A Clinical Conundrum with Diagnostic and Therapeutic Challenge: a Tale of Two Disorders in One Case.

Living organisms are exposed to exogenous and endogenous agents that affect genomic integrity by creating DNA double strand breaks (DSBs). These breaks are repaired by DNA repair proteins to maintain homeostasis. Defects in DNA repair pathways also affect lymphocyte development and maturation, as DSB sites are critical intermediates for rearrangements required for V(D)J recombination. Recent classifications for inborn errors of immunity (IEIs) have listed DNA repair defect genes in a separate group, which suggests the importance of these genes for adaptive and innate immunity. We report an interesting case of a young female (index P1) with mutations in two different genes, DCLRE1C and FANCA, involved in DNA repair pathways. She presented with clinical manifestations attributed to both defects. With the advent of NGS, more than one defect is increasingly identified in patients with IEIs. Familial segregation studies and appropriate functional assays help ascertain the pathogenicity of these mutations and provide appropriate management and genetic counseling.

Clinical, immunological and molecular findings of patients with DOCK-8 deficiency from India.

DOCK8 deficiency affects various cell subsets belonging to both the innate and adaptive immune systems. Clinical diagnosis is challenging, as many cases present with severe atopic dermatitis as the only initial manifestation. Though flow cytometry helps in the presumptive diagnosis of DOCK8-deficient patients by evaluating their DOCK8 protein expression, it requires subsequent confirmation by molecular genetic analysis. Currently, haematopoietic stem cell transplantation (HSCT) is the only curative treatment option available for these patients. There is a paucity of data from India on the clinical diversity and molecular spectrum of DOCK8 deficiency. In the present study, we report the clinical, immunological and molecular findings of 17 DOCK8-deficient patients from India diagnosed over the last 5 years.

Normative data for paediatric lymphocyte subsets: A pilot study from western India.

Background & objectives: Accurate diagnosis of immunodeficiencies requires a critical comparison of values with age-matched controls. In India, the existing reference values for rare lymphocyte subsets are currently not available and we rely on the data originating from other countries for the interpretation of the results. Furthermore, there is limited information on normal variation for these rare-subset parameters in Indian children. So, this study aimed to establish normative values for clinically important lymphocyte subsets in Indian children at different age groups. Methods: 148 children aged ≥16 yr were enrolled in this study. The study population included 61 per cent males and 39 per cent females and was divided into the following groups: cord blood (n=18), 0-6 months (n=9), 6-12 months (n=13), 1-2 yr (n=19), 2-5 yr (n=27), 5-10 yr (n=25) and 10-16 yr (n=37). The absolute and relative percentage of lymphocytes, T, B, natural killer cell, along with activated, naïve and memory subsets, was determined by flow cytometry. Results: Median values and the 10th and 90th percentiles were obtained for 34 lymphocyte sub-populations. The T and B naïve compartments showed a decreasing trend, whereas memory cells showed an increase with age. The activated T cell subset shows an increasing pattern up to one year and then declines gradually. Double negative T cells are relatively stable. TCRgd+T cell percentage increases with age. Interpretation & conclusions: This single-centre pilot study provides preliminary data that justifies the need for future large-scale multi centric studies to generate a reference range for interpreting extended immunophenotyping profiles in the paediatric age group, making it possible for clinicians to assess the immunological status in inborn errors of immunity, infectious and autoimmune diseases.

CD8 + T Cells Exhibit an Exhausted Phenotype in Hemophagocytic Lymphohistiocytosis.

