Profile of 208 Patients With Inborn Errors of Immunity at a Tertiary Care Center in South India

doi:10.21203/rs.3.rs-1425451/v1

Download PDF

Research Article

Profile of 208 Patients With Inborn Errors of Immunity at a Tertiary Care Center in South India

https://doi.org/10.21203/rs.3.rs-1425451/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Primary immune deficiencies better known as Inborn Errors of Immunity (IEI) are a heterogeneous group of disorders that predispose affected individuals to infections, allergy, autoimmunity, autoinflammation, and malignancies.. Inborn Errors of Immunity are increasingly being recognized in the Indian subcontinent. Two hundred and eight patients diagnosed with an IEI during February 2017 to November 2021 at a tertiary care centre in South India were included in the study. The clinical features, laboratory findings including microbiologic and genetic data, treatment & outcome details were analyzed. The diagnosis of IEI was confirmed in a total of 208 patients (198 kindreds) based on relevant immunological tests and/or genetic tests. The male to female ratio was 1.8:1. The most common IEI in our cohort was SCID (17.7%) followed by CGD (12.9%) and CVID (9.1%). We also had a significant proportion of patients with DOCK8 deficiency (7.2%), LAD (6.2%), and six patients (2.8%) with autoinflammatory diseases. Autoimmunity was noted in forty-six (22%) patients during the course of their illness. Molecular testing was performed in 152 patients by exome sequencing on NGS platform and a genetic variant was reported in 132 cases. Twenty-nine children underwent 34 HSCT, and 135 patients remain on supportive therapy such as immunoglobulin replacement and/or antimicrobial prophylaxis. Fifty-nine (28.3%) patients died during the study period and infections were the predominant cause of mortality. Seven families underwent prenatal testing in the subsequent pregnancy. We describe the profile of 208 patients with IEI and to the best of our knowledge, this represents the largest data on IEI from the Indian subcontinent reported so far.

Inborn Errors of Immunity

Immune Deficiency

Hematopoietic stem cell transplant

Severe Combined Immune Deficiency

Primary immune deficiencies (PIDs) better known as Inborn errors of immunity (IEI) are clinically and genetically, a heterogeneous group of disorders characterized by abnormalities of one or more components of the immune system that predispose affected individuals to infections, allergy, autoimmunity, autoinflammation, and malignancies. More than 430 genetically defined single-gene IEI have now been recognized [1]. Depending on the severity of the disease, they can manifest in the neonatal period and early childhood or present during adulthood [2].

Conventionally, IEI were thought to be a rare group of diseases, however, studies report the prevalence rates as high as 1:1200 in the general population [3], and the incidence is expected to be even higher in areas where endogamy is widely practiced. In India, very few centers have the clinical experience and laboratory facilities for the diagnosis and management of these conditions. There is no nationwide data on the prevalence of IEI in India, however, experts in the field believe the prevalence to be more than one million [4, 5]. Due to a lack of awareness about these diseases in the medical fraternity, a vast majority of these patients continue to remain underdiagnosed and undertreated. In this paper, we describe the clinical, immunologic, and genetic profile of 208 patients with IEI at a tertiary care center in South India and discuss the challenges that are unique to a resource-constrained setting. To the best of our knowledge, this represents the largest single-center data on IEI from the Indian subcontinent reported so far.

Our hospital is a tertiary care center that caters to the needs of four states in Southern India. It is one of the few centers specialized in the management of IEI and has all the wherewithal necessary to diagnose and treat these patients. The availability of a trained Pediatric Immunologist well versed in the diagnosis and treatment of IEI has been the strength of our center. In the current study, all patients diagnosed with an IEI between February 2017 to November 2021 at our center were included. Clinical and laboratory data were prospectively entered in a pre-designed excel sheet. Children and adults with immune deficiencies secondary to HIV infection, chemotherapy, or chronic immunosuppressive therapy were excluded. The diagnosis of IEI was confirmed based on the characteristic clinical presentation, relevant laboratory data, and genetic analysis. The study was approved by the Institutional Ethics Committee.

Patient details such as the age at onset of symptoms, clinical presentation, age at diagnosis, family history, laboratory findings including microbiologic data, treatment & outcome details were recorded. Clinical details included both infectious and non-infectious manifestations (allergy, autoimmunity, malignancy, etc) at presentation and in the past. A family history with a focus on consanguineous parentage, sibling death if any, and details of the affected family members were obtained. Family history was considered positive in case of an affected family member. Treatment details including antimicrobial prophylaxis, intravenous immunoglobulin (IVIG) replacement therapy (IRT), and immunosuppression, if any, were noted. In patients who underwent a hematopoietic stem cell marrow transplant (HSCT), the details of the type of transplant, and their outcomes were recorded.

Immunological evaluation

Immunological work-up included serum immunoglobulin profile, nitro blue tetrazolium test, flow cytometry-based analysis of peripheral blood lymphocyte subset and dihydrorhodamine assay, and CD11/18 expression on neutrophils. Dihydrorhodamine assay for the diagnosis of CGD was performed using a modification of the procedure described by O’Gorman and Corrochano [6]. Post phorbol myristate acetate (PMA) stimulated tubes were always processed in duplicate to rule out processing-related causes for a negative DHR assay. Transport control samples were processed in parallel and used for comparison whenever available. Lymphocyte subset enumeration (T cells, B cells, NK cells) was performed on a bead-based single platform assay using a commercial CE-IVD approved test kit (Multitest 6-Color TBNK Reagent, BD Biosciences) and analyzed on an IVD certified 8 color flow cytometer instrument (BD FACS Canto2) using the accompanying software. Immunoglobulin levels were estimated on a nephelometer (Seimens Atelica Neph 630) using a commercially available kit. Appropriate internal and external controls were used for all the above assays.

