I’m just so relieved that you finally know what it is

Mark Taylor

Immunology Advanced Trainee

Westmead Hospital

Primary Immunodeficiency Workshop

27th July 2016

Mrs CY

39 year old femalereferred to WMH Immunology 03/2016

Background:1. Thalassaemia trait2. G4P43. Uterovaginal prolapse – repair 20124. Depression

Doxycycline 100 mg dailyEscitalopram 10 mg dailyIron supplement daily

Born Lebanon – consanguineous parents (first cousins)Immigrated to Australia in 1977, age 9 monthsGrew up in south western Sydney, lived Lebanon three years 1996–99Currently lives with husband and four sons, aged 5, 6, 7 and 8 yearsNot employed – seeking DSPEx-tobacco smoker, ≈10 pack-year smoking history

39 yrs 35 yrsrecurrent

LRTI& cellulitis

?

27 yrs29 yrs36 yrs 24 yrsrec

vaginal discharge

5 yrs8 yrs 6 yrs7 yrs

62 yrsPUD

69 yrsIHDPUD

d. 6 yrs‘leukaemia’

History of disease• Recurrent upper and lower respiratory tract infections since childhood – especially rhinosinusitis- frequent ear infections from age 13 – oral antibiotics- at least three episodes of lower respiratory tract infection 2013–16

• Chronic daily watery diarrhoea ~6 years, 1–2 bowel mvts daily – no blood or mucous noted

• Recurrent RUQ and LUQ abdominal pain

• Painless loss of all secondary molar teeth in early adolescence, ‘crumbled’ painlessly on chewing

• Alopecia totalis age 18 – complete loss of scalp hair and eyebrows – refractory to oral and intradermal steroids - regrowth of scalp hair around age 25, with subsequent alopecia areata

• Bilateral MCP and PIP joint arthralgia, morning back pain and stiffness- sicca symptoms, Raynaud’s phenomenon, intermittent parotid gland swelling and tenderness

• Referred by GP to haematologist August 2015:- anorexia with 6–8 kg unintentional weight loss- daily fevers and chills, drenching night sweats, periods of profound lethargy- pancytopaenia- splenomegaly with multiple hypoechoic hepatic lesions on ultrasound

On review by Immunology

BMI 20

Mild swan-neck deformity bilateral 2nd–5th fingersNo onychomycosis or nail dystrophy, synovitis, rheumatoid nodules, rash or vitiligo

Alopecia areata

No oral candidiasisDry coughBibasal inspiratory crepitations

No cervical, supraclavicular, axillary, inguinal or popliteal lymphadenopathy

Abdomen soft, mild generalised palpation tenderness Smooth hepatosplenomegaly

ALP 70 U/L ( < 35 U/L)GGT 154 U/L ( < 110 U/L)- serum biochemistry otherwise unremarkable- thyroid function and short synacthen tests: normal

Hb 128 g/L (115 – 165 g/L)Neutrophils 1.5 x 109 /L (2.0 – 8.0 x 109/L)Platelets 149 x 109 /L (150 – 400 x 109 /L)

Lymphocytes 440 /μL (1000 – 4000 /μL)CD19+ 13 /μL (3% PBLs) (80 – 430 /μL)CD3+ 348 /μL (650 – 1820 /μL)CD3+CD4+ 273 /μL (380 – 1390 /μL)CD3+CD8+ 70 /μL (200 – 690 /μL)NK cells 70 /μL (85 – 500 /μL)

IgG 3.9 g/L (6.6 – 15.6 g/L) IgA < 0.25 g/L (0.75 – 3.8 g/L) IgM 0.25 g/L (0.40 – 3.1 g/L) - urinary protein loss excluded

pre-vaccine post-vaccinePneumococcus IgG < 3.3 µg/mL < 3.3 µg/mL (> 39 µg/mL)Hib IgG 0.16 µg/mL 0.18 µg/mL (> 0.15 µg/mL)Diphtheria IgG 0.15 IU/mL 0.19 IU/mL (> 0.01 IU/mL)Tetanus IgG 0.26 IU/mL 0.28 IU/mL (> 0.16 IU/mL)

All bacterial & mycobacterial investigations, incl IGRA negativeHBV, HCV and HIV serology negative