PURPOSE: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome mainly caused by uncontrolled activation of antigen presenting cells and CD8 T cells. CD8 T cell exhaustion is a known phenomenon in chronic viral infections and cancer. However, the role of T cell exhaustion is not yet identified in HLH in the background of persistent inflammation. So, currently, we have characterized the CD8 T cells using flow cytometry to understand the phenomenon of exhaustion in these cells in HLH. METHODS: We have comprehensively evaluated lymphocyte subsets and characterized CD8 T cells using immunophenotypic markers like PD1, TIM3, LAG3, Ki67, Granzyme B, etc. in a cohort of 21 HLH patients. Effector cytokine secretion and degranulation by CD8 T cells are also studied. RESULTS: Our findings indicate skewed lymphocyte subsets and aberrantly activated CD8 T cells in HLH. CD8 T cells exhibit significantly increased expression of PD1, TIM3, and LAG3 prominently in primary HLH as compared to controls. PD1 + CD8 T cells express elevated levels of Granzyme B and Ki67. Moreover, CD8 T cells are hypofunctional as evidenced by significantly reduced cytokine secretion and compromised CD107a degranulation. CONCLUSION: The study has revealed that CD8 + cytotoxic T lymphocytes from HLH patients exhibited high expression of exhaustion markers with overall impaired function. To the best of our understanding, this is the first report suggesting functional exhaustion of CD8 T cells in both primary and secondary HLH. Future studies to understand the association of exhaustion with disease outcome are needed for its probable therapeutic implementation.

Spectrum of Inborn errors of immunity in a cohort of 90 patients presenting with complications to BCG vaccination in India.

World Health Organisation recommends the practice of BCG vaccination at birth in countries which have a high incidence of tuberculosis and/or high leprosy burden. The BCG vaccination is considered safe for a competent immune system. However, in children with weakened immune systems cause of which can be primary or secondary, the vaccine may lead to side effects which can be localized or disseminated. In this study, we report a spectrum of inborn errors of immunity (IEI) commonly referred to as primary immunodeficiency disorders (PIDs) diagnosed in a large cohort of patients presenting with complications to BCG vaccination from India. Retrospective data analysis of patients referred to ICMR- National Institute of Immunohematology (ICMR-NIIH) for IEI workup between 2007 and 2019 was done. IEI was identified in n = 52/90 (57.7%) patients presenting with BCG complications. Of these, n = 13(14.4%) patients were diagnosed with severe combined immune deficiency, n = 15(16.7%) with chronic granulomatous disease, n = 19(21.1%) with Inborn errors of IFN-γ immunity, n = 4(4.4%) with Combined immunodeficiency and n = 1(1.1%) with Leucocyte Adhesion Deficiency type1. Majority of cases with BCGosis (88%) had an underlying IEI. This study strongly highlights the need for evaluation of patients with BCG complications for underlying IEI. While disseminated BCGosis strongly predicts underlying IEI, even localized persistent adenitis may be a warning sign of underlying IEI. It is also strongly recommended to record a family history of previous sibling death prior to administration of this live vaccine and deferring live vaccine till the diagnosis of IEI is ruled out in cases with a positive family history.

Approach to Molecular Diagnosis of Chronic Granulomatous Disease (CGD): an Experience from a Large Cohort of 90 Indian Patients.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by mutation in any one of the five genes coding NADPH oxidase components that leads to functional abnormality preventing the killing of phagocytosed microbes by affecting the progression of a respiratory burst. CGD patients have an increased susceptibility to infections by opportunistic and pathogenic organisms. Though initial diagnosis of CGD using a nitroblue tetrazolium (NBT) test or dihydrorhodamine (DHR) test is relatively easy, molecular diagnosis is challenging due to involvement of multiple genes, presence of pseudogenes, large deletions, and GC-rich regions, among other factors. The strategies for molecular diagnosis vary depending on the affected gene and the mutation pattern prevalent in the target population. There is a paucity of molecular data related to CGD for Indian population. METHOD: This report includes data for a large cohort of CGD patients (n = 90) from India, describing the diagnostic approach, mutation spectrum, and novel mutations identified. We have used mosaicism in mothers and the expression pattern of different NADPH components by flow cytometry as a screening tool to identify the underlying affected gene. The techniques like Sanger sequencing, next-generation sequencing (NGS), and Genescan analysis were used for further molecular analysis. RESULT: Of the total molecularly characterized patients (n = 90), 56% of the patients had a mutation in the NCF1 gene, 30% had mutation in the CYBB gene, and 7% each had mutation in the CYBA and NCF2 genes. Among the patients with NCF1 gene mutation, 82% of the patients had 2-bp deletion (DelGT) mutations in the NCF1 gene. In our cohort, 41 different mutations including 9 novel mutations in the CYBB gene and 2 novel mutations each in the NCF2, CYBA, and NCF1 genes were identified. CONCLUSION: Substantial number of the patients lack NCF1 gene on both the alleles. This is often missed by advanced molecular techniques like Sanger sequencing and NGS due to the presence of pseudogenes and requires a simple Genescan method for confirmation. Thus, the diagnostic approach may depend on the prevalence of affected genes in respective population. This study identifies potential gene targets with the help of flow cytometric analysis of NADPH oxidase components to design an algorithm for diagnosis of CGD in India. In Indian population, the Genescan method should be preferred as the primary molecular test to rule out NCF1 gene mutations prior to Sanger sequencing and NGS.

