Novel frameshift variant (c.409dupG) in SLC25A38 is a common cause of congenital sideroblastic anaemia in the Indian subcontinent.

AIMS: Congenital sideroblastic anaemias (CSAs) are a group of rare disorders with the presence of ring sideroblasts in the bone marrow. Pathogenic variants are inherited in an autosomal recessive/X-linked fashion. The study was aimed at characterising the spectrum of mutations in SLC25A38 and ALAS2 genes in sideroblastic anaemia patients, exploring the genotype-phenotype correlation and identifying the haplotype associated with any recurrent mutation. PATIENTS AND METHODS: Twenty probable CSA patients were retrospectively analysed for genetic variants in ALAS2 and SLC25A38 genes by direct bidirectional sequencing. Real-time PCR was used to quantify gene expression in a case with promoter region variant in ALAS2. Three single nucleotide polymorphisms were used to establish the haplotype associated with a recurrent variant in the SLC25A38 gene. RESULTS: Six patients had causative variants in ALAS2 (30%) and 11 had variants in SLC25A38 (55%). The ALAS2 mutated cases presented at a significantly later age than the SLC25A38 cases. A frameshift variant in SLC25A38 (c.409dupG) was identified in six unrelated patients and was a common variant in our population exhibiting 'founder effect'. CONCLUSION: This is the largest series of sideroblastic anaemia cases with molecular characterisation from the Indian subcontinent.

Screening of genetic variants in ELANE mutation negative congenital neutropenia by next generation sequencing.

AIMS: Congenital neutropenia (CN) is a rare inherited disease that results in recurrent, life-threatening bacterial infections due to a deficiency of mature neutrophils. They are usually caused by heterozygous ELANE mutations although mutations in other genes like HAX-1, G6PC3 and GFI1 have also been reported. Identifying the causative mutation aids in the establishment of diagnosis and rules out other secondary causes of neutropenia like autoimmune cytopenia and evolving aplasia. We aimed to identify the molecular defects in CN patients who had no mutations in ELANE gene, by next generation sequencing (NGS) targeting a customised panel of genes. METHODS: DNA samples were sequenced with an Illumina NextSeq sequencer using an in-house customised panel of genes at ≥100× depth. Bioinformatics analysis was carried out and the pathogenic variants were identified using a stepwise filtering and analysis strategy. Specific mutations identified were subsequently validated by Sanger sequencing. RESULTS: The pathogenic variants identified in the study includes previously reported variants in SBDS (compound heterozygous c.258+2T>C and c.1A>T), GATA2 (heterozygous c.1186C>T) and novel variants in WAS (hemizygous c.812T>C), JAGN1 (homozygous c.70G>A) and RTEL1 (heterozygous c.2893G>C) genes. CONCLUSION: This study highlights that the absence of ELANE mutations does not rule out the diagnosis of CN and this NGS based approach with a customised panel will help in diagnostic confirmation in such patients. The early onset of the disease, clinical severity and associated high risk of malignant transformation in CN strongly suggests the need for early diagnosis and therapeutic intervention.

Cure for thalassemia major - from allogeneic hematopoietic stem cell transplantation to gene therapy.

Allogeneic hematopoietic stem cell transplantation has been well established for several decades as gene replacement therapy for patients with thalassemia major, and now offers very high rates of cure for patients who have access to this therapy. Outcomes have improved tremendously over the last decade, even in high-risk patients. The limited data available suggests that the long-term outcome is also excellent, with a >90% survival rate, but for the best results, hematopoietic stem cell transplantation should be offered early, before any end organ damage occurs. However, access to this therapy is limited in more than half the patients by the lack of suitable donors. Inadequate hematopoietic stem cell transplantation services and the high cost of therapy are other reasons for this limited access, particularly in those parts of the world which have a high prevalence of this condition. As a result, fewer than 10% of eligible patients are actually able to avail of this therapy. Other options for curative therapies are therefore needed. Recently, gene correction of autologous hematopoietic stem cells has been successfully established using lentiviral vectors, and several clinical trials have been initiated. A gene editing approach to correct the ß-globin mutation or disrupt the BCL11A gene to increase fetal hemoglobin production has also been reported, and is expected to be introduced in clinical trials soon. Curative possibilities for the major hemoglobin disorders are expanding. Providing access to these therapies around the world will remain a challenge.

