Wide Spectrum of Molecular and Clinical Heterogeneity in HbH Disease in North Indian Patients

Background: Deletional a⁺-thalassemia comprising of -a^3.7 and -a^4.2 deletions is commonly encountered in the Indian sub-continent but HbH disease, resulting from the co-inheritance of a⁺-thalassemia trait and a^o-thalassemia trait, is uncommon with just a few cases reported. Few recent reports on the molecular characterization of patients with HbH disease from India has been published which includes both deletional a-thalassemia and non-deletional a-thalassemia. This report highlights the clinical and molecular heterogeneity of HbH disease from north India.

Materials and Methods: We encountered 34 patients with HbH disease in 28 families from north India over 15 years and performed the molecular analysis to determine the frequency of both deletional and non-deletional alpha gene defects. Patients as well as parents and affected siblings were also studied for automated complete blood cell counts, reticulocyte counts and HbH inclusions using 1% brilliant cresyl blue. Cation exchange HPLC (Bio-Rad Laboratories, Hercules, CA, USA) and hemoglobin electrophoresis at pH 8.6 were performed. Genomic DNA from peripheral blood leucocytes was extracted by the phenol-chloroform method. A multiplex Gap-PCR was carried out as an initial screen which tested for deletions a^3.7, a^4.2, a^SEA, a^MED and a^SA. Southern Blot for alpha and zeta probes with Bam H1 digest was performed on 20 subjects. PCR followed by sequencing for both a1 and a2 genes were done. The a–Globin^XS MLPA kit (MRC Holland) with 35 probes was used to screen for the extent of deletions.

Results: Homozygous Hb Sallanches (a2 codon 104 G>A; Cys →Tyr) in 6 cases in 5 families was found. Homozygous polyadenylation signal mutation of a2(-AA) AATAAA →AATA—was detected in 3 cases. One patient each showed double heterozygosity for -a^3.7/a^{Hb Sallanches} a, a^{76 +T}a/a^{Hb Sallanches} a. ^PolyAa/ a^{Hb Sun Prairie} a, a^PolyAa/ a^{76 +T}a, and -a^3.7/a^{Hb Seal Rock}a and a^{Seal Rock} a/--. Three patients were double heterozygous for -a^3.7/--^SA. Fifteen patients from 12 families had a⁰ deletion of varying lengths in combination with -a^3.7 based on the MLPA analysis. The extent of deletions was variable ranging from a⁰ starts upstream of z gene & extends downstream of a1, deletion of probes 1-18, deletion of probes 1-22 and deletion of probes 8-18.The genotype phenotype correlation illustrating the clinical heterogeneity of HbH disease is detailed in Table 1.

Discussion: This is the largest series of cases of HbH disease encountered in Indians. Since HbH disease is uncommon in India, the clinical recognition of the diagnosis is limited to few centres and molecular characterization is incomplete. Our study shows considerable heterogeneity both at the molecular level as well as the clinical presentation. Alpha2 gene sequencing revealed a novel mutation with addition of +T at codon 76 leading to a frameshift mutation .MLPA will be useful in characterizing the approximate location of the breakpoints and designing simple GAP PCR assays to identify the deletion. Increased awareness amongst the clinicians as well as more diagnostic laboratories will help to identify more patients with HbH disease.