key: cord-0015479-cl3unqmz
authors: Vignesh, Pandiarajan; Rawat, Amit; Kumrah, Rajni; Singh, Ankita; Gummadi, Anjani; Sharma, Madhubala; Kaur, Anit; Nameirakpam, Johnson; Jindal, Ankur; Suri, Deepti; Gupta, Anju; Khadwal, Alka; Saikia, Biman; Minz, Ranjana Walker; Sharma, Kaushal; Desai, Mukesh; Taur, Prasad; Gowri, Vijaya; Pandrowala, Ambreen; Dalvi, Aparna; Jodhawat, Neha; Kambli, Priyanka; Madkaikar, Manisha Rajan; Bhattad, Sagar; Ramprakash, Stalin; CP, Raghuram; Jayaram, Ananthvikas; Sivasankaran, Meena; Munirathnam, Deenadayalan; Balaji, Sarath; Rajendran, Aruna; Aggarwal, Amita; Singh, Komal; Na, Fouzia; George, Biju; Mehta, Ankit; Lashkari, Harsha Prasada; Uppuluri, Ramya; Raj, Revathi; Bartakke, Sandip; Gupta, Kirti; Sreedharanunni, Sreejesh; Ogura, Yumi; Kato, Tamaki; Imai, Kohsuke; Chan, Koon Wing; Leung, Daniel; Ohara, Osamu; Nonoyama, Shigeaki; Hershfield, Michael; Lau, Yu-Lung; Singh, Surjit
title: Clinical, Immunological, and Molecular Features of Severe Combined Immune Deficiency: A Multi-Institutional Experience From India
date: 2021-02-08
journal: Front Immunol
DOI: 10.3389/fimmu.2020.619146
sha: d0d2d36d37d7c52debd87dbd18d2885b972eb360
doc_id: 15479
cord_uid: cl3unqmz

BACKGROUND: Severe Combined Immune Deficiency (SCID) is an inherited defect in lymphocyte development and function that results in life-threatening opportunistic infections in early infancy. Data on SCID from developing countries are scarce. OBJECTIVE: To describe clinical and laboratory features of SCID diagnosed at immunology centers across India. METHODS: A detailed case proforma in an Excel format was prepared by one of the authors (PV) and was sent to centers in India that care for patients with primary immunodeficiency diseases. We collated clinical, laboratory, and molecular details of patients with clinical profile suggestive of SCID and their outcomes. Twelve (12) centers provided necessary details which were then compiled and analyzed. Diagnosis of SCID/combined immune deficiency (CID) was based on 2018 European Society for Immunodeficiencies working definition for SCID. RESULTS: We obtained data on 277 children; 254 were categorized as SCID and 23 as CID. Male-female ratio was 196:81. Median (inter-quartile range) age of onset of clinical symptoms and diagnosis was 2.5 months (1, 5) and 5 months (3.5, 8), respectively. Molecular diagnosis was obtained in 162 patients - IL2RG (36), RAG1 (26), ADA (19), RAG2 (17), JAK3 (15), DCLRE1C (13), IL7RA (9), PNP (3), RFXAP (3), CIITA (2), RFXANK (2), NHEJ1 (2), CD3E (2), CD3D (2), RFX5 (2), ZAP70 (2), STK4 (1), CORO1A (1), STIM1 (1), PRKDC (1), AK2 (1), DOCK2 (1), and SP100 (1). Only 23 children (8.3%) received hematopoietic stem cell transplantation (HSCT). Of these, 11 are doing well post-HSCT. Mortality was recorded in 210 children (75.8%). CONCLUSION: We document an exponential rise in number of cases diagnosed to have SCID over the last 10 years, probably as a result of increasing awareness and improvement in diagnostic facilities at various centers in India. We suspect that these numbers are just the tip of the iceberg. Majority of patients with SCID in India are probably not being recognized and diagnosed at present. Newborn screening for SCID is the need of the hour. Easy access to pediatric HSCT services would ensure that these patients are offered HSCT at an early age.

Background: Severe Combined Immune Deficiency (SCID) is an inherited defect in lymphocyte development and function that results in life-threatening opportunistic infections in early infancy. Data on SCID from developing countries are scarce.

Objective: To describe clinical and laboratory features of SCID diagnosed at immunology centers across India.