Genetic analysis

A total of 152 cases were evaluated via whole-exome sequencing (WES) on the next-generation sequencing (NGS) platform as per standard protocols. Briefly, genomic DNA was extracted and libraries were prepared using a custom capture kit. Paired-End Sequencing was performed with 2x100/2x150 chemistry using NovaSeq 6000 (Illumina Inc., San Diego, CA). Sequenced reads were assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome buildGRCh37/UCSC hg19. Variants were filtered based on clinical history utilizing relevant HPO terms and only variants with a minimum read depth of 10X and Phred score > 30 were included in the analysis. All reported variants were Sanger confirmed. The observed variants were classified according to the American College of Medical Genetics criteria [7]. Only a phenotype-based analysis and reporting was performed for all cases. Incidental findings were not analyzed and reported. Cases with Pathogenic (P)/Likely Pathogenic (LP) variants consistent with the inheritance pattern were considered to have a confirmed diagnosis. Cases with a Variant/s of Uncertain Significance (VUS) in the presence of corroborative ancillary immunological workup were considered to have a putative diagnosis (PD). The diagnostic yield was inclusive of both confirmed and putative cases. Pre-test counseling was done and informed consent was obtained for all the patients under study.

Prenatal screening

The prenatal screening was carried out in seven pregnant mothers with mutation proven IEI in the previous child. Amniocentesis, chorionic villus sampling, and cordocentesis were performed for prenatal diagnosis and their records were analyzed as well.

Statistical methods

Clinical and laboratory data was analyzed using Statistical Package for Social Sciences (SPSS version 20.0). Pearson Chi-square test was performed for the categorical data and a P value < 0.05 was considered to be statistically significant.

A total of 208 patients (198 kindreds) with suspected IEI were evaluated based on relevant immunological tests and/or genetic tests. Of the 208 patients, 72 (34.6%) were < 1 year, 112 (53.8%) were ≤ 18 years, 24 (11.5%) were above 18 years. The mean age at onset of first clinical presentation in children (< 18 yrs) was 1.24 years (range 1 day to 17 years) and in adults (≥ 18 yrs) was 19 years (range 18–62 years). In our cohort, male to female ratio was 1.8:1 (134 males, 74 females). The mean age at diagnosis was 3.9 years in children and 37 years in adults, with a mean lag time in the diagnosis being 4.5 years (2.7 years in children and 18.3 years in adults). Positive family history was noted in 47 (22.5%) patients, with a history of sibling death in 34 (16.3%) cases. Sixty-three children (30.2%) were born from consanguineous parents.

The most common IEI in our cohort was Severe Combined Immunodeficiency (SCID) (n = 37, 17.7%) followed by Chronic granulomatous disease (n = 27, 12.9%) and Common Variable Immune Deficiency (CVID) (n = 19, 9.1%). We also had a significant proportion of patients with DOCK8 deficiency (n = 15,7.2%) and Leukocyte Adhesion Deficiency (n = 13,6.2%). We did not have any patients with phenocopies of IEI. Our cohort also included six patients (2.8%) from five kindreds diagnosed with autoinflammatory diseases such as Blau syndrome (n = 1), Chronic recurrent osteomyelitis (CRMO n = 2), TNF receptor-associated periodic fever syndrome (TRAPS, n = 1), and Familial Cold Autoinflammatory Syndrome (FCAS, n = 2). Details of their clinical manifestations have been tabulated in Table 1.

