The Changing Diagnostic Landscape of Primary Immune Deficiency

Transcription

1 The Changing Diagnostic Landscape of Primary Immune Deficiency Dr. Sabeena Selvarajah Paediatric Immunology Fellow, Princess Margaret Hospital for Children,

2 Term infant girl of non-consanguineous parents Eczema, intermittent diarrhoea, failure to thrive, persistent oral candida and functional asplenia. Six months age: Prolonged admission with PJP complicated by pulmonary hypertension and a enterobacter line infection. Ongoing issues with eczema, possible drug allergies and atypical drug eruptions. Persistent mild CD3/CD8 and CD19 lymphopenia. Good proportion of naïve versus memory cells. T regulatory cells 11% Normal T cell proliferation on two occasions Hypogammaglobinemia (IgG/A/M) at 6 months, replacement commenced. Persistent eosinophilia Raised IgE which subsequently normalised. Dependent on TPN for weight gain.

3 x 1 0 * 6 / L C D 4 A b s o l u t e C o u n t C D 4 A b s o l u t e C o u n t ( x 1 0 * 6 / L ) H H H L L L A p r J u l O c t J a n A p r C A S L E Y, P O P P Y

4 x10*9/l Eosinophils Absolute Eosinophils Absolute (x10*9/l) H H H L 0.0 Jan 2015 Apr Jul Oct Jan 2016 CASLEY, POPPY Apr Jul

5 CLINICAL AND LABORATORY SCID (Hypomorphic ) Dock 8 deficiency MHC Class 1 Deficiency Omenn s Syndrome AR Hyper IgM IPEX-like syndrome Zap-70 deficiency

6 MDM A 6 month old girl with an undefined disorder of her immune system but which is (increasingly) phenotypically in keeping with a dysregulation disorder (akin but not the same as IPEX). This would potentially explain her persistent oral candida, opportunistic infection (PJP), eczema, raised IgE and eosinophilia. Decision to transplant rather than wait for another life limiting infection. This was based entirely on the clinical phenotype. The laboratory phenotype did not correlate. DNA sent to Westmead Children for extended T cell panel however decision made to transplant regardless. The panel was unremarkable as was an non-validated panel conducted at FSH in Perth. Matched unrelated donor transplant on the 12 th November Conditioning regime included treosulfan, fludarabine and alemtuzumab.

7 POST TRANSPLANT ISSUES Engrafted within 2-3 weeks. Full donor chimerism 2 weeks post transplant. Post transplant rash initially attributed to antibiotics Biopsy confirmed atypical skin Grade 2 GvHD Difficulty weaning steroids and stopping mycophenolate. Remains on both medications. Initial issue with ongoing loose stool however GI biopsy normal and stools now formed (Peptijunior) July 2016 Fracture of distal tibia after minimal trauma.

8 PID GENES SELECTED FOR DIAGNOSTIC NGS PANEL Combined Immunodeficiencies Predominantly antibody deficiences Diseases of immune dysregulation Congenital defects of phagocyte numbers, function or both Defects in innate immunity Autoinflammatory Disorders Complement deficiencies Phenocopies of PID ADA DOCK8 BAFFR PRF1 RAC2 IRAK4 NLRP3 C2 NRAS ADAMTS13 IL7R MAGT1 CD19 UNC13D CYBB MYD88 PSTPIP1 C3 KRAS RAG1 STK4 MS4A1 (CD20) STX11 NCF1 UNC93B1 NOD2 CFI C1IN RAG2 TRAC1 CR2 (CD21) STXBP2 NCF2 CARD9 MVK CFH CD40 IL21R CD81 LYST NCF4 IL17RA TNFRSF1A CD46 LIG4 UNC119 ICOS XIAP IL12B MEFV ITGB2 IL2RG IKBKG TACI FOXP3 IL12RB1 PLCG2 CFP DCLRE1C PIK3CD BTK AIRE IFNGR1 NLRP12 CFB TAP1 CD27 NFKB2 TNFRSF6 IFNGR2 CFHR1 TAP2 STAT3 NFKB1 CASP8 STAT1 CFHR3 TAPBP TYK2 AICDA CASP10 GATA2 CFHR5 RFXANK STAT5B UNG FADD CYBA THBD RFXAP IL21 FASLG MPO RFX5 CTLA4 SLC35C1 (FUCT1) CIITA JAK3 ITK CD45 ZAP70 CORO1A PNP CD3 complex CD8A CD3G CD40LG SH2D1A Others ADA, CD27, CD3GCD40, CD40LG, CD8A, CIITA, DCLRE1C, DOCK8, IL21R, IL2RG,IL7R,ITK,, LIG4,MAGT1,PIK3CD,PNP,RAG1,RAG2,RFX5,RFXANK,RFXAP,RMRP,RMRP,SH2D 1A, STK4,TAP1,TAP2, TAPBP, TRAC, UNC119, ZAP70

