Utility of Immunohistochemistry and Immunofluorescence in Determining the Pathogenic Variants of Chronic Granulomatous Disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD. MATERIALS AND METHODS: Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n = 6), tissue blocks from autopsy cases (n = 3), and cellblocks of cell pellet prepared from peripheral blood (n = 4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed. RESULTS: All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing. CONCLUSIONS: Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

A Novel CEBPE Variant Causes Severe Infections and Profound Neutropenia.

PURPOSE: Specific granule deficiency (SGD) is a rare inborn error of immunity resulting from loss-of-function variants in CEBPE gene (encoding for transcription factor C/EBPε). Although this genetic etiology has been known for over two decades, only a few patients with CEBPE variant-proven SGD (type I) have been reported. Herein, we describe two siblings with a novel homozygous CEBPE deletion who were noted to have profound neutropenia on initial evaluation. We aimed to evaluate the immunohematological consequences of this novel variant, including profound neutropenia. METHODS: Light scatter characteristics of granulocytes were examined on various automated hematology analyzers. Phagocyte immunophenotype, reactive oxygen species generation, and Toll-like receptor (TLR) signaling were assessed using flow cytometry. Relative expression of genes encoding various granule proteins was studied using RT-PCR. Western blot analysis and luciferase reporter assay were performed to explore variant C/EBPε expression and function. RESULTS: Severe infections occurred in both siblings. Analysis of granulocyte light scatter plots revealed automated hematology analyzers can provide anomalously low neutrophil counts due to abnormal neutrophil morphology. Neutrophils displayed absence/marked reduction of CD15/CD16 expression and overexpression (in a subset) of CD14/CD64. Three distinct populations of phagocytes with different oxidase activities were observed. Impaired shedding of CD62-ligand was noted on stimulation with TLR-4, TLR-2/6, and TLR-7/8 agonists. We demonstrated the variant C/EBPε to be functionally deficient. CONCLUSION: Homozygous c.655_665del variant in CEBPE causes SGD. Anomalous automated neutrophil counts may be reported in patients with SGD type I. Aberrant TLR signaling might be an additional pathogenetic mechanism underlying immunodeficiency in SGD type I.

Pediatric systemic lupus erythematosus: phagocytic defect and oxidase activity of neutrophils.

BACKGROUND: There are limited data on neutrophil function in pediatric-onset systemic lupus erythematosus (pSLE) patients. This study aimed to evaluate phagocytosis and oxidase activity of neutrophils in patients with pSLE. PATIENTS AND METHODS: Eighty-seven patients with pSLE and 44 controls were enrolled. Phagocytic activity was assayed using pHrodoTMRed E. coli BioParticles Phagocytosis Kit by flow cytometry. Determination of NADPH oxidase activity was carried out by Dihyrdrorhodamine-123 (DHR-123) flow cytometry assay. RESULTS: Phagocytic activity of patients' neutrophils (mean 76.59%) was lower than that in controls (91.30%) (p < 0.001). Median delta median fluorescence intensity (ΔMFI) and stimulation index (SI) in patients (ΔMFI: 0.09; SI: 2.79) were also decreased compared to controls (ΔMFI: 0.18; SI: 5.00) (p < 0.002; p < 0.001 respectively). Disease activity showed an inverse correlation with phagocytic activity. Oxidase activity was also significantly low (SI DHR < 40) in 16% of patients. No significant correlation was found between oxidative burst and disease activity. CONCLUSION: Neutrophil function is impaired in patients with pSLE, as evidenced by the markedly reduced phagocytic activity. Phagocytic activity is also inversely correlated with disease activity. The oxidative activity was also reduced but not significantly. IMPACT: Neutrophil phagocytic function is impaired in pediatric-onset systemic lupus erythematosus (pSLE). There is an inverse correlation between disease activity in pSLE and phagocytic activity. NADPH oxidase activity in patients with pSLE did not show significant correlation with disease activity.

Foliar treatment of potassium nitrate modulates the fermentative and sucrose metabolizing pathways in contrasting maize genotypes under water logging stress.