Poliovirus Excretion in Children with Primary Immunodeficiency Disorders, India.

Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.

Clinical, Immunological, and Molecular Findings of Patients with p47phox Defect Chronic Granulomatous Disease (CGD) in Indian Families.

Chronic granulomatous disease (CGD) is a group of inherited disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex. Reduced or absent oxygen radical synthesis seen in these patients leads to impaired killing of intracellular bacteria and fungi. CGD clinically presents with recurrent and life-threatening infections as well as granulomatous inflammatory responses. p47phox encoded by the NCF1 gene is the most common autosomal recessive form of CGD which is often clinically milder. Here, we are presenting the data on clinical and immunological findings in 21 Indian patients with Del GT mutation in the NCF1 gene. Diagnosis of these patients was based on detailed clinical evaluation, measurement of respiratory burst activity by nitro blue tetrazolium and dihydrorhodamine-1,2,3 assay, expression of p47phox by flow cytometry, and molecular confirmation by GeneScan method. Seventeen male and four female patients with median age of onset of 1 year ranging from 1.5 months to 6 years were included in the study. Sixty-two percent (13 out of 21) of patients belonged to a consanguineous marriage with only one family having a history of a previous sibling death. Significant variability in clinical presentation was observed in spite of identical genetic defect ranging from asymptomatic to very severe presentation leading to early death or requiring transplantation. However, none of these patients showed difference in immunological parameters to account for this variability. Thus, this study highlights the phenotypic heterogeneity seen in these patients with Del GT mutation in the NCF1 gene and its implication in management of these patients.

Rapid Flow cytometric prenatal diagnosis of primary immunodeficiency (PID) disorders.

OBJECTIVES: Primary Immunodeficiency diseases (PID) are a heterogeneous group of inherited disorders of immune system. Immunophenotypic evaluation of PIDs using flowcytometry provides important clues for diagnosis of these disorders, though confirmation requires identification of underlying molecular defects. Prenatal diagnosis (PND) forms an important component of management in families affected with severe PID. However, molecular diagnostic facilities for each of these diseases are not available and may not be possible to perform in all cases. In such scenario we opted for phenotypic prenatal diagnosis by cordocentesis for families with index case having immunophenotypically well characterized PID. METHODS: Normal reference ranges of lymphocyte subsets, CD 18/CD11 integrins on leukocytes, MHC class II expression and oxidative burst activity of fetal neutrophils at 18 weeks of gestation were previously established on 30 cord blood samples. PND was performed in 13 families with PIDs. Maternal contamination was ruled out by VNTR analysis. RESULTS: Out of 13 fetuses, nine were found to be unaffected (three cases with leukocyte adhesion deficiency (LAD-I), four cases with severe combined immunodeficiency diseases (SCID), one with X-linked agammaglobulinemia (XLA), and one with chronic granulomatous disease (CGD)] and three were found to be affected (one with T-B+NK-SCID, one with MHC class II deficiency and one with LAD-I). Diagnosis was confirmed by testing the cord blood samples after delivery and further follow-up of the children. In one family diagnosis could not be offered due to maternal contamination. No procedure related complications were observed. CONCLUSION: Flowcytometry offers rapid and sensitive method for prenatal diagnosis and genetic counseling for selected phenotypically well characterized PID in cases where molecular diagnostic facilities are not available.