Clinical, Hematological and Molecular Analysis of Homozygous Hb E (HBB: c.79G > A) in the Indian Population.

Homozygous Hb E [ß26(B8)Glu→Lys; HBB: c.79G > A] is a clinically mild disease with no significant symptoms. Very few studies are available on clinical variability in Hb E disorders. We report the profile of a series of homozygous Hb E patients in the Indian population. We analyzed various genetic factors that contribute to the heterogeneity in the phenotype of homozygous Hb E patients. Analysis of these parameters further enhances our understanding of the Hb E syndrome.

Characterization of Clinical and Laboratory Profiles of the Deletional α2-Globin Gene Polyadenylation Signal Sequence (AATAAA > AATA- -) in an Indian Population.

α-Thalassemia (α-thal) is characterized by large deletions involving the variable regions of α2 and/or α1 genes. Nondeletional mutations and polyadenylation (polyA) signal sequence motif mutations are less common. In this retrospective study, we describe a fragment length analysis-based polymerase chain reaction (PCR) assay for screening the T(Indian) (AATAAA > AATA- -; HBA2: c.*93_*94delAA) polyA signal deletion along with its clinical and laboratory presentation in 21 patients. Most of the patients were diagnosed in early adulthood with a clinical presentation ranging from asymptomatic in the heterozygous state to severe Hb H disease with a prominent hemolytic component in the homozygous state. On genetic analysis, 14 patients were found to be homozygotes, five were compound heterozygotes and two were heterozygotes. Thus, the T(Indian) polyA signal deletion is common in the Indian population and should be screened for in patients with nondeletional α-thal mutations.

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia.

BACKGROUND: Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. METHODS: Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. RESULTS: Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. CONCLUSION: This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

Cytidine deaminase genetic variants influence RNA expression and cytarabine cytotoxicity in acute myeloid leukemia.

AIM: Cytidine deaminase (CDA) irreversibly deaminates cytarabine (Ara-C), a key component of acute myeloid leukemia (AML) induction and consolidation therapy. CDA overexpression results in Ara-C resistance, while decreased expression is associated with toxicity. We evaluated factors influencing variation in CDA mRNA expression in adult AML patients and normal controls, and how they contributed to Ara-C cytotoxicity in AML cells. MATERIALS & METHODS: CDA mRNA expression in 100 de novo AML patients and 36 normal controls were determined using quantitative reverse-transcriptase PCR. Genetic variants in the CDA gene were screened by direct sequencing. IC50 of Ara-C was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: CDA RNA expression as well as Ara-C IC50 showed wide variation in AML samples and normal controls. Fourteen sequence variants were identified, three of which (-33delC, intron 2 TCAT repeat and the 3´untranslated region 816delC variants) showed significant association with RNA expression and the nonsynonymous coding variant 79A>C was associated with Ara-C cytotoxicity. CONCLUSION: CDA genetic variants explain the variation in RNA expression and may be candidates for individualizing Ara-C therapy.

A novel 26 bp deletion [HBB: c.20_45del26bp] in exon 1 of the ß-globin gene causing ß-thalassemia major.

Molecular characterization of ß-thalassemia (ß-thal) is essential in prevention and in understanding the biology of the disease. Deletion mutations are relatively uncommon in ß-thal. In this report, we describe a novel 26 bp deletion from codon 6 to codon 14 in the ß-globin in a consanguineous family from Tamil Nadu, India. This novel mutation causes a shift in the normal reading frame of the ß-globin coding sequence, and consequently, a premature chain termination of translation due to the creation of a stop codon at the position of codon 21. The identification of this novel deletional mutation adds to the repertoire of ß-thal mutations in India.

Hyperbilirubinemia in homozygous HbE disease is associated with the UGT1A1 gene polymorphism.

Homozygous HbE [beta26(B8)Glu-->Lys] is a clinically mild disorder with no significant symptoms. However, we have frequently noted hyperbilirubinemia among patients with homozygous HbE in the Indian population, with jaundice being the major complaint at presentation. A study of the UGT1A1 gene polymorphism shows that the variant TA7 in the promoter region of the UGT1A1 gene is associated with hyperbilirubinemia in homozygous HbE patients.

Compound heterozygosity for Hb E and Hb Lepore-Hollandia in India; first report and potential diagnostic pitfalls.

