A Screening Approach for Inherited Erythrocytosis due to the VHL:c.598C > T Mutation (Chuvash Polycythemia).

Genetic work-up of unexplained erythrocytosis that is suspected to be inherited in nature currently requires either laborious exon-by-exon gene panel testing by Sanger sequencing or expensive next-generation sequencing. A high prevalence of Chuvash polycythemia (61%) has been previously reported among north Indian erythrocytosis patients. We assessed PCR-RFLP for VHL c.598C > T mutation as a first-line test in 99 persons with JAK2 V617F-negative, unexplained erythrocytosis. We enrolled two groups: Group A (n = 38) had erythrocytosis patients (n = 33) or their first-degree relatives (n = 5), and, Group B with 61 healthy blood donation volunteers who were deferred after the discovery of unexplained high hemoglobin levels. Detailed history and clinical examination, hemogram, erythropoietin levels and PCR-RFLP for the VHL:c.598C > T;p.R200W mutation were done. In Group A, three (8%) persons aged 9, 13 and 30-years were homozygous for VHL:c.598C > T. Two were heterozygous (parents of a known case of Chuvash polycythemia). None of the Group B subjects had the Chuvash mutation. Erythropoietin levels in group A were low in 5/26 cases (19%) and normal in 18/26 (69%). In Group B, seven (11%) donors had normal values while the remaining 54 (89%) had high erythropoietin levels. Despite a lower frequency (8%) compared to literature, our results suggest that the relatively simpler PCR-RFLP for VHL:c.598C > T mutation may be considered for the initial genetic screening of unexplained, suspected congenital erythrocytosis in regions where Chuvash polycythemia comprises a large proportion of inherited erythrocytosis, after polycythemia vera and common acquired secondary causes are excluded. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01668-9.

Hematological and genetic profiles of persons with co-inherited heterozygous ß-thalassemia and supernumerary α-globin genes.

INTRODUCTION: Thalassemias are common monogenic autosomal recessive hemoglobin disorders. The usually asymptomatic heterozygotes (ß-thalassemia traits, ßTT) may rarely develop non-transfusion-dependent-thalassemia (NTDT) due to co-inheritance of supernumerary α-globin genes. Literature on phenotypic/genotypic features of these rare combinations is limited. MATERIALS AND METHODS: We studied the demographic, clinical, and laboratory data from 47 persons with co-inherited ßTT + supernumerary α-globin genes. HBB mutations were tested for by ARMS-PCR and/or Sanger sequencing, ααα(anti3.7) /ααα(anti4.2) and deletional α-thalassemia testing by multiplex gap-PCRs, and Xmn1G γ genotyping by PCR-RFLP. RESULTS: The 47 cases comprised 0.08% of 61 010 hemoglobinopathy screenings during the study period. Mean age was 31.9 ± 14.7 years (range 5.5-83 years), with 57.4% males. Thirty (63.8%) had NTDT-phenotype, 16 (34%) were asymptomatic/minimally symptomatic, and 1 became transfusion-dependent at the age of 20 years. Anemia/pallor and jaundice were the commonest complaints (76% each); 40% had required blood transfusions. Twenty-one had splenomegaly, 14 had hepatomegaly. Mean hemoglobin was 9.0 ± 1.9 g/dl (range 4.0-13.0). HbA2 was 5.1 ± 0.7% (3.4%-6.3%) and HbF% 4.2 ± 3.2% (0.5%-18.4%). Forty-four (93.6%) had αααanti3.7 , while 3 (6.4%) had αααanti4.2 triplications. HBB:c.92+5G>C (47%), HBB:c.27_28insG (14.9%), and HBB:c.47G>A (8.5%) were the commonest ß-globin mutations. One case showed HBB:c.-138C>T (ß++ ), while the rest had ß0 or severe-ß+ mutations. Symptomatic cases had significantly lower hemoglobins and higher HbF% than asymptomatic ones. CONCLUSION: This largest Indian and globally second-largest study reports the ßTT + ααα4.2 state for the first time in such genotypically-complex Indian cases. Supernumerary α-genes should be suspected in all ßTT with disproportionate clinical symptoms, mild-to-moderately elevated HbF, and unexplained anisopoikilocytosis.

A novel ATRX splice variant causing acquired HbH disease in myelodysplastic syndrome with excess blasts-1.

A 60-year-old male with myelodysplastic syndrome with excess blasts-1 had unexplained microcytic hypochromic anemia. The cause of his anemia was revealed on supravital staining, hemoglobin studies and next-generation sequencing to be a novel hemizygous potentially pathogenic missense/splice site variant NM_000489.5:c.6848A>C, (p.Lys2283Thr) in exon 31 of the ATRX gene.