Diagnosis and Management of Infections in Patients with Mendelian Susceptibility to Mycobacterial Disease.

The diagnosis and treatment of patients with mendelian susceptibility to mycobacterial disease (MSMD) pose consistent challenges due to the diverse infection spectrum observed in this population. Common clinical manifestations include Bacillus Calmette-Guérin vaccine (BCG) complications in countries where routine BCG vaccination is practiced, while in non-BCG-vaccinating countries, Non-Tuberculous Mycobacteria (NTM) is prevalent. In tuberculosis-endemic regions, Mycobacterium tuberculosis (MTB) has a high prevalence, along with other intracellular organisms. Isolating these organisms presents a significant challenge, and treatment is often initiated without confirming the specific species. This review primarily focuses on the methods and challenges associated with diagnosing and treating MSMD patients.

Clinical and Genetic Spectrum of Inborn Errors of Immunity in a Tertiary Care Center in Southern India.

OBJECTIVES: To study the incidence, clinical manifestations, and genetic spectrum of primary immunodeficiency diseases (PID)/inborn errors of immunity (IEI) in a tertiary care hospital in Southern India. METHODS: A retrospective analysis of all patients with a clinical suspicion of PID/IEI seen at a tertiary care hospital was performed. All patients had at least one or more warning signs of PID. Serum immunoglobulin levels and other targeted investigations were performed as warranted by the clinical presentation. All families with suspected PID were counseled and offered genetic testing. RESULTS: A total of 225 children were evaluated for PID during the study period of 6 y. Fifty-six of them did not meet the European Society of Immunodeficiencies (ESID) criteria (working definition of clinical diagnosis) and were excluded. An IEI was found in 30/49 (61.2%) patients. The most frequent reason for referral was recurrent/unusual or serious infections (28%), or cytopenia (16%). Group IV diseases of immune dysregulation was the most common category (19%), followed by group III predominant antibody deficiencies in 23/163 (14%), as per the International Union of Immunological Societies (IUIS) classification. CONCLUSIONS: This study highlights the heterogeneity of the present cohort, the underuse of genetic tests, and efforts to provide optimal care for children with possible IEI in this center.

Clinical, immunological and genomic characteristics of children with X-linked agammaglobulinemia from Kerala, South India.

X-linked agammaglobulinemia (XLA) is an X-linked recessive primary immunodeficiency disorder caused due to a pathogenic variant in the Bruton tyrosine (BTK) gene with an incidence of 1:379,000 live births and 1:190,000 male births. Patients affected with XLA present with recurrent infections of the gastrointestinal and respiratory tracts. Here we report the first case series of 17 XLA patients of 10 South Indian families with a wide spectrum of clinical and genetic features. In our cohort, patients presented mainly with recurrent pneumonia, gastrointestinal infection, otitis media, pyoderma, abscesses, empyema, arthritis, and osteomyelitis. Using next-generation and Sanger sequencing we have identified 10 unique pathogenic and likely pathogenic variants in 17 patients. This encompasses three nonsynonymous, two stop-gain, two frameshifts, two structural, and one splicing variant, out of which two of them are novel. Based on the type of variant, patients had variable clinical features and treatment responses. We have also evaluated Btk protein expression for six patients in comparison to the healthy individuals and determined mosaic Btk expression patterns in four mothers. We have also performed family screening in 6 families using Sanger sequencing and identified 19 carriers for the variant. The diagnosis for the patients led to the proper treatment i.e. 15 patients were on intravenous immunoglobulin (IVIG) and the other two had successful hematopoietic stem cell transplantation (HSCT). Unfortunately, two of our patients died due to sepsis, while on IVIG. We envision the present study could help in better understanding of patients with XLA and help in family screening and prenatal diagnosis. To the best of our knowledge, this is the largest case series of patients affected with XLA from South India.