The effect of potassium nitrate on the status of fermentative and sucrose metabolizing pathways was studied in two maize (Zea mays L.) genotypes, viz., LM 5 (relatively susceptible to flooding) and I 167 (relatively tolerant to flooding) under water logging stress. The higher increase in pyruvate decarboxylase, alcohol dehydrogenase and aldehyde dehydrogenase activities in the hypoxic roots of I 167 seedlings over LM 5 showed the former's efficient tolerance mechanism towards anaerobic conditions. Foliar application of KNO3 reduced these enzymatic activities in the roots of both the genotypes. The shoots of I 167 seedlings also showed a parallel increase in alcohol dehydrogenase and pyruvate decarboxylase activities under water logging stress. These enzymatic activities, however, remained unaffected in shoots of water logged LM 5 seedlings. There was a higher decrease in acid and alkaline invertase activities in the hypoxic roots of I 167 seedlings. KNO3 treatment led to higher acid invertase activity in roots of I 167 seedlings than those of LM 5. Sucrose synthase (synthesis) and sucrose phosphate synthase activities decreased, but sucrose synthase (breakdown) activity increased in the roots of both the genotypes, during water logging. KNO3 increased sucrose synthesizing activities with a parallel increase in the sucrose content of the roots. Sucrose synthesis was comparatively unaffected in I 167 shoots under water logging stress while LM 5 shoots showed higher reduction in its sucrose synthase (synthesis) and sucrose phosphate synthase activities. It may thus be concluded that KNO3 induced a network of reactions for improving water logging tolerance. The nitrate ions acted as an alternate electron acceptor and thus reduced the activities of fermentative enzymes. It promoted the funneling of sugars into the glycolytic pathway by inducing the activities of acid and alkaline invertases in the roots and shoots of maize genotypes. It also directed the hexoses towards biosynthetic pathway by increasing the activities of sucrose synthesizing enzymes.

Effect of osmo priming on sucrose metabolism in spring maize, during the period of grain filling, under limited irrigation conditions.

The present study was undertaken to study the effect of osmo priming on sucrose metabolism of spring maize, under limited irrigation conditions. Osmo priming increased the activities of acid invertase, alkaline invertase and sucrose synthase (cleavage) and the contents of reducing sugars and starch in the grains of stressed plants. There was also an increase in sucrose phosphate synthase activity with a parallel increase in sucrose content in leaves of stressed plants in comparison with those of hydro priming treatment. It showed that osmo priming helped in improving sucrose phosphate synthase activity in leaves of plants, leading to higher sucrose content, under stress conditions.

The Appraisement of Antioxidant and Oxidant Status in Women Undergoing Surgical Menopause.

The present study was undertaken to study the impact of surgical menopause on oxidant and antioxidant status in relation to estrogen levels after 3 months of surgery. Total 130 women who had undergone total hysterectomy (TH) with or without bilateral salpingo-oophorectomy (BSO) were included in this study. The oxidant status was assessed by measuring plasma levels of malondialdehyde and antioxidant status was assessed by measuring superoxide dismutase, catalase, glutathione, glutathione peroxidase, estrogen, and Vitamin A, E and C levels. The malondialdehyde level was significantly increased (p < 0.05) in all women who underwent TH with or without BSO. Significant increased levels of superoxide dismutase were observed in women who underwent TH with BSO. The blood glutathione levels were significantly decreased in women after TH only but significantly increased in women who had undergone TH with BSO. The levels of estrogen, vitamin E and vitamin C were significantly decreased in women who underwent TH with BSO. The catalase, GPx and vitamin A did not differ significantly in all groups. The result suggests that surgical menopause is associated with oxidative stress which reiterates the fact that ovaries retain some function even after menopause.

Comparative study of clozapine versus risperidone in treatment-naive, first-episode schizophrenia: A pilot study.

BACKGROUND & OBJECTIVES: Clozapine may be more useful in treatment-naive patients with first-episode schizophrenia for better symptoms control and improving quality of life. The current study was carried out to compare the efficacy and tolerability of clozapine versus risperidone in treatment-naive, first-episode patients of schizophrenia. METHODS: This was a comparative, open-label, six months prospective study of treatment-naive, first-episode patients with schizophrenia between the age group of 18 and 40 yr diagnosed as per the International Classification of Diseases-10 (ICD-10) criteria. A total of 63 patients were recruited and randomly assigned to clozapine group or risperidone group using computer-generated random number tables. Eight patients were lost to follow up. The dosages of the respective drugs were kept in therapeutic range of 200-600 mg/day and 4-8 mg/day orally for clozapine and risperidone, respectively. RESULTS: On general psychopathology score, after six months of intervention, clozapine led to 60.32 per cent mean reduction in Positive and Negative Syndrome Scale (PANSS) for Schizophrenia total score while risperidone led to 56.35 per cent mean reduction in PANSS total score, which meant more improvement with clozapine. Clozapine group was found to have significant improvement in quality of life (P = 0.04339). On Glasgow Antipsychotic Side-effect Scale, clozapine was superior to risperidone. The most common side effects observed in clozapine group were oversedation (78.96%) and dizziness (55.23%), and in risperidone group, common side effects were rigidity (62.36%), sedation (38.69%), tremors (65.69%) and menstrual irregularities in 80.25 per cent of female patients. INTERPRETATION & CONCLUSIONS: The findings of this preliminary study showed clozapine as a better choice than risperidone in terms of efficacy, tolerability and better quality of life in treatment-naive, first-episode schizophrenia. However, further studies need to be done on a larger group of patients to confirm the findings.

