Comparison of telomere length in patients with bone marrow failure syndromes and healthy controls.

INTRODUCTION: During normal aging, telomeric DNA is gradually lost in dividing somatic cells, and critically short telomeres lead to replicative senescence, apoptosis, or chromosomal instability. We studied telomere length in bone marrow failure syndromes (BMFS) compared to normal healthy population. METHODS: Peripheral blood was collected from the participants, and genomic DNA was extracted. Relative telomere length was measured using a quantitative polymerase chain reaction. Statistical analysis was performed using SPSS and GraphPad Prism 8.2 software. RESULTS: The median age of normal Indian population was 31 (0-60) years. As expected, telomere length (TL) showed a decline with age and no difference in TL between males and females. The median age of 650 patients with aplastic anemia (AA) was 30 (1-60) years. TL was significantly shorter in patients with AA compared to healthy controls (p < .001). In FA and MDS patients, TL was significantly shorter than age-matched healthy controls (p = .028; p < .001), respectively. There was no difference between the median TL in age-matched AA and FA patients (p = .727). However, patients with MDS had shorter TL than age-matched AA (p = .031). CONCLUSION: TL in BMF syndrome patients was significantly shorter than age-matched healthy controls.

Treosulfan Exposure Predicts Thalassemia-Free Survival in Patients with Beta Thalassemia Major Undergoing Allogeneic Hematopoietic Cell Transplantation.

A toxicity-reduced conditioning regimen with treosulfan, fludarabine, and thiotepa in patients with high-risk ß-thalassemia major has significantly improved hematopoietic stem cell transplantation (HCT) outcomes. However, complications resulting from regimen-related toxicities (RRTs), mixed chimerism, and graft rejection remain a challenge. We evaluated the dose-exposure-response relationship of treosulfan and its active metabolite S, S-EBDM, in a uniform cohort of patients with ß-thalassemia major to identify whether therapeutic drug monitoring (TDM) and dose adjustment of treosulfan is feasible. Plasma treosulfan/S, S-EBDM levels were measured in 77 patients using a validated liquid chromatography with tandem mass spectrometry method, and the pharmacokinetic parameters were estimated using nlmixr2. The influence of treosulfan and S, S-EBDM exposure, and GSTA1/NQO1 polymorphisms on graft rejection, RRTs, chimerism status, and 1-year overall survival (OS), and thalassemia-free survival (TFS) were assessed. We observed that treosulfan exposure was lower in patients with graft rejection than those without (1,655 vs. 2,037 mg•h/L, P = 0.07). Pharmacodynamic modeling analysis to identify therapeutic cutoff revealed that treosulfan exposure ≥1,660 mg•hour/L was significantly associated with better 1-year TFS (97% vs. 81%, P = 0.02) and a trend to better 1-year OS (90% vs. 69%, P = 0.07). Further, multivariate analysis adjusting for known pre-HCT risk factors also revealed treosulfan exposure <1,660 mg•h/L (hazard ratio (HR) = 3.23; 95% confidence interval (CI) = 1.12-9.34; P = 0.03) and GSTA1*B variant genotype (HR = 3.75; 95% CI = 1.04-13.47; P = 0.04) to be independent predictors for inferior 1-year TFS. We conclude that lower treosulfan exposure increases the risk of graft rejection and early transplant-related mortality affecting TFS. As no RRTs were observed with increasing treosulfan exposure, TDM-based dose adjustment could be feasible and beneficial.

Molecular insights into placental iron transfer mechanisms and maternofetal regulation.

PURPOSE: Adequate iron transportation from the mother across the placenta is crucial for fetal growth and establishing sufficient iron stores in neonates at birth. The past decade has marked significant discoveries in iron metabolism with the identification of new players and mechanisms. Immunohistochemical studies rendered valuable data on the localization of substantial iron transporters on placental syncytiotrophoblasts. However, the function and regulation of maternal-placentofetal iron transporters and iron handling is still elusive and requires more attention. METHODS: A thorough literature review was conducted to gather information about placental iron transfer, the role of regulators and maintenance of iron homeostasis. RESULTS: The role of classical and new players in maternal-fetal iron transport and the regulation in the placenta has been addressed in this review. Animal and human studies have been discussed. The role of placental iron regulation in thalassemia and hemochromatosis pregnancies has been reviewed. CONCLUSIONS: The current advances that highlight the mechanisms of placental iron regulation and transport in response to maternal and fetal signals have been presented.

