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.

Role of Oxidative Stress and the Protective Effect of Fermented Papaya Preparation in Sickle Cell Disease.

Fermented papaya preparation (FPP) is the source of antioxidants that may help in reducing the complications associated with oxidative stress and may improve the quality of life in sickle cell disease patients. In this study, we assessed the in vitro effect of FPP on sickled red blood cells (RBCs) using oxidative stress markers and observed that FPP has the potential to reduce the oxidative stress. Scanning electron microscopy (SEM) and eosin 5' malaemide (E5'M) dye test showed that FPP protects red cell morphology against the oxidative stress. Liquid chromatography mass spectrometry (LCMS) analysis of FPP suggests the presence of essential amino acids, vitamin D3, and its derivatives. Fermented papaya preparation can be of benefit either in reducing oxidative stress parameters or in preventing pathophysiological events in the sickle cell disease patients.

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.

Fabrication of gelatin functionalized silver nanoparticles for blood group profiling.

We report the fabrication of silver nanoparticles (AgNPs) surface functionalized with gelatin at different concentrations (G10/G20/G40 AgNPs) with an average particle size of ∼200 nm, bioconjugated with antisera antibodies (AsAbs) of the major and clinically significant blood groups (CSBGs) at different titres from neat to 1:128. Bioconjugation using ionic interaction at pH 7.4 enabled 'end-on' configuration, with the -NH2 group of the antibody free for interaction with the red blood cell antigen, as confirmed by Fourier transform infrared spectroscopy. The tube agglutination test (TAT) revealed optimum agglutination with G20NPs, while SDS PAGE confirmed the optimal titre as 1:8 for the major blood groups A, B, AB and O. Bioconjugated AgNPs coated onto microtitre assay plates with the major blood groups and CSBGs to enable simultaneous identification, were validated against the TAT on 400 random blood samples for the major blood groups and revealed high accuracy (95%). While similar accuracy was seen for most of the CSBGs with only false negatives, the method was not found to be suitable for the Kell, Kidd and Duffy groups. The absence of false positives reflects high safety, and eliminates the risk of a mismatched blood transfusion. The method uses diluted blood and hence could enable point-of-care detection. The significantly lower AsAb requirement also provides a cost advantage.

Fibrotic Cytokine Interplay in Evaluation of Disease Activity in Treatment Naïve Systemic Sclerosis Patients from Western India.

BACKGROUND: : Systemic sclerosis (SSc) is a demyelinating disease of skin, subcutaneous tissue, muscles and internal organs, with fibrosis as an important pathological event. AIM: : To understand cytokine interplay of IL-1ß, IL-4 and IL-6 and their association with disease activity in treatment naïve active cases of systemic sclerosis from Western India. METHODS: Twenty-five SSc patients as per ACR-EULAR 2013 criteria (classified based on pulmonary fibrosis and generalized fibrosis) and 25 age-sex matched controls were enrolled. Serum cytokine levels of IL-1ß, IL-4 and IL-6 were assessed by multiplex bead based immunoassay. RESULTS: Ten patients had Interstitial lung disease (ILD), whereas, 16 patients had generalized fibrosis. Anti-nuclear antibodies were seen in 22 patients (88%); antiScl70 in 15 patients (60%) and anti-Centromere antibodies in 5 patients (20%). Serum levels of IL-1ß in patients were significantly higher than healthy controls (p=0.0006). IL-4 levels in all SSc patients were marginally raised (p=0.0102), while IL-6 levels were significantly raised (p<0.0001). IL-4 was found to be significantly raised in SSc patients with ILD (p=0.021) as compared to patients without ILD. IL-1ß (p=0.0293) and IL-4 (p<0.0001) were significantly higher in SSc patients with fibrosis. On the contrary, IL-6 levels in patients with fibrosis were found to be lower than in patients without fibrosis. CONCLUSION: Significantly raised cytokine levels among treatment naïve systemic sclerosis patients were found to be associated with higher disease severity in our study. Higher levels of IL-1ß and IL-6 indicated an active inflammatory status, whereas significantly raised IL-4 levels indicated at higher fibrotic activity.

Cleavage-resistant RIPK1-induced autoinflammatory syndrome-A report of three generations with periodic fever and clinical response to colchicine.