Targeted deep resequencing identifies MID2 mutation for X-linked intellectual disability with varied disease severity in a large kindred from India.

We report a novel missense mutation (c.1040G>A, p.Arg347Gln) in MID2, which encodes ubiquitin ligase E3, as the likely cause of X-linked mental retardation in a large kindred. The mutation was observed in all affected and obligate carriers but not in any unaffected males of the family or in population controls (n = 200). When transiently expressed in HEK293T cell line, the mutation was found to abolish the function of the COS domain in the protein. The GFP-tagged mutant protein accumulated in the cytoplasm instead of binding to the cytoskeleton resulting in its altered subcellular localization. Screening of coding exons of this gene in additional 480 unrelated individuals with idiopathic intellectual disability identified another novel variation p.Asn343Ser. This study highlights the growing role of the ubiquitin pathway in intellectual disability and also, the difference in MID2 determined phenotype observed in this study compared with that of its paralogue MID1 reported in literature.

Accuracy of transcutaneous bilirubin measurement in preterm low-birth-weight neonates.

UNLABELLED: The objective of this study was to evaluate the correlation and agreement between transcutaneous and serum bilirubin among preterm low-birth-weight neonates. Neonates born at <35 weeks of gestation with birth weight <2,000 g were enrolled prospectively. Transcutaneous bilirubin (TcB) was measured at forehead, sternum, and abdomen at 24 ± 6 and 72 ± 12 h after birth and when icterus involved arms or legs (Kramer zone 4-5). Serum total bilirubin (STB) was measured by microbilimeter (STB-M) at all these time-points and by high-performance liquid chromatography (STB-H) at one randomly chosen time-point. A total of 1,619 observations were made in 256 neonates (median gestation, 34 weeks (IQR, 32-35), birth weight 1,522 ± 288 g). Overall there was excellent correlation and agreement between TcB and STB-M with TcB on forehead being most accurate (r = 0.84, mean difference, 0.3 ± 1.9 mg/dL) followed by TcB on abdomen (r = 0.73, mean difference, 1.5 ± 2.6 mg/dL) and sternum (r = 0.72, mean difference, 1.5 ± 2.6 mg/dL). TcB performed well at all three points of measurement with best correlations being observed at icterus level 4/5. Correlation between TcB and STB-H measured by high-performance liquid chromatography was less strong but significant (r = 0.59 to 0.69 at different time points of measurement). CONCLUSIONS: TcB has good correlation and agreement with STB in preterm low-birth-weight neonates born at ≥28 weeks of gestation.

Effect of irrigation on wild and inbred maize with relation to the antioxidant status of pollens, flag leaves, and developing grains.

The investigation was carried out to evaluate the net effect of limited irrigation on the antioxidant status of pollens, flag leaves, and developing grains of wild and inbred maize lines. Teosinte pollens showed the highest activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-s-transferase (GST), and peroxidase (POX) under stressful conditions while LM 11 showed a significant decrease in APX, CAT, GR, and GST activities. Limited irrigations increased the contents of superoxide and malondialdehyde (MDA) to maximum levels in LM 11 leaves. The pollens, leaves, and developing grains of teosinte had the highest content of total phenols. Proline was maximum in the developing grains of teosinte and CML 32 while lowest in those of LM 11. Principal component analysis showed that LM 11 genotype and the respective antioxidant enzymes were in completely opposite quadrants. Chord analysis showed that CAT activity and total phenol content in pollens, leaves, and developing grains contributed towards most of the variations observed in teosinte and might be responsible for managing the yield attributes of genotype during stress conditions. The pollens and leaves of teosinte, with significant SOD activity, further helped in optimizing plant yield, under stressful conditions. CML 32 occupied intermediate position owing to the unaffected activities of most of the antioxidant enzymes and high content of antioxidants in its tissues. It may be concluded that the overall antioxidant status of tissues decides the tolerance behavior of plants.