Complex Interactions in Regulation of Haematopoiesis-An Unexplored Iron Mine.

Iron is one of the most abundant metals on earth and is vital for the growth and survival of life forms. It is crucial for the functioning of plants and animals as it is an integral component of the photosynthetic apparatus and innumerable proteins and enzymes. It plays a pivotal role in haematopoiesis and affects the development and differentiation of different haematopoietic lineages, apart from its obvious necessity in erythropoiesis. A large amount of iron stores in humans is diverted towards the latter process, as iron is an indispensable component of haemoglobin. This review summarises the important players of iron metabolism and homeostasis that have been discovered in recent years and highlights the overall significance of iron in haematopoiesis. Its role in maintenance of haematopoietic stem cells, influence on differentiation of varied haematopoietic lineages and consequences of iron deficiency/overloading on development and maturation of different groups of haematopoietic cells have been discussed.

Molecular Characterization of G6PD Deficiency: Report of Three Novel G6PD Variants.

G6PD deficiency is a monogenic, X-linked genetic defect with a worldwide prevalence of around 400 million people and an overall prevalence of 8.5% in India. Hemolytic anemia is encountered in only a small proportion of patients with G6PD variants and is usually triggered by some exogenous agent. Although G6PD deficiency was reported in India more than 50 years ago, there are very few studies on molecular characterization and phenotypic correlation in G6PD deficient patients. We aimed to study the epidemiology and correlate the phenotypic expression with molecular genotypes in symptomatic G6PD deficient patients. All symptomatic hemolytic anaemia patients with a possible etiology of G6PD deficiency based on the clinical, hematological and biochemical parameters and reduced G6PD enzyme levels were included in this study. Molecular analysis of the G6PD gene was done by direct Sanger sequencing. From a total of 38 patients with hemolytic anemia suspected for G6PD deficiency, 24 patients had reduced G6PD enzyme levels and were included for the molecular analysis and mutations in the G6PD gene were identified in 21 of them (83.3%). The different mutations identified in our study include 6 patients with c.131C > G (G6PD Orissa), 3 patients with c.563C > T (G6PD Mediterranean), two patients with c.825G > T (G6PD Bangkok), one patient each with c.208T > C (G6PD Namouru), c.487G > A (G6PD Mahidol), c.949G > A (G6PD Kerala-Kalyan), c.100 G > A (G6PD Chatham), c.1178C > G (G6PD Nashville), c.1361 G > A (G6PD Andalus) and 4 patients with novel mutations (2 patients with c.1186C > T and 1 patient each with c.1288-2A > T and c.1372C > T. No disease causing genetic variants were identified in the other three cases. Co-inheritance of other red cell and hemoglobin disorders can modify the clinical phenotype of G6PD patients and the diagnostic accuracy can be improved by molecular characterization of the variant.

Spectrum of ELANE mutations in congenital neutropenia: a single-centre study in patients of Indian origin.

AIMS: Congenital and cyclical neutropenia are rare inherited diseases that result in recurrent life-threatening bacterial infections due to a deficiency of mature neutrophils. Cyclical neutropenia is usually caused by heterozygous ELANE mutations while congenital neutropenia is genetically heterogeneous with mutations in genes like ELANE, HAX-1, G6PC3 and GFI1. The presence of ELANE mutation aids in the establishment of diagnosis and rules out other secondary causes of neutropenia such as autoimmune cytopenia and evolving aplasia. Further, patients with ELANE mutations are also at a high risk of developing myelodysplasia or acute myeloid leukaemia. Hence it is important to screen for these mutations in patients presenting with neutropenia early in life. METHODS: The study included 52 patients who were evaluated for inherited neutropenia. Genomic DNA was extracted from peripheral blood leucocytes and mutation analysis was done by bidirectional Sanger sequencing. RESULTS: Ten different missense, frameshift or splice site variants in ELANE gene were identified in 11 patients: c.125C>T (p.Pro42Leu), c.164G>A (p.Cys55Tyr), c.169G>A (p.Ala57Thr), c.179T>C (p.Ile60Thr), c.770C>T (p.Pro257Leu), c.367-8C>A, c.597+1G>A along with three novel mutations c.302T>A (p.Val101Glu), c.468G>T (p.Try156Cys) and c.596delT (Phe199Ser fs*13). Family studies were available for three patients and, in all three instances, the mutation had a de novo origin. CONCLUSION: The widespread distribution of mutations suggests the need to screen all the exons in ELANE gene for proper characterisation of the genotype.