9 KEY ISSUES Discordance between clinical phenotype and laboratory results difficulty categorising Decision to transplant without a genetic confirmation How to obtain a moleular diagnosis. The ethics of now pursing a molecular diagnosis. Determine prognosis Treatment including novel treatments (underlying mechanism understood) and conditioning regimes Gene therapy Reproductive choices (family and proband) How to interpret relevant findings, what to do with incidental findings.

10 TRADITIONAL APPROACH Knowledge of molecular pathways/classification systems leading to Sanger sequencing of exons or mutation harboring regions of candidate genes SNP arrays, Mouse models, clinical-pathological correlation Linkage analysis: use SNP flanking pathogenic mutations to define the disease loci shared by affected subjects. Requires large families with multiple affected individuals. (LRBA). Targeted Increased depth of sequencing coverage higher accuracy, improved sensitivity Reduces risk of incidental findings Can be cheaper Low likelihood of identifying novel mutations

11 ARGUMENTS FOR WES/WGS More 240 genes associated with PID, >30 discovered in the last 3 years Clinical heterogeneity and overlap Traditional approach may not be suitable for ambiguous or atypical phenotypes Discovery of novel mutations

12 WHOLE EXOME SEQUENCING The human genome contains 3.2million base pairs. Only 1% represents the exome (coding regions). Pathogenic mutations are commonly contained within the exome WES should therefore be more cost effective than WGS. WES can be less efficient as depth of coverage can be poor. Coverage refers to how many times a base pair is sequenced. WES with homozygosity mapping HM identifies genetic regions most likely to harbour pathogenic mutation based on the assumption that the causative mutation for an ARD occurs with a locus containing clusters of homozygous SNPs specific to the affected subjects and inherited from a common ancestor

13 DISADVANTAGES OF WES How to interpret the data from WGS/WES Public databases of known polymorphism are used to sort common polymorphisms from rare variants False positive rate as high as 36% in some studies Up to 10% of SNV can be previously unreported WES does not detect intronic mutations. Intronic mutations in IL7 and IL2RG have been reported to cause SCID. Intronic mutation in UNC13D (familial HLH), SH2D1 and NEMO have also been reported. A homozygous intronic mutation in ZAP-70 has also been reported to be cause disease.

14 DISADVANTAGES WES/WGS Incidental findings (particularly in the paediatric context) No consensus; no legislation In 2013 the American Society of Genetics and Genomics released a policy statement that constitutional mutations of the genes on the minimum list should be reported regardless of the clinical indication for sequencing. The minimum list includes 56 mutations, some of which occur in adult hood (BRAC1/2, cardiomyopathy, Marfan s syndrome, long QT, polyposis syndromes) It is the responsibility of the requesting physician to provide pre and post test counselling. Opting out is not encouraged. Autosomal dominant disease is more difficult to detects (69% more heterozygous than homozygous variants in ay genome) How to integrate polygenetic causes, epigenetics etc.

15 ACKNOWLEDGMENTS AND REFERENCES Professor Peter Richmond, Dr. Michael O Sullivan, Dr Melanie Wong, Andrew Williams, Dianne De Santis, Pendapala Namundjebo, Linh Truong, A/Prof Lloyd D Orsogna Raje N, Soden S, Swanson D, Ciaccio C, Kingsmore S, Dinwiddie D. Utility of Next Generation Sequencing in Clinical Primary Immunodeficiencies. Curr Allergy Asthma Rep. 2014;14(10) Platt C, Geha R, Chou J. Gene hunting in the genomic era: Approaches to diagnostic dilemmas in patients with primary immunodeficiencies. Journal of Allergy and Clinical Immunology. 2014;134(2): Chou J, Ohsumi T, Geha R. Use of whole exome and genome sequencing in the identification of genetic causes of primary immunodeficiencies. Current Opinion in Allergy and Clinical Immunology. 2012;12(6): Picard C, Dogniaux S, Chemin K, et al. Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity. Eur J Immunol 2009; 39: )