Table 1

Clinical manifestations, organism profile, and outcomes of 208 patients with Inborn Errors of immunity (IEI).
SNo.	IEI	No	Mean age at presentation (years)	Infectious manifestations (n)	Organisms isolated (n)	Non-infectious manifestations (n)	Outcome
1.	SCID	37	0.42	Pneumonia (29) Diarrhea (8) Otitis media (3) Septicemia (18) Rash (4) Abscess (2) BCG adenitis (2) Disseminated BCG (4) Oral thrush(9) Osteomyelitis(1)	Acinetobacter (1) Proteus (1) Mycobacterium bovis (7) CMV (2) Candida (6)	Rib abnormalities (1)	Alive (5) Died (32) HSCT (11)
2.	CGD	27	4.3	Pneumonia (20) Osteomyelitis (3) Lymphadenitis (10) Abscess (2) Cellulitis (1) Pyoderma (2) Disseminated BCG (3) BCG adenitis (2) HLH (1) Lupus vulgaris (1) Hepatitis (1) Pericarditis (1) Sepsis (2) Meningitis (2)	Staphylococcus aureus (6) Aspergillus (1) Burkholderia pseudo mallei (1) Klebsiella (2) Mycobacterium bovis (5) Mycobacterium tuberculosis (3) Cladophialophora (1) Malassezia restricta (1) Salmonella (1) Acinetobacter (1)	Colitis (5) Seizure (1) HLH (1) Sarcoidosis (1) Uveitis (1)	Alive (19) Died (4) HSCT (4) LF (4)
3.	CVID	19	30.4	Pneumonia (7) Diarrhea (6) Abscess (2) Otitis media (4) Sinusitis (4) Lymphadenitis (1) Bronchiectasis (2)	Clostridium difficile (1) Aspergillus fumigatus (1)	Arthritis (2) AIHA (1) ITP (2) Lupus (1) Alopecia areata (1) Nail dystrophy (1) Colitis (3) Non Hodgins lymphoma (1)	Alive (18) Died (1)
4.	DOCK8 deficiency	15	6.4	Pneumonia (7) Diarrhea (1) Otitis media (6) Warts (2) Bronchiectasis (2) Oral thrush (2) Cellulitis (1)		Alopecia areata (1) Atopic dermatitis (10) Mucosal hyperpigmentation (12) AIHA (2) Kawasaki disease (1) Lymphadenopathy (1) Plexiform neurofibroma (1) Vasculitis (1)	Alive (9) Died (4) HSCT (2) LF (2)
5.	XLA	14	7.3	Pneumonia (7) Empyema (1) Recurrent Otitis media (2) Cellulitis (2) Gangrene (1) Diarrhea (2) Pyoderma (2) Septic Arthritis (1) Meningitis (2) Sepsis (2) Bronchiectasis (1)	Haemophilus influenzae (1) Pneumococcus (1) Staphylococcus aureus (1)	TIA (1) Arthritis (1) Kawasaki disease (1)	Alive (13) Died (1)
6.	LAD	13	0.95	Pneumonia (6) Omphalitis (3) Otitis media (6) Cellulitis (1) Abscess (2) Diarrhea (1)	Pseudomonas aeruginosa (2) SARS-CoV-2 (1)	Pyoderma gangrenosum (4) Bleeding diathesis (1) VSD (1) Delayed cord fall (3)	Alive (7) Died (5) HSCT (5) LF (1)
7.	MSMD	7	2.3	Disseminated BCG (4) Lymphadenitis (3) BCG adenitis (3) Pneumonia (1)	Mycobacterium tuberculosis (1) Salmonella typhimurium (1) Candida guilliermondii (1) Enterobacter (1) Chryseobacterium indologenes (1)		Alive (5) Died (1) LF (1)
8.	AD HIES	6	10.6	Pneumonia (3) Cellulitis (1) Pyoderma (3) Orbital cellulitis (1) Oral thrush (5) Lymphadenitis (1) Abscess (1) Bronchiectasis (1)	Aspergillus (1) Mycobacterium abscessus (1)	Allergic rhinitis (1) Eczema (2) Hyperextensible joints (1)	Alive (6)
9.	Cyclic neutropenia	6	5.8	Oral ulcers (5) Lymphadenitis (3) Otitis media (3) Mastoiditis (1) Orbital cellulitis (2) Pneumonia (2) Gingivitis (2) Skin infection (2) Oral thrush (2) Umbilical abscess (1) Diarrhea (1)		Kawasaki disease (1)	Alive (6)
10.	Hyper-IgM syndrome	5	4.9	Pneumonia (5) Otitis media (1) Oral ulcers (1) Lymphadenitis (2) Meningitis (1) Septic arthritis (1) Diarrhea (1)			Alive (2) Died (1) LF (2) HSCT (2)
11.	CMC	5	9.3	Recurrent oral thrush (5) Pyoderma (1) Bronchiectasis (1) Diarrhea (1) Sepsis (1)	CMV (1)	Hypothyroidism (1) Vitiligo (1)	Alive (5)
12.	APDS type 1	3	4.6	Pneumonia (2) Otitis media (2) Skin ulcers (1) Parotid abscess (1) Chronic diarrhea (1)	EBV (1)	Lymphadenopathy (1) Splenomegaly (1)	Alive (3)
13.	CARMIL2 deficiency	3	16.7	Pneumonia (2) Esophageal Candidiasis (1) Oral thrush (1) Bronchiectasis (2) Diarrhea (2)	CMV (1) Candida (1)	Atopic dermatitis (1) Esophageal ulceration (1) Pyloric stenosis (1)	Alive (2) Died (1)
14.	CHAI	3		-	-	Colitis (3) Autoimmune hepatitis (1) AIHA (2) Asthma (2) Lymphadenopathy (1) Splenomegaly (1)	Alive (3)
15.	APECED	2	9	Oral thrush (2) Onychomycosis (1)	Candida (2)	Alopecia areata (1) Hypoparathyroidism (1) Addison’s disease (1)	Alive (2)
16.	AT	2	7.35		-	Telangiectasia (1) Ataxia (2)	Alive (2)
17.	Monogenic Lupus	2	11.5	Abscess (1)	-	Lupus (2) Nephropathy (1) Myelopathy (1)	Alive (1) Died (1) HSCT (1)
18.	IgG2 deficiency	2	37	Sinusitis (1) Pneumonia (1) Mastoiditis (1) Diarrhoea (1)	Streptococcus Pneumoniae (1)	-	Alive (2)
19.	VEO-IBD	2	1.5	Chronic diarrhoea (2)	-	AIHA (1) Colitis (2)	Alive (2) HSCT (1)
20.	VODI	2	0.37	Otitis media (1) Pneumonia (1) Oral thrush (1) Sepsis (1) Hepatitis (1)	-	Seizures (1)	Died (1) LF (1)
21.	LRBA deficiency	2	11.7	-	-	Type I diabetes mellitus (1) Colitis (1) Polyarthritis (1)	Alive (2)
22.	Otulopenia	2	24		-	Pyoderma gangrenosum (2)	Alive (2)
23.	FCAS type 1	2	19.5	-	-	Cold induced urticaria (2) Arthralgia (1)	Alive (2)
24.	CRMO	2	7.7	-	-	Recurrent multifocal sterile osteomyelitis (2)	Alive (2)
25.	ALPS	1	0.33	-	-	Lymphadenopathy (1) Hepatosplenomegaly (1)	Died (1)
26.	STIM1 deficiency	1	1.3	Pneumonia (1)		Hypopigmented hair (1) Hypohydrosis (1) Abnormal teeth (1) Hypotonia (1)	Alive (1)
27.	CID	1	2	Diarrhea (1) Oral thrush (1)	-	-	Died (1) HSCT (1)
28.	Griscelli syndrome	1	11	Recurrent Otitis media (1)		Rash (1) Oculocutaneous albinism (1)	Alive (1)
29.	HPS type 7	1	1	Pre-septal cellulitis		Colitis (1) Oculocutaneous albinism (1)	Alive (1)
30.	Idiopathic CD4 lymphopenia	1	45	Meningitis (1)	Cryptococcus neoformans (1)		Alive (1)
31.	Severe congenital Neutropenia	1	0.41	Pneumonia (1)	-	Delayed cord fall (1)	LF (1)
32.	IPEX	1	7	Otitis media (1) Pneumonia (1) Diarrhea (1)	Streptococcus pneumoniae (1) Staphylococcus aureus (1)	Nephrotic syndrome (1)	Alive (1)
33.	JAK1 GOF	1	9	-	-	ITP (1) Lymphadenopathy (1)	Alive (1)
34.	Netherton syndrome	1	6.9	Otitis media (1)	-	Eczema (1) Ganglioglioma (1)	Alive (1)
35.	ORAI1 deficiency	1	1	Diarrhea (1) Pneumonia (1)	-	-	Died (1)
36.	Roifmann syndrome	1	1	Pneumonia (1)	-	Hypocalcemia (1) Developmental delay (1) Cardiac arrest (1)	Alive (1)
37.	X-linked neutropenia	1	1		-	HLH (1)	Died (1) HSCT (1)
38.	Tricohepatoenteric syndrome	1	0.3	Diarrhea (1) Oral thrush (1)	-		LF (1)
39.	STAT 3 GOF	1	3			Autoimmune hepatitis (1) ILD (1)	Alive (1)
40.	SIFD	1	2	Diarrhea (1)		Periodic fever (1) Cardiomyopathy (1)	Died (1) HSCT (1)
41.	BENTA	1	0.2			Splenomegaly (1)	Alive (1)
42.	WAS	5	6.1	Lymphadenitis (1)	Acinetobacter baumanii (1) Campylobacter jejuni (1) Elizabethkingia meningoseptica (1)	Kawasaki disease (1) Dermatitis herpetiformis (1) Bullous pemphigoid (1) AIHA (1) Intra-cranial hemorrhage (1)	Alive (2) Died (1) LF (2)
43.	PGM3 deficiency	1	0.75	Pneumonia (1) Sepsis (1) Abscess (1) Meningitis (1) Diarrhea (1)	RSV (1) Staphylococcus aureus (1) Candida parapsilosis (1)	Eczema (1)	Alive (1)
44.	Blau syndrome	1	2.5	-	-	Polyarthritis (1) Rash (1)	Alive (1)
45.	TRAPS	1	10.5	-	-	Periodic fever (1) Abdominal pain (1)	Alive (1)
AD HIES – Autosomal Hyper IgE syndrome, APDS – Activated Phosphoinositide 3-Kinase Delta Syndrome 1, ALPS – Autoimmune Lymphoproliferative syndrome, APECED – Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, AT – Ataxia Telangiectasia, BENTA – B-cell expansion with NF-kB and T-cell anergy, CGD – Chronic Granulomatous Disease, CHAI – CTLA4 Haploinsufficiency, CID – Combined Immunodeficiency, GOF – Gain of Function, CMC – Chronic Mucocutaneous Candidiasis, CVID – Common Variable Immunodeficiency, FCAS type 1 – Familial Cold Autoinflammatory syndrome type 1, HPS type 7– Hermansky Pudlak Syndrome type 7, IPEX – Immune dysregulation, polyendocrinopathy, enteropathy, X-linked, LAD – Leukocyte Adhesion deficiency, MSMD – Mendelian susceptibility to Mycobacterial Disease, SCID – Severe Combined immunodeficiency, SIFD – Sideroblastic anemia with B-cell Immune deficiency, Periodic Fevers and developmental delay, TRAPS – Tumor necrosis factor receptor-associated periodic syndrome, VEO IBD- Very early onset Inflammatory Bowel disease, VODI – Hepatic Veno-occlusive disease with immunodeficiency, WAS – Wiskott Aldrich Syndrome, XLA – X-Linked Agammaglobulinemia, CMV- Cytomegalovirus, RSV-Respiratory Syncytial Virus, EBV- Ebstein Barr Virus, BCG- Bacillus Calmette Guerin, VSD- Ventricular Septic Defect, ITP- Immune Thrombocytopenic Purpura, AIHA- Autoimmune Hemolytic Anemia, HLH- Hemophagocytic Lymphohistiocytosis, ILD- Interstitial Lung Disease, HSCT- Hematopoietic Stem Cell Transplant, LF- Lost to Follow up.