Next-Generation Sequencing Based Approach to Identify Underlying Genetic Defects of Glanzmann Thrombasthenia.

Glanzmann thrombasthenia (GT) is an autosomal recessive platelet function disorder characterized by mucocutaneous bleeding as the most common clinical phenotype. Patients with GT have normal platelet counts, platelet morphology but reduced platelet aggregation in response to various agonists. Homozygosity or compound heterozygosity for variants in the ITGA2B/ITGB3 genes is the genetic basis for GT. Establishing a molecular diagnosis is definitive and is important for predictive testing. Using multi-gene panels is an accurate, faster, and cost-effective mode as compared to Sanger sequencing in large genes. We used a targeted resequencing based approach to identify pathogenic variants in eight cases in seven families. These variants were validated using Sanger sequencing in patients as well as family members and were predicted probably pathogenic using in-silico prediction tools. The variants include three missense (3/7 = 43%) (ITGA2B:c.1028 T > C, ITGA2B:c.1186G > A, ITGB3:c.1388G > C), two deletions (ITGA2B:c.559delG, ITGA2B:c.3092delT), one duplication (ITGA2B:c.1424_1427dupAGGT) and nonsense variant (ITGA2B:c.2578C > T, p.Gln860Ter). Except for one case which was compound heterozygous, the rest of the cases were homozygous. We found two novel variants that are reported for the first time in GT. The targeted resequencing based approach revealed varied genetic variants in North Indian patients, including two novels ones. The high yield of our panel indicates its suitability for usage in larger cohorts for the genetic diagnosis of GT patients. This approach is cost-effective and less cumbersome as compared to Sanger sequencing for these large size genes with multiple exons. The information so obtained is helpful in prenatal testing, carrier analysis, and genetic counseling.

A high frequency of Gilbert syndrome (UGT1A1*28/*28) and associated hyperbilirubinemia but not cholelithiasis in adolescent and adult north Indian patients with transfusion-dependent ß-thalassemia.

Hyperbilirubinemia and pigment gallstones are frequent complications in transfusion-dependent ß-thalassemia (TDßT) patients. Bilirubin production and clearance are determined by genetic as well as environmental variables like ineffective erythropoiesis, hemolysis, infection-induced hepatic injury, and drug- or iron-related toxicities. We studied the frequency of the Gilbert syndrome (GS), a common hereditary cause of hyperbilirubinemia in 102 TDßT patients aged 13-43 years (median 26 years). Total and unconjugated hyperbilirubinemia were frequent (81.4% and 84.3% patients respectively). Twenty (19.6%) patients showed total bilirubin > 3.0 mg/dL; 53 (51.9%) had an elevation of either alanine or aspartate aminotransferase, or alkaline phosphatase liver enzymes. Nineteen (18.6% of the 92 tested) were positive for hepatitis B or C, or HIV. The mean total and unconjugated bilirubin levels and AST, ALT, and ALP levels in patients positive for hepatitis B or C were not significantly different from negative cases. Eighteen patients (17.7%) had GS: homozygous (TA)7/7 UGT1A1 promoter motif (the *28/*28 genotype), 48 (47.1%) were heterozygous (TA)6/7. Total + unconjugated bilirubin rose significantly with the (TA)7 allele dose. Fourteen (13.7%) patients had gallstones. There was no significant difference in total/unconjugated bilirubin in patients with/without gallstones and no significant differences in frequencies of gallstones within the three UGT1A1 genotypes. This largest study in Indian TDßT patients suggests that GS should be excluded in TDßT cases where jaundice remains unexplained after treatable causes like infections, chelator toxicity, or transfusion-related hemolysis are excluded. GS was not associated with gallstones, possibly due to a lower incidence of cholelithiasis overall, a younger age cohort, or other environmental factors.

Missing Hb Q-India Peak in a Triple-Heterozygous Patient with Hb D-Punjab/Hb Q-India/ß-Thalassemia Trait.

Interpretation of variant hemoglobins (Hbs) can pose challenges. We describe a puzzling case with multiple variant Hb peaks that was solved by family studies. A 32-year-old female with anemia and jaundice underwent cation exchange high performance liquid chromatography (HPLC), which revealed near-absence of Hb A along with variant peaks in the D- and C-windows (78.9 and 13.3%, respectively) and normal range of Hb F. As the HPLC did not fit any known pattern, family screening was performed. Her mother was heterozygous for Hb D-Punjab (HBB: c.364G>C) and Hb Q-India (HBA1: c.193G>C) with the hybrid αQ-India/ßD-Punjab eluting in the C-window on HPLC. Her sister had ß-thalassemia (ß-thal) trait, while her brother was heterozygous for Hb Q-India. In view of the family study results, the index case was interpreted as a double heterozygote for Hb D-Punjab and ß-thal with coinherited Hb Q-India. The Hb Q-India peak [retention time (RT) 4.7 min.] was absent on her HPLC as there were no normal ß-globin chains available to bind with the αQ-India chains. To the best of our knowledge, such an HPLC pattern with a missing Q-India peak, despite having inherited the αQ-India variant, has not been previously reported. This case illustrates the importance of family screening as an inexpensive and rapid method to resolve difficult and unusual HPLC patterns.

