1. August 2014 SCID … MAJOR TYPES AND EMERGENCY MANAGEMENT Alan Davidson Paediatric Haematology/Oncology Red Cross Children’s Hospital Cape Town, South Africa

2. PRIMARY IMMUNODEFICIENCY Where do we start? PIDs are not as rare as previously thought … Bousfiha et al estimate about 900000 Africans with PID PIDs usually present with recurrent infections but clinical manifestations are very variable A positive family history is the single most important warning sign … was reported in 30% of patients reported to the SA PID Registry Early diagnosis:  [1] facilitates early institution of vital supportive care and immunoglobulin replacement  [2] allows for early consideration of stem cell transplantation

3. SEVERE COMBINED IMMUNODEFICIENCY We’ve come a long way … Human SCID was first described by Eduard Glanzmann and Paul Riniker in 1950. They described Swiss infants who were profoundly lymphopaenic and died of infections before their first or second birthdays. Incidence 1 per 50-100000 births Higher in certain populations e.g. Navajo Within two decades the first successful transplant was performed in Minneapolis (1968)

4. SEVERE COMBINED IMMUNODEFICIENCY Characterized by: Block in T lymphocyte differentiation that is variably associated with abnormal development of B lymphocytes and natural killer cells. To recap T cell development HSCs  lymphoid cells, which differentiate into T, B or NK cells … T cell progenitors migrate to the thymus and elaborate a surface TCR complex..

5. SEVERE COMBINED IMMUNODEFICIENCY TCR function requires: TCR (α and β chains) CD3 (five polypeptides) CD45 (tyrosine phosphatase) TCR development requires: CD1, CD2 and the IL-2 Receptor VDJ rearrangement by recombinase activating genes RAG1 and 2. Mature T cells become MHC II restricted (CD4+) or MHC I restricted (CD8+), while autoreactive cells are removed. CD4+ T lymphocytes require expression of CD40 ligand in order to interact with B cells.

6. SEVERE COMBINED IMMUNODEFICIENCY Activation of the TCR requires: Elevation of Intracellular Ca ++ Lipid mediators such as IP3 and diacylglyerol Activation of protein kinase C Phosphorylation and Activation of TKs … ZAP-70 and Lck Defects in at least 14 different genes cause SCID. In 2007 the IUIS noted that were at least 150 different forms of PID resulting from defects in over 120 distinct genes.

7. CLASSIFYING SCID … Where do we start? Clinical Phenotype Immunophenotype Genotype

8. SCID … THE PHENOTYPES SCID is characterized by: Severe, recurrent infections in infancy Oral thrush, Chronic diarrhoea and failure to thrive Skin rash which may represent Eczema or GvHD Autoimmune disease Death by 18-24 months Manifestations are protean …

9. SCID … THE PHENOTYPES Omenn syndrome Severe form with erythroderma and desquamation, alopecia, chronic diarrhoea, FTT, lymphadenopathy+hepatosplenomegaly, eosinophilia and elevated serum IgE levels. Typically … hypomorphic but not deleterious mutations in genes responsible for VDJ recombination (e.g. RAG 1&2 and Artemis). In the absence of proper regulation clonally expanded T cells promote autoimmune as well as allergic inflammation. May be indistinguishable from SCID with maternal T cell GvHD! Leaky and Variant SCID Which are milder forms resulting from incomplete or unidentified gene defects that usually occur at a later age of onset. May also present with OS!

10. SCID … IMMUNOPHENOTYPES The International Union of Immunological Societies Expert Committee for Primary Immunodeficiency updates it’s classification every two years, and the latest version was published in Frontiers in Immunology in April 2014 … http://journal.frontiersin.org/Journal/10.3389/fimmu.2014.00162/ The current classification (which added 30 new gene defects) divides combined deficiencies into those with (e.g. AT, WAS, dGS) and those without non-immunological abnormalities, which are still classified according to their immunophenotype …