The most common infectious manifestations were recurrent/persistent pneumonia (n = 98, 47%) and otitis media (n = 33, 15.8%) followed by recurrent /chronic diarrhea (n = 31, 14.9%), oral thrush (n = 26, 12.5%), suppurative lymphadenitis (n = 22, 10.5%), skin/visceral abscesses (n = 12, 5.7%), sepsis (n = 18, 3.8%), meningitis (n = 5, 2.4%) and osteomyelitis (n = 4, 1.9%). While pneumonia and septicemia were more common in patients with SCID, suppurative lymphadenitis was more common in patients with CGD. Lupus vulgaris was seen in a patient with CGD. Bronchiectasis was noted in nine patients and the underlying IEI were CARMIL2 deficiency (n = 2), CVID (n = 2), DOCK8 deficiency (n = 2), X-Linked Agammaglobulinemia (XLA, n = 1), and Chronic mucocutaneous candidiasis (CMC, n = 1), AD Hyper-IgE syndrome (AD-HIES, n = 1). Micro-organisms were isolated on 69 occasions {bacteria = 45 (65%), virus = 7 (10.1%) fungi = 17 (24.6%)} in our cohort. Staphylococcus aureus (n = 9) was the most common bacteria and Candida (n = 11) was the most common fungus isolated. Among the viruses, cytomegalovirus (CMV) was identified in four patients, while one patient had SARS-CoV-2 infection. Unusual infections eg. Cladophialophora and Malassezia restricta were noted in a patient with CGD. Infections with BCG were noted in 18 patients. Patients presenting with local BCG infection were diagnosed to have SCID (n = 2), CGD (n = 2), and MSMD (n = 3) whereas, disseminated BCG infection was noted in patients with SCID (n = 4), CGD (n = 3), and MSMD (n = 4). The clinical manifestations and organism profile have been tabulated in Table 1.

Forty-six (22%) patients presented with or developed one or more autoimmune manifestations during the course of their illness. A total of 59 autoimmune manifestations were documented during the study period. The male to female ratio in this subgroup was 2.5:1. Amongst them, autoimmunity was the key or only manifestation in 15 (32.6%) patients. The most common autoimmune manifestation was inflammatory colitis (34.7%) followed by autoimmune cytopenia (23.9%), pyoderma gangrenosum (13%), and arthritis (8.6%). Inflammatory colitis was common among patients with phagocytic disorders (eg: CGD), whereas autoimmune cytopenia was more common in patients with immune dysregulation and CVID. Autoimmune skin manifestations (bullous pemphigoid, dermatitis herpetiformis) were seen in Wiskott Aldrich syndrome (WAS). Autoimmune endocrinopathies (n = 4), arthritis (n = 4), systemic lupus erythematosus (n = 3), and Kawasaki disease (n = 3) were also reported. Amongst those with autoimmunity, mutations were frequently noted in CYBB, NCF1, NCF2, LRBA, CTLA-4, DOCK8, WAS, and IL10RA. Details of the autoimmune manifestations in various IEI have been tabulated (Table S1). Patients received immunosuppression in the form of steroids (n = 34), azathioprine (n = 5), cyclosporine (n = 2), mesalamine (n = 3), dapsone (n = 1), methotrexate (n = 2), rituximab (n = 3), cyclosporine (n = 2), sirolimus (n = 1), adalimumab (n = 1). The mean delay in arriving at a diagnosis was 8.2 years in the ‘autoimmune cohort’ and this was statistically higher as compared to the rest of the study population (p < 0.001). The overall survival was 76% in the “autoimmune” cohort.