A compound heterozygous state of Hb E [beta26(B8)Glu-->Lys] with Hb Lepore is rare with very few cases reported in the literature. This report describes the first such case from India. The clinical features and hemoglobin (Hb) analysis mimic Hb E-beta-thalassemia (thal) but with a mild phenotype. Detection was made possible in this case because DNA analysis gave discrepant results suggestive of homozygous Hb E. As this was inconsistent with the clinical phenotype and Hb analysis, further evaluation was undertaken that confirmed the presence of Hb Lepore. This study shows that cases of Hb E/Lepore may remain undetected unless family studies and/or detailed DNA analyses in patients diagnosed to have Hb E-beta-thal are performed.

Hb Showa-Yakushiji [beta110(G12)Leu-->Pro] in four unrelated patients from west Bengal.

A T-->C mutation in the beta-globin gene at codon 110 that produces the hyper unstable variant Hb Showa-Yakushiji was identified in four unrelated individuals in India. It was found in a compound heterozygous state with other mutations producing beta-thalassemia (thal) or Hb E [beta26(B8)Glu-->Lys]. The mutation producing this abnormal hemoglobin (Hb) was found on the same haplotype in all these patients but differed from the Japanese haplotype, indicating its independent origin in India.

High frequency of the 1896 precore mutation in patients and blood donors with hepatitis B virus infection from the Indian subcontinent.

AIM: Hepatitis B virus (HBV) e antigen (HBeAg)-negative variants are reported to harbor 1896 precore mutants, and predict a worse clinical outcome. The aim of this study was to estimate the incidence of a precore mutation (1896) in both patients with chronic hepatitis B (CH-B) infection and blood donors in a tertiary care hospital in south India. METHODS: One hundred and twenty-two consecutive HBV DNA-positive CH-B patients (group I) and 102 HBsAg-positive 'healthy' blood donors (group II) were recruited. Samples found to be positive for HBV DNA were further studied. A nested PCR was used for the detection of HBV DNA. The 1896 precore mutation was detected using PCR-restriction fragment length polymorphism (RFLP). Nucleotide sequencing was performed on representative samples to confirm PCR-RFLP findings. The study population was stratified comprising: group IA: 17 HBeAg-positive CH-B patients; group IB: 105 HBeAg-negative CH-B patients; group IIA: 12 HBeAg-positive blood donors; and group IIB: 55 HBeAg-negative blood donors. RESULTS: There was no significant difference in the HBeAg-positive status between groups I and II. Significantly higher levels of alanine transaminase (ALT) were seen in groups IA and IB than in groups IIA and IIB, respectively (p = 0.033; p = 0.004). A significantly higher proportion of CH-B patients (32.7%) were positive for anti-HBc IgM compared with the blood donor groups (10.4%; p = 0.0006). Among the HBeAg-negative subjects, 69% of the CH-B patients and 65% of the blood donors showed evidence of 1896 precore mutant. This infection included the 1896 mutant exclusively or mixed infection involving the 1896 mutant and 1896 wild-type. DISCUSSION: The absence of detectable HBeAg in most of the viremic blood donors and patients emphasizes the need for HBV DNA testing irrespective of HBeAg status. Mixed infection was detected in a higher proportion (42.6%) of CH-B patients than in blood donors (26.8%; p = 0.031). Among those with mixed infection, a significant proportion (44.2%) of CH-B patients, had ALT levels greater than the upper limit of normal (ULN), as compared with the blood donor groups (16.6%; p = 0.036). CONCLUSIONS: The majority of CH-B patients and blood donors were negative for HBeAg despite their positive HIV DNA status. About two-thirds of the HBsAg-positive blood donors were viremic. Mixed infection was detected more frequently in CH-B patients and appears to be associated with more pronounced liver damage, as indicated by increased ALT levels.

Glutathione S-transferase M1 polymorphism: a risk factor for hepatic venoocclusive disease in bone marrow transplantation.