Whole genome sequencing identifies novel structural variant in a large Indian family affected with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA, OMIM #300755) is a primary immunodeficiency disorder caused by pathogenic variations in the BTK gene, characterized by failure of development and maturation of B lymphocytes. The estimated prevalence worldwide is 1 in 190,000 male births. Recently, genome sequencing has been widely used in difficult to diagnose and familial cases. We report a large Indian family suffering from XLA with five affected individuals. We performed complete blood count, immunoglobulin assay, and lymphocyte subset analysis for all patients and analyzed Btk expression for one patient and his mother. Whole exome sequencing (WES) for four patients, and whole genome sequencing (WGS) for two patients have been performed. Carrier screening was done for 17 family members using Multiplex Ligation-dependent Probe Amplification (MLPA) and haplotype ancestry mapping using fineSTRUCTURE was performed. All patients had hypogammaglobulinemia and low CD19+ B cells. One patient who underwent Btk estimation had low expression and his mother showed a mosaic pattern. We could not identify any single nucleotide variants or small insertion/ deletions from the WES dataset that correlates with the clinical feature of the patient. Structural variant analysis through WGS data identifies a novel large deletion of 5,296 bp at loci chrX:100,624,323-100,629,619 encompassing exons 3-5 of the BTK gene. Family screening revealed seven carriers for the deletion. Two patients had a successful HSCT. Haplotype mapping revealed a South Asian ancestry. WGS led to identification of the accurate genetic mutation which could help in early diagnosis leading to improved outcomes, prevention of permanent organ damage and improved quality of life, as well as enabling genetic counselling and prenatal diagnosis in the family.

Clinical Profile of Hyper-IgE Syndrome in India.

Introduction: Hyper-IgE Syndrome (HIES) is a rare inborn error of immunity (IEI) characterized by a constellation of symptoms related to susceptibility to Staphylococcal skin and pulmonary infections, eczema, raised serum IgE (>2,000 IU/ml), craniofacial anomalies, and recurrent bone fractures. Data on HIES from the Indian subcontinent is scarce and restricted to small case series and case reports. This is the first compilation of national data on HIES. Materials and Methods: A total 103 cases clinically diagnosed and treated as HIES were analyzed from nine centers. Cases with clinical and/or molecular diagnosis of DOCK8 deficiency were not included. Patients were divided into two groups: group I for whom a heterozygous rare variant of STAT3 was identified, and group II, with clinical features similar to those of AD STAT3 deficiency, but without any genetic diagnosis. Results: Genetic diagnosis was available in 27 patients (26.2%) and all harbored rare variants in the STAT3 gene. Majority of these STAT3 HIES patients presented with recurrent skin abscesses (77.7%) or pneumonia (62.9%) or both (59.2%). Other features included eczema (37%), candidiasis (55.5%), facial dysmorphism (55.5%), recurrent fractures (11.1%), and retained primary teeth (7.4%). Mycobacterial infections were seen in a significant 18.5%. Mortality was seen in three subjects (11.1%). A similar trend in the clinical presentation was observed when all the 103 patients were analyzed together. Twenty percent of patients without a rare variant in the STAT3 gene had an NIH score of ≥40, whereas, 51.9% of STAT3 HIES subjects had scores below the cut off of ≥40. TH17 cell numbers were low in 10/11 (90.9%) STAT3 HIES tested. Rare variants observed were 8 in exon 21; 8 in exon 13; 3 in exon 10; 2 in exon 15, and one each in exon 6, 16, 17, 19, 22, and splice site downstream of exon 12. Seven variants were novel and included F174S, N567D, L404Sfs*8, G419 =, M329K, T714I, R518X, and a splice site variant downstream of exon 12. Conclusions: The report includes seven novel STAT3 variants, including a rare linker domain nonsense variant and a CC domain variant. Mycobacterial diseases were more frequent, compared to western literature.

The Spectrum of Clinical, Immunological, and Molecular Findings in Familial Hemophagocytic Lymphohistiocytosis: Experience From India.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.