Exome sequencing with CNV calling was performed in 152 probands after informed consent and 132 cases were identified with 144 genetic variants (Table 2). Among the 132 cases, 42 cases were initially identified with VUS, and with the help of ancillary immunological workup, the diagnosis was confirmed in 23 cases (putative diagnosis). The diagnosis was confirmed in 88 cases (including the CNVs) and a putative diagnosis made in 23 cases totalling to a diagnostic yield of 73.02% (111 cases). A total of 126 unique set of variants belonging to 60 genes were identified in the present study cohort, which included 6 unique exon deletions and a microdeletion on chromosome 1 resulted as part of the integrated CNV calling pipeline in our analysis. The observed CNVs were confirmed appropriately via alternate methods like MLPA and microarray. Apart from the exon deletions and microdeletion, the distribution of 117 variants based on their functional consequence is as follows - Acceptor Splice Site Mutation (6), Donor Splice site Mutation (4), Frameshift (17), Inframe Deletion (3), Insertion (3), Missense (54), Missense-Splicesite (1), Noncoding region (2), Nonsense (24), Start loss (2), Synonymous mutation affecting Splice site (1). These 117 variants were classified as per ACMG guidelines into likely pathogenic (60), pathogenic (17) and variants of uncertain significance (42). Of the total variants identified, 90 were found to be novel (absent in population genetic databases or not reported in the literature) and 36 are previously reported in the literature and/or clinical variation database.

WES was performed in 19 patients with CGD and a genetic diagnosis was confirmed in 15 cases. Exome sequencing did not yield any significant variant in three cases of CGD with reduce p47phox expression as well as two cases of LAD who had an absent CD18 expression on neutrophils. The diagnosis was genetically confirmed in 23 of 26 patients with SCID. Pathogenic variants were identified in ITGB2 in six of eight patients with LAD type 1, while two patients with a negative genetic test had absent CD18 expression on neutrophils. In addition to the above cases, the following are the unique (single) cases identified in the study: BENTA, Blau syndrome, CID due to MAP3K14 deficiency, CMC due to STAT1 GOF, IL17RA mutation, TRAF3IP2 mutation; Griscelli syndrome, HPS type 7, IPEX, JAK1 GOF, LAD type 3, PGM3 mutation, Roifmann syndrome, Severe congenital Neutropenia, SIFD, STAT3 GOF, STIM1 deficiency, TRAPS, Trichohepatoenteric syndrome-1, VEO-IBD due to mutation in RIPK1 and IL10RA deficiency, and X-linked neutropenia.

A total of 135 (64.9%) patients remain on supportive therapy such as immunoglobulin replacement, antimicrobial prophylaxis, and/or specific therapy. At the time of writing this paper, 29 patients continue to receive regular IVIG therapy. Co-trimoxazole was the most commonly prescribed prophylactic agent, while itraconazole/fluconazole was used for antifungal prophylaxis. One child with CTLA-4 haploinsufficiency (CHAI) had severe diarrhea that responded to sirolimus. Another child with DOCK8 deficiency had rituximab-refractory autoimmune hemolytic anemia that resolved on sirolimus therapy. Infliximab was used to treat refractory colitis in a child with VEO-IBD due to an IL10RA defect.

Fifty-nine (28.3%) patients died during the study period and infections (pneumonia/sepsis) were the predominant cause of mortality. Mortality was highest amongst patients with SCID. Eighty-nine percent (89%) of patients with SCID (including six patients who underwent an HSCT) died during the follow-up. Mortality was also significant in patients with LAD (5/13, 38.4%), DOCK8 deficiency (5/15, 26.6%) and CGD (4/27, 14.8%). Majority of patients with XLA and CVID continue to remain well on IVIG replacement therapy. Fourteen patients (6.7%) were lost to follow-up.

Twenty-nine children (13.9%) underwent 34 HSCT (11-SCID, 5-LAD type 1, 4-CGD, 2-DOCK8 deficiency, 2-Hyper IgM syndrome, 1-MAP3K14 deficiency, 1-SIFD, 1-VEO IBD, 1-X-linked neutropenia, 1-C1q deficiency). Amongst these, two children with DOCK8 deficiency were transplanted elsewhere and expired due to transplant-related complications. Out of the 32 transplants performed at our center, the majority were haplo-identical (24/32), while three patients underwent Matched Sibling Donor (MSD), four patients underwent Matched Related Donor (MRD) and only one patient had a Matched Unrelated Donor (MUD transplant. Fifteen children underwent a successful HSCT, of which one patient continues to have moderate graft versus host disease (GVHD) and the others are on regular follow-up with stable chimerism whereas 13 children (48%) died during or after transplant due to transplant-related complications. Causes of death included infections (6/13, 4 bacterial sepsis, 1 disseminated BCG, 1 disseminated CMV), severe GVHD (n = 5), cardiotoxicity (n = 1), and reactivation of HLH (n = 1). Four of the six deaths due to infections occurred in patients with SCID who had infections prior to starting HSCT. Four patients died within 30 days (all due to infection) and eight patients died between 30–100 days post-transplant (GVHD being the dominant cause − 5/8) and one patient died at 336 days after transplant with acute onset respiratory distress and died before he could reach the local hospital during the COVID lockdown and cause could not be established. He had grade 3 gut GVHD which was under control with steroids being weaned, and compliance and follow-up were poor and affected during the lockdown. Autoimmunity was a predominant manifestation in seven of these patients and possibly contributed to a poor outcome as three of them died post-transplant.

Prenatal testing was performed in seven affected families. The diagnosis in the proband was SCID (n = 3), X-linked neutropenia with HLH (n = 1), XLA (n = 1), LAD (n = 1) and DOCK8 deficiency (n = 1). Amniocentesis (n = 3), chorionic villus sampling (n = 3) and cordocentesis (n = 1) were performed for prenatal diagnosis which confirmed the presence of an affected fetus in four families and 4/7 of these pregnancies were terminated. Three unaffected fetuses were born healthy and continue to remain well.