Bordetella pertussis DNA detected in sputum 11/2015Human metapneumovirus detected in sputum 04/2016EBV detected serum 04/2016 – below level of quantificationFaecal microbiological studies: negative

Memory B cells 7.9% B cells (NR > 11.0%)Switch memory B 0.08% PBLs (NR > 0.4%)CD21-lo B cells 54% B cells (NR < 20%)→ Freiberg phenotype Ia

TACI and BAFF receptor expression normalT-regs 3.9% CD4 T-cells (NR 4.5 – 12.3%)

Genetics:- PIK3CD exons 10, 11, 24 + intron/exon boundaries- CTLA4 exons 1–4 + intron/exon boundaries NAD - awaiting whole genome sequencing

Bone marrow biopsy (02/09/15): - mildly hypercellular marrow, occasional atypical lymphocytes- no clonal lymphoid population detected by flow cytometry

Liver biopsy (17/09/15):- necrotising granulomatous inflammation – no organisms seen

Gastroscopy and colonoscopy (27/11/15): unremarkable macroscopically- chronic gastritis- small bowel and colonic biopsies:

no inflammation, parasites, dysplasia or malignancy

PET 29/03/16:- small pulmonary nodules - non FDG avid - no FDG avid lymphadenopathy - marked splenomegaly, mod hepatomegaly, w/o suspicious hypermetabolism

CT neck/chest/abdomen/pelvis (07/09/15):- multiple hepatic hypodensities (to 15 mm) and lung nodules (4-10 mm)- para-aortic, mesenteric and inguinal lymphadenopathy (to 11 mm)- marked hepatosplenomegaly with evidence portal hypertension - thickened gastric antrum

Brother, GG

35 year old male

1. Obesity Class III, current weight 153.6 kg, BMI ~ 50 kg/m2

2. Obstructive sleep apnoea – BIPAP

3. Asthma – since childhood – multiple ICU admissions but never intubated4. Recurrent bronchitis as a child5. Upper and lower respiratory tract infections most winters from early 20s6. Perineal abscess 1992 7. Septic shock – unknown cause – two separate hospital presentations with ICU admission 20138. Cellulitis lower limbs ~ 3x annually

9. Secondary polycythaemia 201310. BM bx: dyserythropoiesis – myeloid and platelet precursors NAD11. Lymphopaenia 200912. Thrombocytopaenia 201313. Hypogammaglobulinaemia (2011–13) IgG 4.45–6.64, IgA 0.40 and IgM < 0.18 g/L 14. CT chest-abdomen-pelvis 2013:

splenomegaly, posterior mediastinal lymphadenopathy and mild left upper lobe bronchiectasis

Brother, GG

IgG 6.9 g/LIgG1 4.966, IgG2 0.554, IgG3 0.206, IgG4 < 0.003 g/LIgA 0.36 g/L (0.75 – 3.8 g/L)IgM < 0.10 g/L (0.40 – 3.10 g/L)

Hb 180 g/L (115 – 165 g/L)Platelets 115 x 109 /L (150 – 400)Neutrophils 2.4 x 109 /L Lymphocytes 550 /μL (1000 – 4000 /μL)CD19+ 72 /μL (80 – 430 /μL)CD3+CD8+ 143 /μL (200 – 690 /μL)CD3+CD4+ 226 /μL (380 – 1390 /μL)NK 82 /μL (85 – 500 /μL)

Switch mem B cells 0.07 % PBMC (NR > 0.4% PBMC)CD21-lo B cells 28 % total B cells (NR < 20% PBMC) → Freiberg phenotype Ia

No clonal B cell populations by flow cytometry

Summary

• Recurrent sinopulmonary infections

• Daily watery diarrhoea

• Pulmonary nodules and granulomatous hepatic nodules with splenomegaly

• Autoimmune phenomena, including possible autoimmune periodontitis, alopecia, arthralgia small joints of hands

• Hypogammaglobulinaemia

• B, T and NK lymphopaenia with Frieburg 1a immunophenotype, and decreased T reg cells

• Consanguinous parentage, brother with milder clinical phenotype, similar immunophenotype