Methods: A detailed case proforma in an Excel format was prepared by one of the authors (PV) and was sent to centers in India that care for patients with primary

Severe Combined Immune Deficiency (SCID) is an inborn error of immunity characterized by defect in T lymphocyte development and function. Children with SCID often develop life-threatening opportunistic fungal, bacterial, or viral infections in early infancy. SCID is considered a medical emergency and affected children often succumb to severe infections if diagnosis and definitive treatment are delayed. The estimated incidence of SCID is 1 in 50,000 to 100,000 live births (1) . Recent data also suggest an incidence of SCID as high as 1 in 3,000 live births in countries with high consanguinity rates (2) . However, due to lack of awareness and diagnostic facilities in developing countries, diagnosis is often missed. Hematopoietic stem cell transplantation (HSCT) is the definitive management for SCID. Early diagnosis and management are essential for successful outcomes. Several countries such as United States of America, Israel, Germany, Switzerland, Sweden, Norway, Iceland, New Zealand, and Taiwan have initiated newborn screening for SCID based on quantification of T-cell receptor excision circles (TRECs) to facilitate early diagnosis (3) .

Opportunistic infections in SCID are recurrent, typically start in early infancy, and result in failure to thrive. Common infection patterns seen in SCID include oral thrush, disseminated BCGosis, disseminated cytomegalovirus, and lifethreatening bacterial and fungal infections. Non-infective manifestations of SCID include Omenn syndrome (OS), graft versus host reaction, autoimmunity, and hemophagocytic lymphohistiocytosis (4) . CD3+ T lymphocyte numbers are usually decreased in SCID (T-). However, in cases of maternal T-cell engraftment or OS, CD3+ T cell numbers can be normal or increased. The expanded T cells are autoreactive in OS, whereas, they are alloreactive in cases with transplacentalacquired maternal T-cell engraftment. T lymphocyte function and naïve T cell numbers are reduced in such cases. T-SCID can be classified based on presence or absence of B lymphocytes and natural killer cells as T-B-NK+, T-B-NK-, T-B+NK-, and T-B+NK+. Combined immunodeficiencies (CID) are also characterized by presence of opportunistic infections and immune dysregulation; however, the age of onset is little older and have a milder immunodeficiency compared to SCID (5) .

Until date, 58 different monogenic defects have been identified to result in immunodeficiencies affecting both cellular and humoral immunity and 18 amongst these are known to result in SCID (5) . Molecular defects in SCID can be broadly classified as abnormalities in VDJ recombination (RAG1, RAG2, DCLRE1C, NHEJ1, LIG4, PRKDC), abnormalities of cytokine signaling (IL2RG, JAK3, IL7RA), toxic metabolite accumulation (ADA, PNP), defective survival of hematopoietic precursors (AK2, RAC2), abnormalities of T-cell receptor and signaling (PTPRC, CD3D, CD3E, CD3Z, LAT), and abnormalities of actin cytoskeleton (CORO1A). While X-linked SCID due to defect in IL2RG is considered to be the commonest form of SCID in the US, Canada, and Europe, autosomal recessive form of SCID due to defects in RAG1/2 are the commonest forms of SCID in countries where consanguinity rates are high (6) (7) (8) . However, after initiation of newborn screening program, defects in RAG1/RAG2 are now increasingly being identified even in countries like US and Canada where consanguinity rates are low (9) .

Reports of clinical data and outcomes of SCID from developing nations are scarce. Being a tropical nation with universal coverage of BCG vaccination in newborns, microbiological pattern of infections in SCID in India is expected to be different from other cohorts. Molecular spectrum is also expected to be different considering high rates of consanguinity and endogamous marriages in India (6) (7) (8) . A recent cohort of 57 patients from Mumbai, India showed a high incidence of autosomal recessive forms of SCID with RAG1/2 defects being the commonest (7) . We aim to describe the clinical, immunological, and molecular features of children with SCID in this large multicentric cohort from India.

A detailed case proforma in an Excel format was prepared by one of the authors (PV) and was sent to centers that are recognized as Foundation for Primary Immunodeficiency Diseases (FPID) centers for care of primary immunodeficiencies in India. The format was also sent to tertiary-care centers that manage patients with primary immunodeficiency diseases (PIDs). Information on clinical, laboratory, and molecular details of patients with SCID and their outcomes was sought and collated. Twelve (12) centers provided details of 319 patients that were then compiled and analyzed. Fifteen (15) patients from 2 other centers with either flow-cytometry or mutation-proven SCID are not included in final analysis as data were incomplete. Twenty-three (23) children did not fulfil the criteria for clinical definition for SCID and were not included for analysis. Duplicate entries (n=4) were also noted and excluded.