Maternal serum median levels of alpha-foetoprotein, human chorionic gonadotropin & unconjugated estriol in second trimester in pregnant women from north-west India.

BACKGROUND & OBJECTIVES: Triple test as prenatal screening procedure does not form a part of routine health care of pregnant women in India. Hence, median values of triple test biomarkers are lacking for Indian population. This study was undertaken to establish population-specific medians for biomarkers viz. alpha-foetoprotien (AFP), human chorionic gonadotropin (hCGß), and unconjugated estriol (uE3) for detection of Down's syndrome, Edward's syndrome and neural tube defects (NTDs) in pregnant women in north-west India. METHODS: Serum biomarker values were derived from 5420 pregnant women between 15-20 wk of gestation who were enrolled for triple test investigations at Department of Gynecology and Obstetrics, Government Medical College and Hospital, Chandigarh, India, between January, 2007 to December, 2009. Median values were calculated for rounded weeks using database comprising pregnancies with normal outcomes only. Simple statistical analysis and log-linear regression were used for median estimation of the biomarker values. RESULTS: The levels of the three biomarkers were found to be ranging from 1.38 to 187.00 IU/ml for AFP, 1.06 to 315 ng/ml for hCGß, and 0.25 to 28.5 nmol/l for uE3. The age of women ranged from 18 to 47 yr and mean weight was 57.9 ± 9.8 kg. Data revealed that AFP, hCGß and uE3 medians in our study population were not significantly different from those reported from other countries or when compared ethnically. INTERPRETATION & CONCLUSION: The population-specific median values for the three biomarkers (AFP, hCGß, uE3) may be used as reference values during prenatal screening in Indian pregnant women.

A Community-Based Study of Antenatal and Neonatal Risk Factors in Autism Spectrum Disorder.

Objective: To assess children in the community for autism spectrum disorder (ASD) and associated risk factors.Methods: In this 2-stage, cross-sectional study, children between 1.5 and 10 years of age were screened using the Chandigarh Autism Screening Instrument. Those with a score above the cutoff of 10 were assessed in detail using the Childhood Autism Rating Scale and the Autism Diagnostic Interview-Revised, and a detailed pediatric assessment was conducted. Risk factors were evaluated, and karyotype and fragile X genetic testing was done for those diagnosed with ASD. The study was conducted from July 2014 to December 2017.Results: Compared to the control group, mothers of ASD children had more pregnancy-induced hypertension (PIH) and bleeding per vaginum (BPV) during the antenatal period. In the multivariate analysis, there was 6.3 times higher odds of having history of PIH (P = .02) and 7.7 times higher odds of BPV (P = .011) among children with ASD. There were much higher odds of having birth asphyxia (OR = 12.6), cardiorespiratory problems (OR = 10), metabolic abnormalities (hypoglycemia/ hypocalcemia) (OR = 12), and neonatal sepsis (OR = 16) in the ASD group compared to controls.Conclusions: ASD patients experienced more antenatal and neonatal problems compared to controls.Trial Registration: Clinical Trials Registry-India (CTRI/2017/02/007935).

Telomerase enzyme activity in patients with major depressive disorder: A pre and post-treatment study.

BACKGROUND: Telomerase is a cellular enzyme that prevents telomere shortening and promoting viability. The literature has reported shortened telomere length in patients with major depressive disorder (MDD). METHODS: 35 patients with diagnosis of major depressive disorder (MDD) fulfilling DMS-5 criteria in the age range of 18-60 years, treatment-naïve after assessing the severity on HAM-D and HAM-A and 35 age and sex matched healthy controls were included in the study. Baseline peripheral blood monocytes (PBMC) telomerase enzyme was assessed in cases and controls and repeated in cases of MDD at 8th week after intervention with escitalopram for 8 weeks. RESULTS: Pretreatment telomerase activity (TA) was elevated in cases as compared to controls and it was also significantly correlated to the severity of depression (p = 0.00). There was a significant positive difference in telomerase activity between non-responders (higher TA) and responders at baseline (p = 0.001) and 8th week (p = 0.012). The TA did not vary significantly amidst pretreatment and post-treatment, although it was slightly lower in the post-treatment group. LIMITATIONS: The study has few limitations in the form of small sample size, shorter duration of follow-up, and leucocyte telomeres length (LTL) was not assessed. CONCLUSION: The index study concludes that TA is higher in drug naïve patients with MDD than age and sex matched healthy control. The non-responders had significantly higher TA as compared to responders at baseline and post-treatment which indicates TA as a potential biomarker in the underlying biological mechanism of MDD and in response to antidepressant pharmacotherapy.