During the study period of February 2017 to November 2021, a total of 208 patients were diagnosed with an IEI. A study by Gupta et al reported 122 cases of IEI from a center in Mumbai (2007–2010) and a total of 153 cases (1992–2010) from a center in Chandigarh [8]. Ours, is by far the largest cohort of patients with IEI reported from a tertiary care center in India.

Males (64.4%) were predominantly affected in this study. A similar trend was reported by centers in Europe [9], India [10, 11], and Sri Lanka [12]. Interestingly, in our cohort males were frequently affected even if X-linked diseases were excluded. However, the reason for this could not be ascertained. The possibility of bias towards seeking medical attention in the male child in our setting cannot be ruled out. The age at onset of the first clinical symptom varied depending on the underlying PID, with a mean age of onset being 1.24 yrs in children and 19 yrs in adults which was in keeping with the studies from South India (mean age-10 months) and North India (2 days to 43 years) (Table S2) [8, 13] .

Lack of awareness regarding IEI amongst the medical fraternity leads to a significant delay in the diagnosis of these conditions. Gupta et al reported a mean delay in diagnosis of 5 years which was similar to that noted in our cohort (4.5 years) [8]. However, in the study reported by Sivasankaran et al, the delay in diagnosis was much lesser (11 months) [13]. The delay in diagnosis in our cohort was maximum in patients with CTLA-4 haploinsufficiency (23.3 years). Three patients from a single kindred were diagnosed to have CTLA-4 haploinsufficiency with the age of the eldest affected member being 55-years. Adults with IEI often fail to get diagnosed for years highlighting the need to raise awareness amongst adult physicians.

High rates of consanguinity and endogamous marriages contribute to the increased prevalence of autosomal recessive diseases in our setting. As compared to the northern part of the country, consanguineous marriages are much higher in South India [14]. In another study from South India, up to 58% of patients had a consanguineous parentage [13], whereas consanguinity was noted in 30.2% of families in our cohort. In correlation with this, a significant proportion of patients were noted to have autosomal recessive inheritance (42.3%), which contrasts with the European and North American studies, wherein X-linked inheritance is more common [15, 16]. Positive family history was noted in 47 (22.5%) patients which were similar to that reported by Gupta et al (23%) from a North Indian cohort and Sivasankaran et al (28%) from South India [8, 13]. In two families (CTLA4 haploinsufficiency = 1, AD HIES = 1), parents were diagnosed as an aftermath of the child’s diagnosis.

The most common IEI in our cohort was SCID (n = 37, 17.7%) whereas most centers from India and the world reported antibody deficiency disorders as the most common PID [13, 11, 17]. Disorders of immune dysregulation were more common in the cohort from West India (33.8%) [4], whereas, combined cellular and humoral immunodeficiency (29%) and phagocytic defects (29%) were predominantly described from North India [18]. Sivasankaran et al in a study from South India reported congenital phagocytic defects to be more common at their center [13]. The variance observed in our population could be attributed to the differences in the genetic constitution of the population in different parts of the country, higher rates of consanguineous marriages (much higher in Western and Southern India), and a referral bias.

Infections are the key manifestations of IEI and the most common infection in our cohort was pneumonia (47%). In a study on CGD by Rawat et al, pneumonia (71%) was the most common infection followed by lymphadenitis (31.6%) and subcutaneous abscesses (23.7%) and mycobacteria (44,18.6%) was the most common organism isolated [19]. In our cohort mycobacteria (8, 29.6%) was the most common organism isolated in patients with CGD. BCG-related complications were seen in 17/236 patients with CGD [19] and 27/254 children with SCID [20] had disseminated BCG infection in the large multicentric cohorts reported from India. We had 18 patients with complications related to BCG vaccination in our cohort. BCG vaccine is being administered to all newborns in India and in the absence of a newborn screening program, infants with SCID often present with disseminated BCG. This was an additional factor contributing to the dismal outcome of babies with SCID in our cohort. Candida was reported in 11 patients and was most commonly noted in babies with SCID (n = 6) in our cohort. Similarly, 16 patients had Candida infection in the recently reported multicentric study on 254 SCID patients from India [20].

Interestingly, autoimmunity (22%) was observed in a significant proportion of our patients. Studies on autoimmunity in patients with IEI from Kuwait [21] and France [22] reported an incidence ranging from 20–26%. Autoimmune cytopenia was reported as the most common manifestation in these studies [21, 22]. In comparison, we had a significant proportion of patients with inflammatory colitis and this could possibly be related to a larger number of patients with CGD in our cohort. The longer delay in diagnosis in patients with autoimmune manifestations compared to the rest, is likely due to the less severe non-infectious complications. There is an urgent need to raise awareness regarding the autoimmune facet of IEI amongst the primary care physicians.

We had six patients with autoinflammatory diseases in our study. The multicentric data on autoinflammatory diseases published from India reported 78 patients with various autoinflammatory diseases. Thirty patients (38%) belonged to non-inflammasome-related conditions such as TRAPS, DIRA, and PAPA. Interestingly, we had a family with FCAS-1 with 10 affected family members across 4 generations! [23].

The Indian health care system being an out-of-pocket expenditure type, genetic testing was only opted for by 152 patients. A diagnostic yield of 73.02% in our study is higher as compared to those reported previously from India [13, 24]. Elsink et al reported a diagnostic yield of 24.6% (pediatric patients) and 9% (adult patients) in a Dutch cohort with IEI [25]. Our is by far the largest genetically confirmed cohort of IEI from India. A putative diagnosis was made in 24 patients. The identified variants corroborated with the clinical phenotype and preliminary immunological work -up. Of these four variants were present in disorders known to have dominant de novo inheritance, however lack of parental testing prevented further classification. We would like to mention here two siblings (IEI 100, 101) wherein the identified variant in Otulin gene has been classified as Uncertain significance due to an atypical presentation. Both affected siblings presented at 6 & 13 years of age with pyoderma gangrenosum like lesions. This is a relatively milder presentation as compared to what has been reported previously [26]. Further functional studies are underway which would help us arrive at a diagnostic conclusion.