Data of 277 children who had a clinical profile suggestive of SCID were taken for final analysis (Supplementary Table 1 ). Children were categorized as SCID/OS/CID/atypical SCID as per the European Society for Immunodeficiencies (ESID) working definition (10) . Three (3) patients were classified as possible SCID as they did not fulfil the complete ESID definition, however, the treating team had a high index of suspicion based on clinical and immunological features ( Table 1) .

Clinical profile of all patients was obtained along with family history and other demographic details. Clinical features included number of infections, type of infections, site of infections, organism involved, age of presentation, age of onset, presence of skin rash, BCG ulceration, history of administration of vaccines and complications, if any. Basic hematological, biochemistry, and immunological investigations including immunoglobulin profile and lymphocyte subsets were also recorded. Analysis of lymphocyte subsets by flow cytometry had been carried out in most patients. Methodology for laboratory assay of lymphocyte subsets, naïve, memory T cells, HLA-DR expression, CD132 expression, CD127 expression, and lymphocyte proliferation assays at Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh and National Institute of Immunohematology (NIIH), Mumbai have been previously described (11, 12) . Other centers performed conventional lymphocyte subsets (CD3, CD19, CD4, CD8, CD56) by flow cytometry in private laboratories.

Adenosine deaminase (ADA) levels and percentage of deoxyadenosine nucleotides (%dAXP) from dried blood filter paper spot were assayed at Duke University, North Carolina for patients with ADA deficiency SCID who were diagnosed at PGIMER, Chandigarh.

Before the facility for in-house next-generation sequencing was made available in 2018, centre at PGIMER, Chandigarh had established academic collaborations with centers at Hong Kong (The University of Hong Kong), Japan (Kazusa DNA Research Institute, Kisarazu, Chiba; National Defense Medical College, Saitama), and USA (Duke University, North Carolina) for molecular work-up of patients. The centre at Hong Kong provided final molecular diagnosis for 12 patients (Pt. 8-10, Pt. 14-19, Pt. 21, Pt. 50-51) ( Table 1 ). Molecular diagnosis for 4 patients was established at Kazusa DNA Research Institute, Japan (Pt. [3] [4] [5] [6] . Thirty-four (34) patients (Pt. 59-90, pt. 119, pt. 127) with SCID were worked-up for molecular diagnosis using NGS at National Defense Medical College, Saitama and Tokyo Medical and Dental University, Tokyo, Japan (Kato T et al. manuscript in submission) . Final molecular diagnosis of a patient with ADA defect (pt. 22) was also established at Duke University, North Carolina.

Sanger sequencing for IL2RG and RAG1/2 genes were initiated at PGIMER, Chandigarh (North India) in 2016. Sanger sequencing for patients with SCID at NIIH, Mumbai (West India) was previously described by Aluri et al. (7) . Methodology for NGS at Christian Medical College, Vellore (South India) was described previously (13) .

Next-generation sequencing (Ion Torrent, Thermo Fisher Scientific India Pvt. Ltd.) for clinical care was started in July 2018 at the Advanced Pediatrics Centre, PGIMER, Chandigarh. A targeted PID gene panel comprising 44 genes was used that covered 6 genes for SCID-ADA, RAG1, RAG2, IL2RG, IL7RA, and LIG4. Preparation of DNA target amplification reaction using 2-primer pools, amplification of target, combination of target amplification reactions, ligating adaptors to the amplicons and their purification was carried out as per the manufacturer's protocol using Ion AmpliSeq ™ Library kit plus (Catalog numbers 4488990, A35121 A31133, A31136, A29751, 4479790). Amplified library was quantified using Qubit ™ 2.0 fluorometer instrument. Dilution that results in a concentration of~100pm was then determined. Template preparation on Ion One Touch ™ Instrument, recovery, washing and enrichment of template-positive ISPs was done as per the manufacturer's protocol using Ion 520 ™ and Ion 530 ™ Kit-OT2 (catalog number A27751). Ion S5 ™ sequencer instrument was then initialized. Annealing of primers to enriched ISPs and chip loading was carried out using Ion 520 and 530 Loading Reagents OT2 Kit. Sequencing run was initiated and Torrent Browser was used to review results. Raw data were analyzed on Ion Reporter software and on integrative genome viewer. NGS using a targeted gene panel was also performed for some patients (n = 6) in private laboratories (Medgenome Labs Pvt. Ltd., India).