Hepatic venoocclusive disease (HVOD) in bone marrow transplantation (BMT) is attributed to toxicity of cytoreductive agents, especially busulfan and cyclophosphamide, in the conditioning therapy. Busulfan, as well as the metabolites of cyclophosphamide, are conjugated with glutathione (GSH), catalyzed by enzymes of the glutathione S-transferase (GST) family. To assess the impact of polymorphisms of the GST genes, GSTM1 and GSTT1, on the risk of HVOD, we evaluated 114 consecutive patients with beta-thalassemia major undergoing BMT. There was a significantly increased incidence of HVOD in patients with the GSTM1-null genotype compared with those with the GSTM1-positive genotype (46.5% vs 18.3%; P =.001). Pharmacokinetic analysis in these patients showed that the clearance of busulfan was higher and first-dose steady-state concentration was lower among those with HVOD (0.403 +/- 0.06 vs 0.33 +/- 0.071 L/h/kg, Student t test P value =.000 01; and 508 +/- 125 vs 656 +/- 255 ng/mL, t test P value =.001, respectively). We conclude that the GSTM1-null genotype predisposes to HVOD, and the sinusoidal endothelial cells and hepatocyte damage may be mediated by metabolites of busulfan through depletion of the cellular GSH pool.

Prenatal diagnosis of Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive disorder of platelet function, which results in major morbidity due to persistent, spontaneous, mucocutaneous bleeding and menorrhagia in women. Platelet transfusions are often needed to control the bleeding. Glanzmann thrombasthenia results from mutations in the genes located on chromosome 17q21-23, encoding the platelet glycoprotein (GP) IIb/IIIa receptor. METHODS: This report describes, for the first time in India, the prenatal diagnosis performed in a family who had a child with GT. As the molecular defect had not been identified at the time of chorionic villus sampling (CVS), prenatal diagnosis was done by linkage assessment. Haplotype analysis was performed using polymorphic markers on chromosome 17q 12-21, which included the dinucleotide repeat polymorphisms (CA)n in BRCA1 gene and locus D17S579 and (CT)n within GP IIIa intron 6, and the known restriction fragment length polymorphism (RFLP) markers Fok I (GP IIb exon 26), Taq I (GP IIIa exon 8) and Sma I (GP IIIa exon 9). The specific mutation in this family was subsequently confirmed. RESULTS: Both parents and the foetus were heterozygous for all the dinucleotide repeat polymorphisms and the affected child was homozygous. Both parents and the affected child were homozygous for Fok I RFLP. The father was heterozygous, and the mother, affected child and foetus were homozygous for Taq I and Sma I. The Fok I RFLP was identical for all the family members and hence did not provide any information for haplotype analysis (foetus not tested). CONCLUSION: The findings from dinucleotide repeat polymorphisms in BRCA1, D17S579, and GP IIIa intron 6 and the Sma I and Taq I RFLPs in GP IIIa strongly suggested that the foetus had inherited the father's mutant and the mother's normal alleles. Hence, the foetus was diagnosed to be a heterozygous carrier of GT by haplotype analysis. A private sequence alteration was later identified in the affected child in GP IIIa IVS1 (-14C --> A). The parents and foetus were heterozygous for this mutation. This confirmed the findings of the haploytpe analysis.

HIV-2 subtype circulating in India (south).

The genetic types and subtypes of HIV vary from region to region. This study looked at the relative prevalence of HIV-2 subtypes in south India. Nucleotide sequencing of the HIV-2 envelope V3 region of strains from 11 individuals infected with HIV-2 and one individual who had dual infection with HIV-1 and HIV-2 was performed. Phylogenetic analysis showed that all individuals were infected with HIV-2 subtype A. These strains were from individuals who live in different regions of south India. In all, up to present, 17 strains inclusive of the authors' 12 have been sequenced, and subtype A of HIV-2 is seen in both monoinfections and dual infections with HIV-1 in India.

Rapid detection of beta-globin gene mutations and polymorphisms by temporal temperature gradient gel electrophoresis.

BACKGROUND: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. METHODS: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 beta-thalassemia genes from homozygous beta-thalassemia patients and unrelated individuals with heterozygous beta-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE. RESULTS: We found common mutations in 753 beta-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the beta-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning beta-globin gene were suspected. CONCLUSIONS: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.

Hb Lepore in the Indian population.

A study of the spectrum of beta-thalassemia mutations in 230 patients with thalassemia major and 90 patients with thalassemia intermedia revealed mutations producing Hb Lepore in four patients. Two were homozygous and two were compound heterozygous for Hb Lepore and beta-thalassemia. Among the six delta beta fusion genes found in these four patients five were those producing Hb Lepore-Hollandia and one producing Hb Lepore-Washington-Boston. We also describe a possible misdiagnosis in the heterozygous state of Hb Lepore, as Hb Lepore and Hb A2 are not distinctly separated by cation exchange high performance liquid chromatography.