ß-Thalassemia Intermedia Caused by Compound Heterozygosity for Hb Lepore-Hollandia and ß-Thalassemia is Rare in the Indian Population.

Compound heterozygosity for one of the Hb Lepore mutations and ß-thalassemia (ß-thal) is a rare cause of non transfusion-dependent thalassemia. We report a 4-year-old boy who presented clinically as homozygous/compound heterozygous ß-thal intermedia (ß-TI), an impression that was corroborated by the initial hemoglobin (Hb) high performance liquid chromatography (HPLC). However, the correct diagnosis of a rare compound heterozygous Hb Lepore-Hollandia/ß-thal was revealed after parental studies and molecular analyses including ß-globin gene sequencing. Our patient highlights the importance of a logical stepwise multi modality approach and the vital importance of parental screening and molecular studies in accurate characterization of complex hemoglobinopathies. Correct diagnosis is especially crucial if pre natal detection is anticipated for future pregnancies. Molecular analyses alone may not compensate for the unavailability of parental testing. This is because the molecular results may be misinterpreted, especially if limited tests are conducted. The infrequent prior reports of this combination from distant parts of the Indian subcontinent suggests that the origin of Hb Lepore-Hollandia from sporadic mutations occurs in isolated families.

Hb M-Iwate in an Indian family.

BACKGROUND: High performance liquid chromatography in a newborn girl with congenital cyanosis and a unilateral cleft palate revealed a variant hemoglobin with retention time of 4.8 min, similar to hemoglobin Q-India. Since hemoglobin Q-India did not explain the cyanosis, further investigations were initiated. METHODS: Sequencing of α-globin genes revealed hemoglobin M-Iwate ([α87 (F8) His → Tyr]) that was confirmed on restriction enzyme analysis. RESULTS: Hemoglobin M-Iwate is a rare methemoglobinemic variant formed due to a point mutation in the α-globin gene. Primarily reported from the Iwate prefecture of Japan, there have been occasional case reports from other regions as well. Inherited methemoglobinemia finds only rare mention in Indian literature while hemoglobin M-Iwate has not been reported from India. CONCLUSIONS: This case illustrates the step-wise logical diagnostic approaches necessary to elucidate the cause of methemoglobinemia in an otherwise healthy child with cyanosis.

High prevalence of VKORC1*3 (G9041A) genetic polymorphism in north Indians: A study on patients with cardiac disorders on acenocoumarol.

Coumarin derivatives such as warfarin and acenocoumarol are used in various disorders such as deep venous thrombosis, pulmonary embolism, atrial fibrillation and artificial heart valves. They have improved prognosis of patients with thromboembolic disease. An individual's response to coumarins depends on several factors. The non-genetic factors include age, gender, body mass index, diet and interacting drugs. Among the genetic factors, the cytochrome P450 system and vitamin K epoxide reductase complex subunit 1 play a key role in drug metabolism. This was a prospective hospital based study in which allele and genotypic frequencies of CYP2C9 gene polymorphisms; 430C>T and 1075A>C and VKORC1 gene polymorphisms; 1639G>A, 9041G>A and 6009C>T in 106 alleles of north Indian patients with valve replacement on acenocoumarol were determined and their effect on acenocoumarol dosing was studied. To the best of our knowledge, this is first report of VKORC1 9041G>A and 6009C>T gene polymorphisms and their effect on acenocoumarol dosing from north India. In 53 patients with valve replacement on acenocoumarol with stable INR, the allele frequency of CYP2C9*2 and CYP2C9*3 gene polymorphisms was 0.05 and 0.17 respectively and that of VKORC1 *2,*3 and *4 gene polymorphisms was 0.15, 0.72 and 0.11 respectively. The presence of CYP2C9*3 or VKORC1*2 gene polymorphism were associated with decrease in acenocoumarol dose requirements (p values 0.03 and 0.02 respectively).This study confirmed the association of lower mean weekly dosages of acenocoumarol in patients with CYP2C9*3 and VKORC1*2 gene polymorphisms. An unusually high frequency of 9041A polymorphism in VKORC1 was found in study population.