Other centers in India obtained molecular testing results from private laboratories (Medgenome Labs Pvt. Ltd., India; Strand Genomics Pvt. Ltd., India; Neuberg Anand Diagnostics Pvt. Ltd., India). Illumina platform was used for sequencing in private laboratories with coverage of >80X. Sanger sequencing was used to confirm variants obtained by NGS.

SALSA MLPA probe-mix P368 DCLRE1C kit was used in this protocol. MLPA was performed according to the instructions provided by the manufacturer (MRC Holland). 50-100ng/µL of DNA was denatured in thermocycler and hybridized with 1.5 µL of probe-mix along with 1.5µL of MLPA buffer. Content was mixed and incubated for 1 min at 95°C followed by incubation at 60°C for 18 h. After hybridization, probes were ligated using a ligase mix at 54°C for 15 min. Ligase was inactivated at 98°C for 5 min. PCR was performed using PCR primers, polymerase, buffers and required amount of water. Following conditions were used for amplifications-95°C for 20 s, 65°C for 80 s, for 35 cycles, followed by a final extension for 20 min at 72°C. ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used for capillary electrophoresis. Later, 0.7µL of PCR reaction, 8.9µL of HI-DI formamide, and 0.4µL of DNA standard LIZ 600 provided by GeneScan were mixed and then denatured for 2 min at 95°C. The sample was then loaded and MLPA data were analyzed using a Coffalyser software.

Current study included data of patients diagnosed and managed at centers in Northern, Southern, and Western parts of India. Amongst the 277 patients, 254 were categorized as SCID (208 -SCID; 17atypical SCID; 26 -OS; 3possible SCID) and 23 as CID ( Table 1) Male-female ratio was 196:81 ( Table 1) . Median [interquartile range (IQR)] age of onset of clinical symptoms and diagnosis was 2.5 months (1, 5) and 5 months (3.5, 8), respectively. Consanguinity was noted in 78 families (28.2%), and was noticeably more in Southern region (32.3%) of our country compared to Northern (22.4%). Family history of early childhood deaths was noted in 120 children (43.3%). Median (IQR) age at diagnosis in children who had a positive family history was 4.5 months (3, 6) compared to 6 months (4, 9) in children who did not have a family history, p<0.05 (Mann-Whitney U test).

Opportunistic infections were the presenting manifestation in most patients. These included pneumonia (82%), diarrhoea (43.7%), oral thrush (18.4%), BCG site ulceration (17%), otitis media (12.6%), and meningitis (4%) (Figures 2, 3) . Bloodculture proven septicemia was seen in 63 children (23%)-Candida sp. Disseminated cytomegalovirus (CMV) infection was documented in 23 (8.3%) children and 6 amongst these had evidence of CMV retinitis. Intestinal lymphangiectasia due to CMV was noted on autopsy of a child with X-linked SCID (pt.8).

Prolonged excretion of vaccine-derived poliovirus was documented in a child with leaky SCID at Mumbai (14, 15) . Vaccine-associated paralytic poliovirus strain was also isolated in a child with RAG1 defect at Mumbai. He had presented with persistent diarrhea, developmental delay, and hypotonia. Clinical features of OS were seen in 33 children (11.9%)classical OS in 11 and incomplete OS in 22 (Figure 3) . Molecular defects associated with OS include RAG1 (7), RAG2 (5), ADA (2),

(1), and RFXANK (1). Two children with IL2RG defect had features of engraftment of transplacental-acquired maternal T cells that mimicked clinical features of OS (Figure 4) . Warm autoimmune hemolytic anemia (AIHA) requiring immunosuppressive medications was observed in 5 children. While anemia responded to intravenous (IV) methylprednisolone pulses in 2 patients (RAG1 and NHEJ1 defect each), pt.42 with STK4 defect received IV rituximab (375 mg/m 2 2 doses) for control of AIHA and she did not have further relapse of AIHA for next 1.5 years. Transfusion-associated graft-vs-host reaction was documented in 4 patients (2 X-linked SCID; 2 AR-SCID); all had development of rash and transaminitis following transfusion of non-irradiated blood products. Four (4) children had features of hemophagocytic lymphohistiocytosis (HLH). Possible triggers for HLH included disseminated BCG (2) and H1N1 (1) infections. The child with SP110 defect did not have any identifiable trigger for HLH (pt.104). Hodgkin lymphoma and intra-cranial B cell lymphoma were noted in children with RAG1 and CORO1A defects, respectively.