Exome sequencing failed to identify a pathogenic variant in four and two children with SCID and LAD type 1 respectively. Similarly, no significant variants were detected in three cases of CGD with a reduced p47phox expression. This can be attributed to the low coverage of NCF1 gene (54.43%) due to high homology with pseudogene [27] as well as lack of consent for deletion/duplication analysis. Reduced p47phox expression on flow cytometry corroborated with a high likelihood of these patients being affected with CGD. The immunological tests (lymphocyte subsets, DHR, and CD18 expression on neutrophils) helped clinched the diagnosis in these cases. Hence our study reflects the importance of performing both immunological as well as genetic testing in patients suspected with IEI.

IVIG replacement therapy (IRT) is one of the most effective and commonly used treatments in patients with IEI, especially patients with XLA and CVID. In our cohort, a total of 19 children and 10 adults remain on regular monthly replacement immunoglobulin therapy. Several states now support IVIG replacement therapy, and this has been a welcome move over the last five years.

While HSCT was indicated in a large majority of our patients, only 29 children could be offered the same due to financial and social constraints. In the absence of a matched family or unrelated donors, haplo-identical transplants seem to be the only feasible solution in conditions with high fatality (eg. SCID). This could have possibly contributed to the poor outcomes seen in our cohort. Despite access to matched unrelated donor registries, only one patient underwent a MUD transplantation. Recently, Raj R et al reported 228 children with IEI who underwent HSCT from seven major centers in India. Forty-four percent of children in this cohort underwent a haploidentical donor HSCT with an overall survival of 62.8% whereas the survival was superior in children receiving HSCT from a MSD (78%) [28]. While providing a timely diagnosis is crucial in improving outcomes, we need better social and financial support systems to cater to the financially weaker sections of the society. Strengthening the regional donor registries will go a long way in improving access to MUD transplants.

Though HSCT is a curative option, it comes at a prohibitive cost. Maintaining lifelong supportive treatment with IVIG is associated with poor quality of life and poses a significant financial burden. In families with an affected child, genetic counseling and prenatal testing are the cornerstones of preventive management. Yadav et al [29] reported 112 families who underwent prenatal testing and the affected fetus was confirmed in 32 families. Our center also contributed to this nation-wide data. In the current study, seven families opted for prenatal testing, and an informed decision to discontinue the pregnancy was made (4/7) after appropriate genetic counselling.

Around 12% of our patients were adults, while the global report on IEI by Modell Foundation in 2018 had 36% adults [30]. This could be attributed to the referral bias as we are primarily a Pediatric Unit. However, the glaring contrast compared to the world literature also highlights the probable lack of awareness on IEI amongst adult physicians, pulmonologists and gastroenterologists in the country. Needless to say, there is a dire need to raise awareness amongst adult physicians and other related specialties to ensure a timely diagnosis in adults with IEI.

We hereby report the largest single-center cohort of IEI from the Indian subcontinent. Clinical and laboratory data were prospectively collected, which is a strength of our study. Genetic testing was performed in 73% of our patients and is a reflection of better availability and accessibility of exome sequencing in our setup. However, we acknowledge our limitations. The lack of a genetically confirmed diagnosis in all patients, the inability to perform parental analysis in all cases and functional/protein expression assays to better delineate the variants of uncertain significance noted on NGS are some of the limitations of the current study.

> Acknowledgements - We thank National Institute of Immunohaematology (NIIH),Mumbai for helping us with p47phox assay.

> Funding - The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

> Conflicts of interest/Competing interests - The authors have no relevant financial or non-financial interests to disclose.

> Availability of data and material - The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

> Authors' contributions –

All authors contributed to the study conception and design.

*SB and *RSM have contributed equally to the manuscript and be considered as co-first authors

SB - Management of Patients, data collection and analysis ,Final review of the manuscript

RSM- Management of patients, data collection and analysis ,Initial draft of manuscript and literature review

SR, RC, FP - Bone marrow transplant, final review of the manuscript

UK , PR - Genetic testing and analysis of genetic data, final review of the manuscript

AJ - Immunological testing, final review of the manuscript

CG, HK, JU, GC, SK, DP, SJ - Clinical management of patients, final review of the manuscript

PJ, AM - Fetal testing, final review of the manuscript

> Ethics approval - This is an observational study. The Institutional Ethics Committee has confirmed that no ethical approval is required.

> Consent to participate - Written informed consent was obtained from all individual participants in the study. In case of minors, consent was obtained from parents.

> Consent for publication - The authors affirm that the participants provided informed consent for publication