The clinical syndrome caused by cleavage-resistant RIPK1 is known as CRIA (Cleavage-resistant RIPK1-induced autoinflammatory) syndrome. We present a family with three generations affected by CRIA syndrome. Our index patient (P1), a boy born of a non-consanguineous marriage, developed recurrent episodes of fever after 5 months of age, with variable periodicity. His father (P2) and paternal grandmother also had periodic fever. At 23 months of age, P1 was diagnosed with renal biopsy-proven steroid-responsive nephrotic syndrome. His first visit to our center was at 2 years of age. At presentation, he had failure to thrive, microcytic hypochromic anemia, and elevated inflammatory markers and interleukin-6 levels. Amyloid A protein was elevated, serum creatinine was normal, and proteinuria resolved after addition of steroids. Next-generation sequencing showed heterozygous mutation (c.970G>A, p.Asp324His) in RIPK1. This mutation has been reported to cause CRIA syndrome. P2 and P1's asymptomatic younger brother had the same mutation. All the affected members showed variability with respect to frequency and duration of periodic fever as well as the age of onset. Both P1 and P2 had elevated amyloid A, with no evidence of renal dysfunction. P1 and P2 showed improvement in the intensity of fever spikes with colchicine treatment; however, both continue to have periodic fever.

Wiskott Aldrich Syndrome: A Multi-Institutional Experience From India.

Background: Wiskott Aldrich syndrome (WAS) is characterized by bleeding manifestations, recurrent infections, eczema, autoimmunity, and malignancy. Over the last decade, improved awareness and better in-house diagnostic facilities at several centers in India has resulted in increased recognition of WAS. This study reports collated data across major primary immunodeficiency diseases (PID) centers in India that are involved in care of children with WAS and highlights the varied clinical presentations, genetic profile, and outcomes of patients in India. Methods: Request to share data was sent to multiple centers in India that are involved in care and management of patients with PID. Six centers provided requisite data that were compiled and analyzed. Results: In this multi-institutional cohort, clinical details of 108 patients who had a provisional diagnosis of WAS were received. Of these, 95 patients with 'definite WAS' were included Fourteen patients were classified as XLT and 81 patients as WAS. Median age at onset of symptoms of patients was 3 months (IQR 1.6, 6.0 months) and median age at diagnosis was 12 months (IQR 6,48 months). Clinical profile included bleeding episodes (92.6%), infections (84.2%), eczema (78.9%), various autoimmune manifestations (40%), and malignancy (2.1%). DNA analysis revealed 47 variants in 67 cases. Nonsense and missense variants were the most common (28.4% each), followed by small deletions (19.4%), and splice site defects (16.4%). We also report 24 novel variants, most of these being frameshift and nonsense mutations resulting in premature termination of protein synthesis. Prophylactic intravenous immunoglobulin (IVIg) was initiated in 52 patients (54.7%). Hematopoietic stem cell transplantation (HSCT) was carried out in 25 patients (26.3%). Of those transplanted, disease-free survival was seen in 15 patients (60%). Transplant related mortality was 36%. Outcome details were available for 89 patients. Of these, 37% had died till the time of this analysis. Median duration of follow-up was 36 months (range 2 weeks- 12 years; IQR 16.2 months- 70 months). Conclusions: We report the first nationwide cohort of patients with WAS from India. Bleeding episodes and infections are common manifestations. Mortality continues to be high as curative therapy is not accessible to most of our patients.

Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients.

Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing congenital haemolytic anaemia (CHA). Diagnosis of GPI deficiency by the biochemical method is unpredicted. Molecular diagnosis by identifying genetic mutation is the gold standard method for confirmation of disease, but causative genes involved in CHA are numerous, and identifying a gene-by-gene approach using Sanger sequencing is also cumbersome, expensive and labour intensive. Recently, next-generation targeted sequencing is more useful in the diagnosis of unexplained haemolytic anaemia. We used targeted next-generation sequencing (NGS) clinical panel for diagnosis of unexplained haemolytic anaemia in two Indian patients which were pending for a long time. All possible causes of haemolytic anaemia were found within normal limit. NGS by clinical exome panel revealed homozygous novel missense mutation in exon 12, c.1009G>A (p.Ala337Thr) in both patients. We further confirm by measuring red blood cell GPI activity in the patients and showed deficiency whereas parents were having intermediate activity. c.1009G>A mutation was also confirmed by Sanger sequencing of exon 12 of GPI gene. The structural-functional analysis by bioinformatics software like Swiss PDB, PolyPhen-2 and PyMol suggested that this pathogenic variant has a direct impact on the structural rearrangement at the region near the active site of the enzyme. This rapid and high-performance targeted NGS assay can be configured to detect specific CHA mutations unique to an individual defect, making it a potentially valuable method for diagnosis of unexplained haemolytic anaemia.

