference has shown post-

natal microcephaly.

As a neonate she was hypotonic, predominantly truncal, and she has profound global

developmental delay with no localizing neurological signs. She has never had seizures. Her

parents report a high pain threshold. She crawled age 7 years, pulls to stand and rides a horse


unaided. She did not communicate with words or signs when seen age nearly 8 years. Her brain

MRI at 8 months showed reduction in white matter bulk, underdevelopment of the cochlea and

semi-circular canals with normal cochlear nerve.

Several diagnoses has been considered, namely Mowat Wilson syndrome as an infant, Odho at

a later stage and more recently a phenotypic overlap with Koolen de Vries syndrome was

considered.

She has an elevated HbF level of 5.2% with normal HbA2 and no abnormal haemoglobins

detected.

Patient 2

During pregnancy there was little fetal movement. Delivery was spontaneous at 37+1 weeks,

APGAR score 5/6/8. The child was hypotonic, pale and wheezy. Because of potential asphyxia

she was given oxygen and an infusion with sodium bicarbonate and glucose. Birth weight was

2190 grams (P3), length 46 cm (P10) and skull circumference 33 cm (P10). She had a few

saturation dips from which she spontaneously recovered. Facial dysmorphisms at that time

included hypertelorism, upslanted palpebral fissures, broad nasal tip with a median ridge and

low set ears.

Investigations by the cardiologist and ophthalmologist revealed no anomalies. On follow up she

appeared to have an amblyopia of the left eye, small papillae, refraction anomaly (+3.5/+4.5)

and convergent strabismus of the left eye. She had recurrent otitis and respiratory tract

infections. An umbilical hernia was surgically treated at the age of 2 years.

From 4 years she had seizures, occipital with secondary generalization. After treatment with

depakin, since 2013 seizures have stopped and the EEG has been normal. From the age of 14

years she became psychotic, heard voices and was treated by the child psychiatrist. She walks

somewhat unstable without clear ataxia.

From birth on there was a delay in growth and psychomotor development. She could sit from 10

months and walk without support from 2,5 years. At the age of 14 years length was 142 cm (-

3,41 SD), weight 32,5 kg (-3,22 SD) and skull circumference 48 cm (-4.01 SD). Her total IQ was

60.

Facial dysmorphisms consisted of asymmetric eyes, upslanted palpebral fissures, periorbital

fullness, broad nasal tip with a median ridge. She has a lordosis, cutis marmorata, thin muscles

and small feet with proximally implanted 4th rays.

Metabolic and hormonal investigations all normal.

Last MRI of the brain (2005) revealed no anomalies.

Genetic investigations including karyotyping of lymphocytes and fibroblasts, subtelomeric

probes, BAC-and SNP array were all normal.


Patient 3

She was the first child of these non-consanguineous Caucasian parents. There were no

previous spontaneous abortions, family history was unremarkable. At the end of the pregnancy

there was suspicion of growth delay. Delivery was at 39+5 weeks with face presentation,

APGAR score 2/7. Birth weight 2015 grams (below P2.3), length 42 cm (below P3), skull

circumference 33 cm (P2), temperature 33.4˚C. Physical examination revealed upslanting

palpebral fissures, broad nasal tip and retrognathia. The 4th ray of the right foot was short. She

had minor respiration problems which resolved after administration of oxygen. Ultrasound of the

brain and cerebral function monitor (CFM) were normal.

From day 3 there was hypertension. Abdominal ultrasound showed extensive thrombosis of the

abdominal aorta and arteries. At day 9 the heart decompensated and at day 11 there was liver

failure, skin necrosis and extension of the thrombosis after which was decided to terminate

treatment.

Laboratory investigations on thrombotic risk factors only revealed a paternally inherited factor V

Leiden.

Karyotyping of lymphocytes and BAC array analysis showed no anomalies.

References:

1. Monti, E. & Hennekam, R.C. Prenatal and postnatal growth retardation, depressed nasal tip, oligodactyly, and mental retardation. Clin Dysmorphol 19, 40-2 (2010).


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