Bousfiha A, Jeddane L, Picard C, Al-Herz W, Ailal F, Chatila T, et al. Human inborn errors of immunity: 2019 update of the IUIS phenotypical classification. Journal of clinical immunology. 2020 Jan;40(1):66-81.
Picard C, Al-Herz W, Bousfiha A, Casanova JL, Chatila T, Conley ME, et al. Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015. Journal of clinical immunology. 2015 Nov;35(8):696-726.
Boyle JM, Buckley RH. Population prevalence of diagnosed primary immunodeficiency diseases in the United States. Journal of clinical immunology. 2007 Sep;27(5):497-502.
Madkaikar M, Mishra A, Desai M, Gupta M, Mhatre S, Ghosh K. Comprehensive report of primary immunodeficiency disorders from a tertiary care center in India. Journal of clinical immunology. 2013 Apr;33(3):507-12.
Gupta S, Madkaikar M, Singh S, Sehgal S. Primary immunodeficiencies in India: a perspective. Annals of the New York Academy of Sciences. 2012 Feb;1250(1):73-9.
O'gorman MR, Corrochano V. Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease. Clinical Diagnostic Laboratory Immunology. 1995 Mar;2(2):227-32.
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015 May;17(5):405-24.
Gupta D, Thakral D, Kumar P, Kabra SK, Lodha R, Kumari R, et al. Primary immunodeficiency disorders among north Indian children. The Indian Journal of Pediatrics. 2019 Oct;86(10):885-91.
Gathmann B, Grimbacher B, Beauté J, Dudoit Y, Mahlaoui N, Fischer A, et al. The European internet-based patient and research database for primary immunodeficiencies: results 2006–2008. Clinical & Experimental Immunology. 2009 Sep;157(Supplement_1):3-11.
Chinnabhandar V, Yadav SP, Kaul D, Verma IC, Sachdeva A. Primary immunodeficiency disorders in the developing world: data from a hospital-based registry in India. Pediatric Hematology and Oncology. 2014 Apr 1;31(3):207-11.
Jindal AK, Pilania RK, Rawat A, Singh S. Primary immunodeficiency disorders in India—a situational review. Frontiers in Immunology. 2017 Jun 19;8:714.
de Silva NR, Gunawardena S, Rathnayake D, Wickramasingha GD. Spectrum of primary immunodeficiency disorders in Sri Lanka. Allergy, Asthma & Clinical Immunology. 2013 Dec;9(1):1-9.
Sivasankaran M, Munirathnam D, Balasubramanian S, Agrawal S, Deshpande S, Bharadwaj R, et al.Diagnostic spectrum and clinical profile of primary immunodeficiency disorders at a tertiary care children hospital in Southern India. Indian Pediatrics. 2021 Mar;58(3):246-9.
Kuntla S, Goli S, Sekher TV, Doshi R. Consanguineous marriages and their effects on pregnancy outcomes in India. International Journal of Sociology and Social Policy. 2013 Jul 19.
Van den Berg JM, van Koppen E, Ahlin A, Belohradsky BH, Bernatowska E, Corbeel L, et al. Chronic granulomatous disease: the European experience. PLoS One. 2009;4(4):e5234.
Lugo Reyes SO, Ramirez-Vazquez G, Cruz Hernandez A, et al. Clinical features, non-infectious manifestations and survival analysis of 161 children with primary immunodeficiency in Mexico: a single center experience over two decades. J Clin Immunol. 2016 Jan;36(1):56–65.
Ballow M. Primary immunodeficiency disorders: antibody deficiency. Journal of Allergy and Clinical Immunology. 2002 Apr 1;109(4):581-91.
Gupta D, Thakral D, Kumar P, Kabra SK, Lodha R, Kumari R, et al. Primary immunodeficiency disorders among north Indian children. The Indian Journal of Pediatrics. 2019 Oct;86(10):885-91.
Rawat A, Vignesh P, Sudhakar M, Sharma M, Suri D, Jindal A, et al. Clinical, immunological, and molecular profile of chronic granulomatous disease: a multi-centric study of 236 patients from India. Frontiers in immunology. 2021 Feb 25;12:54.
Vignesh P, Rawat A, Kumrah R, Singh A, Gummadi A, Sharma M, et al. Clinical, immunological, and molecular features of severe combined immune deficiency: a multi-institutional experience from India. Frontiers in immunology. 2021 Feb 8;11:3747.
Massaad MJ, Zainal M, Al-Herz W. Frequency and manifestations of autoimmunity among children registered in the Kuwait National Primary Immunodeficiency Registry. Frontiers in immunology. 2020 Jun 2;11:1119.
Fischer A, Provot J, Jais JP, Alcais A, Mahlaoui N, Adoue D, Aladjidi N, Amoura Z, Arlet P, Armari-Alla C, Bader-Meunier B. Autoimmune and inflammatory manifestations occur frequently in patients with primary immunodeficiencies. Journal of Allergy and Clinical Immunology. 2017 Nov 1;140(5):1388-93.
Suri D, Rawat A, Jindal AK, Vignesh P, Gupta A, Pilania RK, Joshi V, et al. Spectrum of Systemic Auto-Inflammatory Diseases in India: A Multi-Centric Experience. Front Immunol. 2021 Mar 19; 12:630691.
Lashkari HP, Madkaikar M, Dalvi A, Gupta M, Bustamante J, Sharma M, et al. Clinical and Genetic Spectrum of Inborn Errors of Immunity in a Tertiary Care Center in Southern India. Indian journal of pediatrics. 2021 Nov 26:1-0.
Elsink K, Huibers M, Hollink IH, Simons A, Zonneveld-Huijssoon E, Leavis H, et al. Implementation of Early Next-Generation Sequencing for Inborn Errors of Immunity: A Prospective Observational Cohort Study of Diagnostic Yield and Clinical Implications in Dutch Genome Diagnostic Centers. Frontiers in immunology. 2021 Jan;12.
Damgaard RB, Elliott PR, Swatek KN, Maher ER, Stepensky P, Elpeleg O, et al. OTULIN deficiency in ORAS causes cell type‐specific LUBAC degradation, dysregulated TNF signalling and cell death. EMBO molecular medicine. 2019 Mar;11(3): e9324.
Kulkarni M, Hule G, de Boer M, van Leeuwen K, Kambli P, Aluri J, et al.Approach to molecular diagnosis of chronic granulomatous disease (CGD): an experience from a large cohort of 90 Indian patients. Journal of clinical immunology. 2018 Nov;38(8):898-916.
Raj R, Aboobacker FN, Yadav SP, Uppuluri R, Bhat S, Choudhry D, et al. Multicenter outcome of hematopoietic stem cell transplantation for primary immune deficiency disorders in India. Frontiers in Immunology. 2021 Jan 8; 11:3136.
Yadav RM, Gupta M, Dalvi A, Bargir UA, Hule G, Shabrish S, et al. Prenatal Diagnosis for Primary Immunodeficiency Disorders—An Overview of the Indian Scenario. Frontiers in immunology. 2020 Dec 7; 11:3163.
Modell V, Orange JS, Quinn J, Modell F. Global report on primary immunodeficiencies: 2018 update from the Jeffrey Modell Centers Network on disease classification, regional trends, treatment modalities, and physician reported outcomes. Immunol Res. 2018 Jun;66(3):367-380.

Table 2 is available in the Supplementary Files section.

Download PDF

Version 1

posted

You are reading this latest preprint version

Profile of 208 Patients With Inborn Errors of Immunity at a Tertiary Care Center in South India

Status:

Version 1

Abstract

Introduction

Methods

Immunological evaluation

Genetic analysis

Prenatal screening

Statistical methods

Results

Discussion

Declarations

References

Table

Supplementary Files

Status:

Version 1