11. Immunophenotypes in SCID. Gaspar H B et al. Blood 2013;122:3749-3758 ©2013 by American Society of Hematology

12. SCID … IMMUNOPHENOTYPES From the point of view of severity … Typical SCID – describes cases with fewer than 300 autologous T cells per litre Leaky SCID – describes cases due to incomplete mutation(s) in a typical SCID gene, T cells ranging from 300– 1,500 per litre and may have a later age of onset of clinical symptoms Variant SCID – describes cases with no known gene defect and a persistence of 300–1,500 T cells/L that have impaired function

13. SEVERE COMBINED IMMUNODEFICIENCY One Child’s Journey Diagnosis of SCID made … WBC 7.63 Neutrophils 5.29 Lymphocytes 0.83 IgG 8.39, IgM 0.01, IgA 0 >>> 10/07/2012 >>> Dear Colleagues Result (CD subsets) of baby attached. Kind regards

14. SEVERE COMBINED IMMUNODEFICIENCY One Child’s Journey T-cell Subsets were repeated at the Red Cross Children’s Hospital … T- B+ NK- confirmed

15. SCID … THE GENOTYPES 170 consecutive cases over 3 decades at Duke University Medical Centre in North Carolina …

16. SCID … THE GENOTYPES ImmunophenotypeDiseaseInheritanceClinical Features T-B+ SCID NK-Common γ-chain deficiency (46%) Janus kinase-3 deficiency (6%) XL AR Growth failure. (GHrec failure) May be OS / LPD with HD features. NK+IL-7 receptor α-chain deficiency (10%) CD45 deficiency CD3  /CD3  /CD3  deficiency Coronin-1A deficiency AR May be OS. Autoimmmune manifestations. Mediates Tcell migration ex Thymus so the organ is still present. T-B- SCID NK-Adenosine deaminase deficiency (15%) ic accumulation of phosphorylated adenosine/ deoxyadenosine which is toxic to lymphoid precursors Reticular dysgenesis Adenylate kinase 2 gene AR AR Deafness, Behavioural and cognitive problems, liver toxicity. Wide spectrum of phenotypes. Severe SCID with sensorineural deafness. NK+RAG-1 / RAG-2 deficiency DCLRE1C (Artemis) deficiency DNA PKcs deficiency Cernunnos deficiency AR Commonly OS (EBV lymphoma). Radiosensitive. May be OS. Radiosensitive. Radiosensitive. Dev delay and microcephaly. RAG = recombination activation gene DNA PKcs = DNA-dependant protein kinase catalytic subunit DCLRE1C = DNA cross-link repair 1C

17. SCID … THE GENOTYPES ImmunophenotypeDiseaseInheritanceClinical Features T-B+ SCID NK-Common γ-chain deficiency (46%) Janus kinase-3 deficiency (6%) XL AR Growth failure. (GHrec failure) May be OS / LPD with HD features. NK+IL-7 receptor α-chain deficiency (10%) CD45 deficiency CD3  /CD3  /CD3  deficiency Coronin-1A deficiency AR May be OS. Autoimmmune manifestations. Mediates Tcell migration ex Thymus so the organ is still present. T-B- SCID NK-Adenosine deaminase deficiency (15%) ic accumulation of phosphorylated adenosine/ deoxyadenosine which is toxic to lymphoid precursors Reticular dysgenesis Adenylate kinase 2 gene AR AR Deafness, Behavioural and cognitive problems, liver toxicity. Wide spectrum of phenotypes. Severe SCID with sensorineural deafness. NK+RAG-1 / RAG-2 deficiency DCLRE1C (Artemis) deficiency DNA PKcs deficiency Cernunnos deficiency AR Commonly OS (EBV lymphoma). Radiosensitive. May be OS. Radiosensitive. Radiosensitive. Dev delay and microcephaly. RAG = recombination activation gene DNA PKcs = DNA-dependant protein kinase catalytic subunit DCLRE1C = DNA cross-link repair 1C The RAG and ARTEMIS family DS DNA breaks are introduced by RAG1 and RAG2 … this creates hairpin structures which are repaired by the Nonhomologous End Joining (NHEJ) pathway: Artemis (whose endonucleolytic activity is promoted by DNA-dependant protein kinase) DNA ligase Cernunnos The RAG and ARTEMIS family DS DNA breaks are introduced by RAG1 and RAG2 … this creates hairpin structures which are repaired by the Nonhomologous End Joining (NHEJ) pathway: Artemis (whose endonucleolytic activity is promoted by DNA-dependant protein kinase) DNA ligase Cernunnos