Morphological and antioxidant responses of Cicer arietinum L. genotypes exposed to combination stress of anthracene and sodium chloride.

Various mutagenic, carcinogenic pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs) are released into the environment posing a negative effect on plant metabolism. All the pollutants that are emitted into the atmosphere, ultimately find their way into the plant. Soil salinity stress is one of the major determinants of crop productivity. Different plants respond differently to different abiotic stress present alone or in combination. One such combination of abiotic stress is PAHs and salinity stress. The present research aims to study the effect of the application of NaCl and Anthracene alone and in various combinations on two chickpea genotypes GPF2 and PDG4. A 21 days laboratory experiment was conducted in petriplates and growth pouches. Different concentrations of NaCl and Anthracene were given to two chickpea genotypes viz. GPF2 and PDG4, alone as well as in combinations to study morphological, physiological and antioxidant responses. Results obtained were further analyzed by using various statistical measures such as Principle Component Analysis and Two-way ANOVA. Results indicated that under the dual presence of NaCl and Anthracene, GPF2 exhibited higher activities of antioxidant enzymes and was shown to have a negative correlation with plant height and chlorophyll content. Based on the results of the present investigation, it was concluded that GPF2 was a better performing chickpea genotype towards the combined presence of Anthracene and NaCl as compared to PDG4.

Maternal serum second trimester screening for chromosomal disorders and neural tube defects in a government hospital of North India.

OBJECTIVE: Down syndrome (DS) has major resource implications especially in developing countries being third most important cause of mental handicap. Maternal serum screening for chromosomal aneuploidies and neural tube defects (NTDs) is practiced worldwide in many countries and has been integrated into mainstream health care, while it is gradually gaining momentum in Asian countries. METHODS: This prospective cohort study was carried out in pregnant women undergoing triple screening test between January 2007 and December 2010 after informed consent. Biomarkers alpha-fetoprotein, human-chorionic-gonadotropin and unconjugated-estriol were tested, and risk of pregnancy being affected with DS, Edward's syndrome or NTDs were calculated. Screen-positive patients were referred for detailed ultrasonography and confirmatory amniocentesis. Follow-up record was maintained until delivery. RESULTS: Of 7400 pregnant women enrolled, 419(5.7%) were screen-positive, including 339 positive for DS, two for trisomy 18, and 62 for NTDs. Total eight cases of DS were eventually diagnosed in the population (prevalence of DS = 1 : 925), seven of which were detected in utero following diagnostic evaluation for positive serum screen (DR of DS screen = 87.5%). Total five cases of NTD were observed, yielding NTD prevalence of 0.67/1000. CONCLUSIONS: Triple screening in the second trimester is reasonably effective for the detection of major chromosomal defects and NTDs, and can be implemented successfully also in India.

Frequency distribution of Q188R, N314D, Duarte 1, and Duarte 2 GALT variant alleles in an Indian galactosemia population.

Classical galactosemia is a genetic disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The Q188R and N314D mutations are the most frequently cited GALT gene mutations. N314D is further associated with two variants, Duarte 1 and Duarte 2. Nevertheless, no reports are available on the clinical and molecular spectrum of galactosemia from the Indian population. The present study was designed to establish the frequency of these two most common mutations and their variants in Indian galactosemia patients so as to determine a single most common mutation/polymorphism for establishing the DNA-based diagnosis of galactosemia. Three alleles were found to be present at a frequency of 0.036 (Q188R), 0.40 (N314D), and 0.39 (D2); no D1 alleles were found. A significantly higher frequency of the Duarte 2 allele in our population suggests the presence of a milder form of galactosemia, which can be well managed by early diagnosis and dietary management.

Awakening a Molecular Mummy: The Inter-and Intramolecular Photochemistry of Pyromellitic Diimides with Alkyl Carboxylates.