Four of 18 children with ADA defect were noted to have radiographic abnormalities-scapular spurring and flattening of lower end of scapula (Figure 3) . Glomerular involvement was seen in 4 children-3 children with OS and 1 with atypical/leaky SCID. Nephrotic range proteinuria was noted in 3 patients and one child (pt.13) had features of mesangial sclerosis on autopsy. Another child (pt. 12) with OS had features of focal segmental glomerulosclerosis on autopsy. One child (pt.10) with IL7RA defect had features of distal renal tubular acidosis and nephrocalcinosis. This patient had deletion of exons 2-5 of CAPSL along with exon 4-8 deletion of IL7RA in chromosome 5p13.2. A child with PNP defect (pt.14) had evidence of horseshoe kidney at autopsy (16) .

Median (IQR) absolute lymphocyte count (ALC) observed was 1.33 × 10 9 /L (0.6, 2.5). Normal ALC (≥ 3 × 10 9 /L) was observed in 51 children (18.4%)-of these 26 had OS, 2 had transplacental-acquired maternal T-cell engraftment, and 23 had leaky SCID/combined immunodeficiency. Eosinophilia was observed in 37 children, and 26 amongst these had features of OS. One child (Pt. 105) with RAG1 defect had unexplained monocytosis (2.7-3.0 × 10 9 /L) that resolved after HSCT. Results of immunoglobulin profile was available for 198 children. Table 2 ). We observed decreased naïve (CD3+CD45RA+) and elevated memory (CD3+CD45RO+) CD3 lymphocytes in 24 children with OS. We noted elevated HLA-DR expression in CD3+ lymphocytes in 15 children with OS. CD132 expression by flow cytometry showed reduced expression in lymphocytes or monocytes in 8 children with suspected X-linked SCID ( Table 2 ) (17) (18) (19) . Levels of ADA and %dAXP were measured in 7 children with ADA SCID and 2 heterozygous carriers of ADA mutation ( Table 3) . Table 4, Supplementary Table 3 ) (7, 13, . A novel variant in RAG1 (c.1758_1760delinsGAATC) was identified in 2 unrelated North Indian families. Deletion of exons 1-3 (8947bp) in DCLRE1C was observed in 11 children (9 from North and 2 from South India). MLPA confirmed EX1_EX3del in DCLRE1C in 7 children from North India ( Figure 5) . Targeted clinical exome sequencing by NGS did not identify pathogenic variants in 25 patients. Whole exome sequencing was performed in 5 children, and pathogenic variants were detected in 2 amongst these (pt. 50 and 51).

Majority of patients (n=198) in this cohort succumbed to overwhelming infections as HSCT could not be carried out in them ( Figure 6 ). Twenty-three patients (8.3%) underwent hematopoietic stem cell transplantation (HSCT) and 11 are doing well post-HSCT. The centre at South India (Apollo Children's Hospitals, Chennai) has performed HSCT for 32 children with SCID until now and 17 are alive and doing well on follow-up. However, only 4 children are included in this analysis, as flow cytometry and mutation details were not available for other children. Another centre in South India (Aster CMI Hospitals, Bengaluru) has carried out HSCT for 9 children with SCID in the last 3 years ( Table 5 ).

We describe the largest multi-centric cohort of patients with SCID from India. We included patients from 12 different tertiary care centers located in Northern, Southern, and Western parts of India. Patients from Eastern parts of India are usually referred to the centers located in other areas of India due to lack of availability of facilities for immunological investigations in that region. We witnessed an exponential rise in the number of cases with SCID after 2013 at multiple centers across India. We attribute this steady increase in cases to 2 factors-establishment of Indian Council of Medical Research Centers for Advanced Research in PIDs at PGIMER, Chandigarh (North India) and NIIH, Mumbai (West India) and expansion of laboratory facilities for pediatric immunology at other centers. The Pediatric Immunology and Bone Marrow Transplant Unit at Aster CMI Hospital, Bengaluru (South India) was established in 2017. Twenty-seven cases of SCID (Pt. were diagnosed between 2017 and 2020, reflecting rise in awareness amongst referring pediatricians and better availability of diagnostic facilities at Bengaluru (South India).