Lymphopenia and Severe Combined Immunodeficiency (SCID) - Think Before You Ink.

OBJECTIVES: Severe combined immunodeficiency (SCID) represents one of the most severe forms of Primary immunodeficiency (PID) disorders, characterized by T cell lymphopenia (TCL) and lack of cellular and humoral immune responses. However, not all patients with low T cell lymphocyte counts may have an abnormal T cell immunity and the observed TCL may be a temporary suppression resulting from transient lymphopenia secondary to severe infections. In such cases, it is necessary to estimate the severity of the observed TCL by assessing thymic capabilities. METHODS: In this study, patients clinically suspected of SCID were evaluated for lymphocyte subsets analysis, naïve T cells and T cell receptor excision circles (TREC). RESULTS: Patients with transient lymphopenia had detectable TREC levels and normal naïve T cells subsets. Normalization of absolute lymphocyte counts, and T cells was seen in the patients after a short duration. CONCLUSIONS: The authors highlight the importance of detailed immunological investigations in an infant with severe infections and lymphopenia before labeling the infant as SCID.

Red cell adenylate kinase deficiency in India: identification of two novel missense mutations (c.71A>G and c.413G>A).

Adenylate kinase (AK) deficiency is a rare erythroenzymopathy associated with hereditary nonspherocytic haemolytic anaemia along with mental/psychomotor retardation in few cases. Diagnosis of AK deficiency depends on the decreased level of enzyme activity in red cell and identification of a mutation in the AK1 gene. Until, only eight mutations causing AK deficiency have been reported in the literature. We are reporting two novel missense mutation (c.71A > G and c.413G > A) detected in the AK1 gene by next-generation sequencing (NGS) in a 6-year-old male child from India. Red cell AK enzyme activity was found to be 30% normal. We have screened a total of 32 family members of the patient and showed reduced red cell enzyme activity and confirm mutations by Sanger's sequencing. On the basis of Sanger sequencing, we suggest that the proband has inherited a mutation in AK1 gene exon 4 c.71A > G (p.Gln24Arg) from paternal family and exon 6 c.413G > A (p.Arg138His) from maternal family. Bioinformatics tools, such as SIFT, Polymorphism Phenotyping v.2, Mutation Taster, MutPred, also confirmed the deleterious effect of both the mutations. Molecular modelling suggests that the structural changes induced by p.Gln24Arg and p.Arg138His are pathogenic variants having a direct impact on the structural arrangement of the region close to the active site of the enzyme. In conclusion, NGS will be the best solution for diagnosis of very rare disorders leading to better management of the disease. This is the first report of the red cell AK deficiency from the Indian population.

Study of pathophysiology and molecular characterization of congenital anemia in India using targeted next-generation sequencing approach.

Most patients with anemia are diagnosed through clinical phenotype and basic laboratory testing. Nonetheless, in cases of rare congenital anemias, some patients remain undiagnosed despite undergoing an exhaustive workup. Genetic testing is complicated by the large number of genes that are involved in rare anemias, due to similarities in the clinical presentation. We sought to enhance the diagnosis of patients with congenital anemias by using targeted next-generation sequencing. The genetic diagnosis was performed by gene capture followed by next-generation sequencing of 76 genes known to cause anemia syndromes. Genetic diagnosis was achieved in 17 of 21 transfusion-dependent patients and undiagnosed by conventional workup. Four cases were diagnosed with red cell membrane protein defects, four patients were diagnosed with pyruvate kinase deficiency, one case of adenylate kinase deficiency, one case of glucose phosphate isomerase deficiency, one case of hereditary xerocytosis, three cases having combined membrane and enzyme defect, two cases with Diamond-Blackfan anemia (DBA) and 1 with CDA type II with 26 different mutations, of which 21 are novel. Earlier incorporation of this NGS method into the workup of patients with congenital anemia may improve patient care and enable genetic counselling.