Pyromellitic acid diimides are not as chemically unreactive as conjecturable (and presupposed) from their numerous applications as electron acceptor units or electron carriers in molecular donor-acceptor dyads or triads. Similar to the corresponding phthalimides, electronically excited pyromellitic diimides oxidize alkyl carboxylates in aqueous solution via intermolecular electron transfer (PET) processes, which eventually results in radical-radical combination products, e.g., the benzylation product 6 from N,N'-dimethyl pyromellitic diimide 5. The analogous product 7 was formed with pivalic acid as tert-butyl radical source. One additional product 8 was isolated from alkylation/dearomatization and multiple radical additions, respectively, after prolonged irradiation. In intramolecular versions, from N-carboxyalkylated pyromellitic diimides 9a-e (C1 to C5-spaced), degradation processes were detected, e.g., the cyclization products 10 from the GABA substrate 9c. In sharp contrast to phthalimide photochemistry, the green pyromellitic diimide radical anion was detected here by UV-vis absorption (λabs = 720 nm), EPR (from 9d), and NMR spectroscopy for several intramolecular electron transfer examples. Only the yellow 1,4-quinodial structure is formed from intermolecular PET, which was deduced from the absorption spectra (λabs = 440 nm) and the subsequent chemistry. The pyromellitimide radical anion lives for hours at room temperature in the dark, but is further degraded under photochemical reaction conditions.

Features of Hemophagocytic Lymphohistiocytosis in Infants With Severe Combined Immunodeficiency: Our Experience From Chandigarh, North India.

Background: Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled and excessive inflammation leading to high mortality. Aetiology of HLH can be primarily due to genetic causes or secondarily due to infections or rheumatological illness. However, rarely T-cell deficiencies like severe combined immunodeficiency (SCID) can develop HLH. Objective: To describe clinical and laboratory features of SCID cases who developed HLH. Methods: We collected clinical, laboratory, and molecular details of patients with SCID who developed HLH at our center at Chandigarh, North India. Results: Of the 94 cases with SCID, 6 were noted to have developed HLH-like manifestations. Male-female ratio was 5:1. Median (inter-quartile range) age of onset of clinical symptoms was 4.25 months (2-5 months). Median (inter-quartile range) delay in diagnosis was 1 month (1-3.5 months). Family history of deaths was seen in 4 cases. Molecular defects in IL2RG were seen in 5 out of 6 cases. Documented infections include disseminated bacillus calmette-guerin (BCG) infection (n=2), blood stream infections (n=3) with Staphylococcal aureus (n=1), Klebsiella pneumonia (n=1), and Pseudomonas aeruginosa (n=1), pneumonia (influenza H1N1 strain, and K. pneumoniae (n=1). Conclusion: Children with SCID can present with HLH-like manifestations secondary to fulminant infections. A high index of suspicion of SCID is needed in infants who present with HLH who have an associated infection or a suggestive family history. Occurrence of HLH-like manifestations in SCID suggests that T-lymphocytes may not have a significant role in immunopathogenesis of HLH.

Utility of targeted next generation sequencing for inborn errors of immunity at a tertiary care centre in North India.

Inborn errors of immunity (IEI) are a heterogeneous group of monogenic disorders that include primary immunodeficiency's and other disorders affecting different aspects of the immune system. Next-Generation Sequencing (NGS) is an essential tool to diagnose IEI. We report our 3-year experience in setting up facilities for NGS for diagnosis of IEI in Chandigarh, North India. We used a targeted, customized gene panel of 44 genes known to result in IEI. Variant analysis was done using Ion Reporter software. The in-house NGS has enabled us to offer genetic diagnoses to patients with IEI at minimal costs. Of 121 patients who were included pathogenic variants were identified in 77 patients. These included patients with Chronic Granulomatous Disease, Severe Combined Immune Deficiency, leukocyte adhesion defect, X-linked agammaglobulinemia, Ataxia Telangiectasia, Hyper-IgE syndrome, Wiskott Aldrich syndrome, Mendelian susceptibility to mycobacterial diseases, Hyper-IgM syndrome, autoimmune lymphoproliferative syndrome, and GATA-2 deficiency. This manuscript discusses the challenges encountered while setting up and running targeted NGS for IEI in our unit. Genetic diagnosis has helped our patients with IEI in genetic counselling, prenatal diagnosis, and accessing appropriate therapeutic options.