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.

Hb Mizuho (HBB: c.206T>C): Pitfalls of Screening Tests in an Unstable Hemoglobin Variant Diagnosed after Targeted Next-Generation Sequencing.

Hyperunstable hemoglobins (Hbs) are challenging to diagnose and may be missed on conventional hemolytic anemia work-up. Here, we report the case of a 2-year-old Indian boy with infancy-onset severe hemolytic anemia. Its etiology was revealed by targeted next-generation sequencing (NGS) to be the rare Hb Mizuho (HBB: c.206T>C). This variant had been missed on the initial routine laboratory investigations (heat and isopropanol tests for unstable Hbs) owing to its hyperunstable nature.

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.

Genetic basis of unexplained erythrocytosis in Indian patients.

OBJECTIVE: To evaluate the spectrum of genetic defects in Indian patients with unexplained erythrocytosis. METHODS: Fifteen families (18 patients) with unexplained erythrocytosis were enrolled after excluding polycythemia vera and secondary erythrocytosis. Focused Sanger sequencing from genomic DNA was performed for EPOR (exon 8), VHL (exons 2-3), EGLN1 (exons 2-5), EPAS1 (exon 12), and all exons of HBB, HBA1, and HBA2 genes. RESULTS: Eleven of the 18 patients (including two pairs of brothers) had Chuvash polycythemia, that is, homozygosity for VHL:c.598C > T (p.Arg200Trp). Three patients (two of whom were brothers) had HBB mutations associated with increased oxygen-affinity hemoglobin-one had a heterozygous Hb McKees Rocks HBB:c.438T > A (p.Tyr146*), and two brothers showed heterozygous Hb Rainier HBB:c.437A > G (p.Tyr146Cys). No pathogenic variants were found in the remaining four cases. CONCLUSION: A gene-by-gene Sanger sequencing approach could determine a genetic basis for erythrocytosis in 11 of the 15 (73%) Indian families, with homozygous VHL:c.598C > T (p.Arg200Trp) being the commonest pathogenic variant. This first study from the Indian subcontinent provides a rationale for analyzing this variant in patients with suspected congenital erythrocytosis from this region. Rare first occurrences of Hb McKees Rocks and Hb Rainier in Indians are also being reported.

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.

An intriguing high performance liquid chromatogram of a double heterozygosity for Hb Q-India/Hb D-Punjab.

Cation exchange high performance liquid chromatography (HPLC) is commonly utilized as the first method of screening for thalassemias and hemoglobinopathies worldwide. This method of diagnosis requires knowledge of the clinical background and complete blood counts as well as skill and experience in interpreting the sometimes complex results produced. An asymptomatic 27-year-old pregnant North Indian woman was found to have a highly unusual chromatographic pattern with multiple unexpected peaks during routine antenatal screening. Most concerning was a C-window peak as Hb C (HBB: c.19G>A) is rare in ethnic Asian Indian populations. Cellulose acetate electrophoresis at alkaline pH (8.6) and parental screening were performed. These revealed the correct diagnosis to be a double heterozygosity for Hb Q-India (HBA1: c.193G>C) (an uncommon asymptomatic α-globin chain variant) plus Hb D-Punjab (HBB: c.364G>C) (a ß-globin chain variant that is common in this region and is asymptomatic in the heterozygous state). The unexpected C-window peak was the hybrid of the abnormal α-Q-India and ß-D-Punjab globin chains. Another small peak was explained as a variant Hb A2 formed by the combination of α-Q-India and δ-globin chains. Hematopathologists should be cognizant of the complex pattern resulting from coinheritance of both α- and ß-globin structural variants. Second-line testing and parental testing are invaluable in resolving unknown peaks, especially if rare or unexpected variants are being considered. Although both Hb Q-India and Hb D-Punjab are relatively common in northwestern India, to the best of our knowledge, only two recent reports describe a total of three cases of such diagnostically puzzling coinheritance.

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.

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.

Short Research Communication Anti-Spike Antibody Response to COVISHIELD™ (SII-ChAdOx1 nCoV-19) Vaccine in Patients with B-Cell and Plasma Cell Malignancies and Hematopoietic Cell Transplantation Recipients.

Introduction: There is limited data on the serologic antibody responses after the ChAdOx1 vaccine in patients with hematological malignancies and hematopoietic cell transplantation recipients. There is no data on the safety and efficacy of the Indian COVISHIELD™ vaccine in this population. Methods: This study reports the anti-S antibody response to the COVISHIELD™ vaccine in a prospective cohort of patients with B-cell and plasma cell malignancies and HCT recipients at a single center. The quantitative antibodies to the SARS-CoV-2 S protein receptor-binding domain in human plasma were determined by the validated Roche Elecsys Anti-SARS-CoV-2 S kit. Results: A total of 118 patients were included over the study period from April 2021 to August 2021. The seropositivity rate at baseline and after the first and second dose of the vaccine was 39%, 66%, and 79%, respectively (p < 0.0001). The seronegative cohort had a higher median age (65 vs. 60 years, p = 0.03), were more likely to be males (81% vs. 42%, p = 0.009), had a diagnosis of B-CLPD (100% vs. 42%, p < 0.001) and were more likely to be on ibrutinib therapy (56% vs. 15%, p = 0.001). Conclusions: This study confirms the safety and efficacy of the COVISHIELD™ vaccine in patients with hematological malignancies.

A 37-Year-Old Man With Bronchial Asthma and Unexplained Hypoxemia.

CASE PRESENTATION: A 37-year-old man presented with breathlessness and wheeze of 3 weeks' duration. There was no chest pain, cough, palpitation, pedal edema, or fever. For the past 12 years, he had been experiencing episodic breathlessness and wheeze, which improved with inhaled salbutamol. He also had symptoms of nasal obstruction, nasal discharge, and sneezing. There was no history of smoking, substance abuse, or the use of any over-the-counter medication. The current episode of bronchial asthma exacerbation was managed with bronchodilators and systemic glucocorticoids. Despite symptomatic relief and clinical improvement, his oxygen saturation remained at 75% to 80%, and he was referred to our facility for further evaluation.