18. SCID …MAKING THE GENETIC DIAGNOSIS By convention immunophenotyping guides the choice of genetic tests in order to: [1] confirm the diagnosis, especially where the phenotype is unusual [2] facilitate genetic counselling. Nowadays some geneticists are encouraging the use of genetic testing to screen for PIDs (Morra et al IACNA 2008) but screening for specific defects can result in false negatives (presence of maternal T cells) or false positives (novel variants). It must be kept in mind that any genotype can be modified by: (1) penetrance (2) modifier genes (3) environmental factors.

19. SCID … a new kid on the block … NEONATAL GENETIC SCREENING One important (and unique) benefit would be the ability to withhold BCG in these children, which is otherwise not feasible because it’s a critical component of the campaign to control TB. COST? DoH requires an ICER (incremental cost-effectiveness ratio) of US $10000 per DALY (disability-adjusted life year)

20. TREC (Tcell receptor excision circle) testing on neonatal dried blood spots is an excellent biomarker of T lymphopaenia … either due to poor production or increased loss. 70% of maturing thymocytes that will express αβ-TCRs form a circular DNA TREC from the excised TCR δ gene that lies within the TCRα gene locus. They do not increase following cell division and thus represent newly formed T cells. They can be measured by PCR. They are being rolled out in several US states.

21. IT’S SCID! … WHAT NEXT? Pursue Transplantation as the only cure! Provide Meticulous Supportive Care Infection control practices Adhering to vaccination policies Rigorous Surveillance Antibiotic Prophylaxis Treat infections aggressively Don’t forget Nutritional Support Adhere to Blood Product policies

22. PURSUING TRANSPLANTATION EARLY Dx and SCT IS CRUCIAL! [1] Large European SCID Cohort (n=475) SCT under 6 months was better, and patients with active infections like viral pneumonitis did worse (p<0.0001). [2] London and Newcastle Units (n=60) Compared SCID diagnosed antenatally or at birth between 1982 and 2010 with those for 48 family members (mostly siblings) previously diagnosed with SCID between 1979 and 2009. Probands: median age 143.5 days; 35% died pre-SCT; OS of 40%. Relatives: median age 0 days ; 1 patient died pre-SCT; OS was 90%. The improvement was independent of donor source, the conditioning regimen used and the underlying diagnosis.

23. PURSUING TRANSPLANTATION Alert the relevant transplant centre … Arrange tissue typing (SABMR) If there are no full siblings … or … any full siblings are HLA non-identical … consider an alternative donor source: Matched unrelated donors Umbilical Cord Blood Haploidentical donor When, how and what tomorrow …

24. SUPPORTIVE CARE: INFECTION CONTROL Isolation in (positive pressure) laminar flow facility Strict contact precautions Limit visitors, and no infectious caregivers and doctors Dedicated nursing staff

25. SUPPORTIVE CARE: VACCINATION AND SURVEILLANCE Avoid all live vaccines, including BCG if possible … This may be possible if there is a family history If BCG has been given rule out disseminated BCG infection … which can occur in at least 1/3 of cases. Rule out mTB Monitor for active EBV and CMV by PCR Do an NPA for RV panel and PJP in the event of any LRTI

26. SUPPORTIVE CARE: PROPHYLAXIS AND TREATMENT Septran, Fluconazole and Acyclovir prophylaxis Immunoglobulin replacement (1g/kg per week) Treat any systemic infections early and aggressively with broad spectrum antibiotics Pay attention to nutrition … resort to TPN if necessary Blood products must be filtered AND irradiated in order to prevent GvHD.

27. SCID RESOURCES … You can get help from: Immunology colleagues NHLS, Labs and the SABMR Transplant Centres (Cape Town and Pretoria) There a number of regional and local organisations …