Based on data from Sample Registration System of India, we estimated around 221 million live births from January 2011 to June 2020 (50) . An estimated 257 patients with SCID have been diagnosed in this time period, which suggests a rough incidence of SCID at 0.12 per 100,000 live births. Though we have included data from most of the centers that care for patients with SCID in India, the estimated incidence from this study may not reflect true incidence of the country because of retrospective nature of the study and some patients diagnosed at other centers may have been missed. Nation-wide registry for SCID is needed for an accurate estimation of incidence. Nevertheless, if we extrapolate our current data on to the U.S. incidence figures of SCID (i.e. 1:58,000 live births), estimated number of children with SCID in India would be around 3,822 during the period 2011-June 2020 (1). Moreover, incidence of SCID in India is expected to be even higher than the U.S. considering high rates of consanguinity within the country. This suggests that though we have been increasingly diagnosing these children over the last few years, the diagnosis is still missed in almost 93% of these children. This is clearly unacceptable and mandates urgent intervention of health care professionals.

We observed a higher incidence of autosomal recessive forms of SCID (78.4%) compared to X-linked SCID. This is similar to reports from several other countries where consanguinity rates are high (Table 6) (7, 8, (51) (52) (53) (54) (55) (56) (57) (58) (59) . Though consanguinity rate of 28.2% observed in our study is lower than that of Saudi Arabia and Iran, practice of endogamous and intra-community marriages is, perhaps, responsible for high proportion of autosomal recessive forms of SCID in India (2, 6) . Median age at diagnosis of SCID in our study is 5 months. This is similar to reports from other countries such as China, Turkey, and U.S.A ( Table 6) . Children who had a family history of SCID had an earlier age of diagnosis (median:4.5 months) compared to children who did not have a suggestive family history (median:6 months). Our observation is similar to the report by Luk et al. that suggested the importance of family history for an early diagnosis of SCID (17) .

Opportunistic infections in SCID are life-threatening and must be identified and treated adequately before HSCT. We documented a higher incidence of microbiologically-proven infections in our cohort compared to a previous report published from India (7) . Amongst the bacterial infections, BCG was the commonest organism isolated. BCG-site ulceration has been noted in 47 children, however, disseminated BCGosis could be proven in 27 children only. BCG adenitis was noted in one child at D+90 post-HSCT as a part of immune reconstitution inflammatory syndrome. Lack of microbiological confirmation of BCG infection in many patients could have accounted for low rates of disseminated BCGosis in our cohort ( Table 6 ) (51, 52). Infants with SCID who had received BCG vaccination and had not developed disseminated infection, are generally started on prophylactic medications-isoniazid and rifampicin at ageappropriate doses, that is generally continued until successful engraftment following HSCT. Septicemia due to unusual organisms such as W. confusa and A. faecalis was also noted in our cohort. These are environmental bacteria and usually do not cause invasive infections in immunocompetent hosts. We also noted a high rate of disseminated CMV infection (8.3%) in our cohort. However, several amongst these were identified only on autopsy (60) . This underscores the importance of vigilant screening and preventive measures for CMV infection in children with SCID. Cytomegalovirus infection, in our cohort, was possibly transfusion-acquired as most of the children received blood transfusions that are not always leuko-depleted and screened for active CMV infection. Though many patients had clinical features suggestive of P. jirovecii pneumonia (tachypnea, hypoxemia, interstitial pneumonia) and were treated for the same, microbiological or histopathological confirmation was possible in only 8 of them.

Thirty-three children had features of OS in our cohort. One child with OS (pt.54) was being treated as severe eczema for 3 years with multiple topical and systemic immunosuppressive agents, and diagnosis of SCID was made only after he developed severe infections. This highlights the importance of early identification of clinical phenotype of OS based on clinical features (generalized erythematous rash with scaling and partial loss of scalp hairs and eyebrows) and referral for appropriate immunological workup. Twenty-eight (28) children with OS had normal or high ALC and 2 children with transplacental-acquired maternal T-cell engraftment had elevated ALC. Laboratory assay of naïve T cells, memory T cells, and HLA-DR expression in T lymphocytes necessary for the diagnosis of OS are currently being performed only in two centers (PGIMER, Chandigarh and NIIH, Mumbai).