• Initial genetic studies at CHW: PIK3CD and CTLA4 exon studies NAD

• Awaiting STAT1 and STAT3 exon sequencing and WGS

Bogaert et al, J Med Genet 2016

Genes associated with CVIDs23

> 5017

18

> 50

LPS-responsive beige-like anchor protein (LRBA) deficiency

2012

16 families with putative recessive inheritance of severe, early-onset humoral immune deficiency with autoimmunity- genome-wide SNP typing, genetic linkage analysis, DNA sequencing

four distinct homozygous mutations in the gene encoding LRBA protein – chromosome 4q31

Gámez-Díaz et al,JACI 2016

22 LRBA deficient patients

Age of onset symptoms0.6 – 17 years

Gámez-Díaz et al,JACI 2016

4 pts B-cell counts < 1% of total lymphocytes

+ adequate T-cell proliferation to mitogens

+ partial redn or total absence specific Ab titers after vaccination

LPS-responsive beige-like anchor protein (LRBA) deficiency

Homozygous deleterious mutations in 10 patients and compound heterozygous mutations in 12 patients lack of LRBA protein expression

LRBA protein - 2851 AA residues - one of largest intracellular proteins- cytosolic protein expressed in hematopoietic, neural, GI and endocrine cells- implicated in intracellular vesicle regulation- regulates the post-translational expression of CTLA4

LRBA deficient patients decrease in total intracellular and cell-surface CTLA4- similar phenotype to CTLA4 haploinsufficiency

LRBA-deficient B-cells defective autophagy, increased apoptosis andarrested proliferation

Treatment of autoimmune/inflammatory phenomena with abatercept & hydroxychloroquine

Gámez-Díaz et al, JACI 2016Alkhairy et al, J Clin Immunol 2016Lo et al, Science, 24 July 2015

NF-κB 1&2 deficiencies

Autosomal dominant NF-κB1 deficiency

Fliegauf et al,Am J Hum Genet, 2015

Autosomal dominant NF-κB2 deficiency

Cook et al, Blood, 2014

Chen K et al, Am J Hum Genet, 2013

NF-κB signaling B cell maturation, survival, differentiation, class switching and tolerance to self-antigens

Missense mutation in NF-κB2 failure p100 phosphorylation blocks processing to p52

appears to be due to disruption of canonical and noncanonicalNF-κB pathways by mutant p100 molecule

Proband - complete B-cell deficiency - diagnosed with CVID age 40 - long history chronic sinusitis, bacterial pneumonia, recurrent intestinal giardiasis, periodontitis- alopecia areata at age 14 years- all 3 affected individuals exhibited alopecia areata, with no other evidence of other autoimmunity

17 patients total: RTI, GI infections, localised herpes simplex infections, onychomycosis, bronchiectasis, pituitary hormone deficiencies (mainly ACTH deficiency), AI (mainly involving skin, hair and nails).

Autosomal dominant NF-κB2 deficiency

Alopecia? NF-κB2 important for central T-cell tolerance mediated by negative selection and T reg induction

18 patients from three different families with AD inheritance CVIDDutch-Australian, German and New Zealand

RTI, GI infections, bacterial skin infections

AI (mainly involving blood cells, gut, hair and thyroid gland),pyoderma gangrenosum, bronchiectasis, chronic lungdisease, LIP, BLH, splenomegaly, hepatomegaly, malignancy3 patients had alopecia!

NF-κB1 mutations also identified in relatives withmilder forms of Ab deficiency (eg, sIgAD, IgG subclass deficiency) and even in some clinically healthy relatives.

Am J Hum Genet, 2015

Thanks!

Dr Ming Wei Lin

Dr Jocelyn Jiang

Dr Melanie Wong and Dr Andrew Williams

Prof Stuart Tangye and CIRCA consortium

Discussion points

(ACTH levels, pulmonary function tests, T cell immunophenotyping and functional studies)

Other clinical screening investigations in our patient?

Any other targeted exome studies to consider, NF-κB 1 & 2?

To what extent should we investigate her brother GG?

And her children?

Thanks!

Dr Ming Wei Lin

Dr Jocelyn Jiang

Dr Melanie Wong and Dr Andrew Williams

Prof Stuart Tangye and CIRCA consortium