Pharmacokinetics and Pharmacodynamics of Treosulfan in Patients With Thalassemia Major Undergoing Allogeneic Hematopoietic Stem Cell Transplantation.

A treosulfan (Treo)-based conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) has been successfully used in treating hematological malignant and nonmalignant diseases. We report Treo pharmacokinetics (PK) in patients with thalassemia major undergoing HSCT (n = 87), receiving Treo at a dose of 14 g/m2 /day. Median Treo AUC and clearance (CL) was 1,326 mg*h/L and 10.8 L/h/m2 , respectively. There was wide interindividual variability in Treo AUC and CL (64 and 68%) which was not explained by any of the variables tested. None of the Treo PK parameters were significantly associated with graft rejection or toxicity; however, Treo CL <7.97 L/h/m2 was significantly associated with poor overall (hazard ratio (HR) 2.7, confidence interval (CI) (1.09-6.76), P = 0.032) and event-free survival (HR 2.4, CI (0.98-5.73), P = 0.055). Further studies in a larger cohort are warranted to identify the factors explaining the variation in Treo PK as well as to establish a therapeutic range of Treo for targeted dose adjustment to improve HSCT outcome.

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.

Identification of rare and novel deletions that cause (뫧)0-thalassaemia and hereditary persistence of foetal haemoglobin in Indian population.

OBJECTIVES: Hereditary persistence of foetal haemoglobin (HPFH) and (δß)(0) -thalassaemia are conditions caused by large deletions that involve δ- and ß-globin genes in the ß-globin cluster, and they are characterized by increased haemoglobin (HbF) levels in adults. Significant phenotypic diversity is observed between the different mutations that cause these conditions. Molecular characterization of these deletions is important for accurate molecular diagnosis, and they will also provide the information on the cis-acting genetic regulatory elements present in the ß-globin cluster. METHODS: We performed gap-PCR, multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescent multiplex PCR (QF-MPCR) and DNA sequencing to detect and characterize the deletions in the ß-globin cluster. RESULTS: We characterized six different deletions resulting in (δß)(0) -thalassaemia or HPFH in 51 unrelated families. CONCLUSION: With the help of multiple genetic tools, we performed comprehensive genetic analysis of HPFH and (δß)(0) -thalassaemia in Indian population and could define the molecular basis of these conditions in this population. We also identified two novel HPFH mutations, 49.98 kb (HPFH-9) and 86.7 kb (HPFH-10) deletions, in this population.

A novel δ-globin gene mutation (HBD: c.323G>A) masking the diagnosis of ß-thalassemia: a first report from India.

An elevated HbA(2) (α2δ2) level (>3.5%) is a well-established diagnostic test for heterozygous ß-thalassemia. Mutations in the δ-globin gene can cause decreased expression of HbA(2), resulting in heterozygous ß-thalassemia with normal levels of HbA(2). In this report, we describe a novel missense mutation in δ-globin (HBD: c.323G>A, Gly > Asp) in an Indian family with heterozygous ß-thalassemia with normal HbA(2) levels.

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.

Iron homeostasis: new players, newer insights.

Although iron is a relatively abundant element in the universe, it is estimated that more than 2 billion people worldwide suffer from iron deficiency anemia. Iron deficiency results in impaired production of iron-containing proteins, the most prominent of which is hemoglobin. Cellular iron deficiency inhibits cell growth and subsequently leads to cell death. Hemochromatosis, an inherited disorder results in disproportionate absorption of iron and the extra iron builds up in tissues resulting in organ damage. As both iron deficiency and iron overload have adverse effects, cellular and systemic iron homeostasis is critically important. Recent advances in the field of iron metabolism have led to newer understanding of the pathways involved in iron homeostasis and the diseases which arise from alteration in the regulators. Although insight into this complex regulation of the proteins involved in iron homeostasis has been obtained mainly through animal studies, it is most likely that this knowledge can be directly extrapolated to humans.

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.