Twenty-three children in our cohort who did not have OS had normal ALC (>3 × 10 9 /L). However, lymphocyte subsets and naïve T cell estimation revealed diagnosis of SCID in them, thereby highlighting the importance of clinical suspicion and immunological investigations in infants with severe and lifethreatening infections even if ALC is normal. Expansion of B cells or NK cells, engraftment of transplacental-acquired maternal T cells, or partial genetic defects allowing selective clone of T cell expansion could be the possible reasons for normal ALC in SCID. Aluri et al. have previously highlighted the importance of assessment of naïve T helper and cytotoxic T cells in children with severe infections and normal ALC to characterise MHC class II and ZAP70 defects, respectively (7) . A child with IL7RA defect in our cohort had a T-B+NK-phenotype, similar to the report by Aluri et al. (7) . Also, two children with IL2RG defect had a T+B-NK-phenotype (1-OS, 1-transplacental-acquired maternal T cell engraftment). A possible explanation for low B cells is the depletion of B cells due to high inflammatory milieu secondary to OS and severe infections (61) .

CD132 expression by flow cytometry is currently carried out at only two centers-PGIMER, Chandigarh (North India) and NIIH, Mumbai (West India). At PGIMER, Chandigarh, we found low CD132 expression in lymphocytes by flow cytometry as an inexpensive and rapid method of confirmation of diagnosis of X-linked SCID in 7 children. Two (2) children with X-linked SCID and previously reported variants in IL2RG (pt. 25 and pt. 78) had a normal expression of CD132 in lymphocytes ( Table 2) . We could not assay phosphorylated STAT5 in activated T-cells by flow cytometry to determine the functionality of IL2Rg in many patients due to absent or very low amounts of T cells, however, naïve T cells by flow cytometry and TREC levels by RT-PCR have been assayed in some of them ( Table 2) . Only a handful centers in India (e.g. PGIMER, Chandigarh, North India, and NIIH, Mumbai, West India) have the wherewithal to perform functional studies. Both the centers have performed flow cytometry tests for samples received from other centers, however, timely transportation of viable blood samples from far off places, especially during hot summers remains a significant problem (11, 12) . Lack of stateof-the-art facilities to do functional assays in all patients with SCID is one of the limitations of our study. Establishment of more clinical immunology laboratories, training of necessary manpower, and improvement in existing laboratory services are needed to overcome these barriers (11, 12) . Genetic confirmation of diagnosis of SCID is necessary for identification of pattern of inheritance and genetic counselling of affected families. Eighty-two (82) patients did not undergo a molecular analysis for confirmation of diagnosis due to lack of easy access to molecular diagnostics and financial difficulties. With the establishment of commercial NGS laboratories and reduction in costs involved for genetic sequencing over last few years, NGS-based diagnostics have become feasible in India (7, 13) . In-house NGS facility for molecular diagnosis of PID is currently available only at PGIMER, Chandigarh (North India) and Christian Medical College, Vellore (South India). Most of the patients with SCID present in a critically-ill state and convincing families for genetic studies is often challenging due to significant financial and social constraints. It must be noted that expenses for molecular diagnosis are borne by the families in India most of the times as it is not covered by state or insurance schemes. Despite these challenges, we have been able to perform genetic studies in 195 patients. Academic collaborations with institutes at Hong Kong, Japan, and USA helped the centre at PGIMER, Chandigarh (North India) to get free molecular diagnosis for the families who cannot afford for costly molecular tests. We prefer to store blood samples in terminally-ill patients and later call the family for counselling to undergo genetic tests, as confirmation of molecular diagnosis has helped the families to undergo antenatal testing in subsequent pregnancies.

Defects in RAG1/2 were found to be commonest in our cohort followed by IL2RG, DCLRE1C, and ADA. This is similar to the previous reports from Turkey, Iran, and Serbia ( Table 6 ) (8, 52, 54) . MHC Class II defect and defects in STIM1, DOCK2, SP110, ZAP70, and STK4 genes are categorized as combined immunodeficiencies as per the 2019 International Union of Immunological Societies Expert Committee classification of human inborn errors of immunity (IEI) (5) . However, we have included children with these defects in our cohort because they had severe infections from early infancy mimicking the clinical presentation of SCID (7) .

Clinical phenotype of patients with RAG1/2 defects in our cohort was very heterogenous. This included classical SCID, OS, atypical/leaky SCID phenotype, autoimmunity in form of AIHA, and development of hematological malignancy such as Hodgkin lymphoma. Wide spectrum of clinical manifestations could be due to difference in VDJ recombination activity or influence of other genetic or environmental factors (34, 62) . Other reported clinical phenotypes in RAG1/2 such as cutaneous granulomas, CVID-like phenotype or elevated gd T cells were not seen in our cohort.

Low or undetectable ADA levels and elevated %dAXP levels were seen in 7 and 5 children with ADA defect, respectively. We noted that %dAXP levels in 2 children (pt. 31 and 36) were lower compared to other 3 children. While pt.31 had a milder clinical phenotype, pt.36 had features of OS. This suggests that low levels of accumulation of dAXP with residual ADA activity in lymphocytes may play a role in development of restricted Tcell clones that could be responsible for partial immunity and development of OS (63). One child with ADA defect (pt. 36) had evidence of nephrotic syndrome along with OS. Renal abnormalities described with ADA defect (such as diffuse mesangial sclerosis) could result in nephrotic syndrome (64) . However, renal involvement in OS manifesting as nephrotic syndrome has also been previously reported (65) . We could not perform renal biopsy in this child due to severe ascites. Two other children with OS in our cohort also had renal involvement at autopsy-focal segmental glomerulosclerosis and mesangial sclerosis. Both of them also had severe infections-disseminated CMV in one and invasive aspergillosis in other. Whether the renal abnormalities are the result of genetic defect, inflammatory phenotype of OS, or severe infections is not clear and further research is needed in this regard.

Identification of radiosensitive forms of SCID is essential in B-NK+ SCID before HSCT as these children are prone to toxicity by chemotherapeutic drugs and radiation (66) . Amongst the radiosensitive forms of SCID, molecular defects are predominantly noted in DCLRE1C in our cohort. Moreover, only mutation in DCLRE1C observed in North Indian children (n=9) was EX1_EX3 del. Initial MLPA screening for DCLRE1C A significant proportion of children (n=254) could not be subjected to HSCT due to medical and social reasons and succumbed to the illness. Presence of fulminant infections at time of diagnosis and lack of financial support dissuaded many families to undergo a costly procedure like HSCT. At present, facilities for pediatric HSCT for IEI are available at very few centers in India. Two centers in India have carried out most of the transplants for SCID -Apollo Children's Hospitals, Chennai (South India) and Aster CMI Hospitals, Bengaluru (South India). Establishment of such dedicated pediatric HSCT units and development of manpower for HSCT services across the country are the need of the hour to ensure easy access to these services for affected patients. Provision of financial support from the government to affected families to undergo HSCT will also be required for successful outcomes. Studies from Western countries have shown that children with SCID transplanted below the age of 3.5 months of age had a significantly better outcome compared to children who underwent transplantation later (58) . Though the age at diagnosis in our cohort is similar to countries where newborn screening has not been initiated, delayed referrals, presence of life-threatening infections at presentation, and lack of easy access to pediatric HSCT accounted for the unacceptable mortality rates in our cohort ( Figure 6 ) ( Table 6 ) (51, 52). We also realise that diagnosis of SCID is still being missed in most babies in India. Institution of universal newborn screening for SCID would provide more accurate estimates of incidence of SCID in our country and would also facilitate early diagnosis and treatment. However, financial implications and cost-effectiveness of implementing such a programme in a country as large, and as diverse, as India need to be worked out by health planners (69) .

To conclude, we describe the largest multicentric cohort of SCID from India and document several novel mutations. Number of children with molecular diagnosis and those who BCG-related complications in 10 children (23.8%) -6 had localized reaction; 3 had regional adenitis; 3 had disseminated BCGosis. Oral thrush (12), CMV (2), and PJP (2) (3) 20 had proven molecular defect (50%). ADA commonest (10) have undergone HSCT has increased significantly in last decade. However, we are only too aware of our limitations. Improvement in awareness amongst physicians and pediatricians, expansion of diagnostic laboratories, institution of newborn screening, development of pediatric HSCT services, and financial support to the families to undergo HSCT are essentially needed for a better diagnosis and outcome of affected patients in the country.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article. 

Laboratory work-up of patients; provided necessary laboratory results for compilation. KG-Provided necessary histopathology details

PRKDC (1), PNP (1), and AK2 (1) Mortality observed in 47 children (82.5%). Four (4) underwent HSCT and none survived

OS noted in 33 children. AIHA and lymphoreticular malignancy observed in 5 and 2 children

PRKDC (1)

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Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

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The authors thankfully acknowledge Centre d'Etude des Defcits Immunitaires, Hopital Necker-Enfants Malades, Paris, France for carrying out the molecular analysis for one of the patients (Case 152).The authors also thankfully acknowledge Mr. Jitendra Kumar Shandilya, Ms. Jhumki Das, and Ms. Kanika Arora, PhD students in Allergy Immunology Unit, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India for assisting in flow cytometry experiments.