Molecular basis of non-deletional HPFH in Thailand and identification of two novel mutations at the binding sites of CCAAT and GATA-1 transcription factors

Abstract

High Hb F determinants are genetic defects associated with increased expression of hemoglobin F in adult life, classified as deletional and non-deletional forms. We report the first description of non-deletional hereditary persistence of fetal hemoglobin (HFPH) in Thailand. Study was done on 388 subjects suspected of non-deletional HPFH with elevated Hb F expression. Mutations in the ^Gγ- and ^Aγ-globin genes were examined by DNA analysis and rapid diagnosis of HPFH mutations were developed by PCR-based methods. Twenty subjects with five different mutations were identified including three known mutations, - 202 ^Aγ (C>T) (n = 3), - 196 ^Aγ (C>T) (n = 3), and - 158 ^Aγ (C>T) (n = 12), and two novel mutations, - 117 ^Aγ (G>C) (n = 1) and - 530 ^Gγ (A>G) (n = 1). Interaction of the - 117 ^Aγ (G>C) and Hb E (HBB:c.79G>A) resulted in elevation of Hb F to the level of 13.5%. Two plain heterozygous subjects with - 530 ^Gγ (A>G) had marginally elevated Hb F with 1.9% and 3.0%, whereas the proband with homozygous - 530 ^Gγ (A>G) had elevated Hb F of 11.5%. Functional prediction indicated that the - 117 ^Aγ (G>C) and - 530 ^Gγ (A>G) mutations dramatically alter the binding of transcription factors to respective γ-globin gene promotors, especially the CCAAT and GATA-1 transcription factors. Diverse heterogeneity of non-deletional HFPH with both known and new mutations, and complex interactions of them with other forms of thalassemia are encountered in Thai population.

Figure 1

DNA sequencing profiles of heterozygous for − 117 ^Aγ (G>C) (A) and identification of the − 117 ^Aγ (G>C) by allele-specific PCR assay (B). M represents the VC 100 bp plus DNA Ladder (Vivantis Technologies Sdn Bhd). Lanes 1 and 2 are subjects with negative and positive for the mutation, respectively.

Figure 2

DNA sequencing profiles of homozygous − 530 ^Gγ (A>G) (A) and identification of the − 530 ^Gγ (A>G) by allele-specific PCR assay (B). M represents the VC 100 bp plus DNA Ladder. Lanes 1 and 2 are subjects with negative and positive for the mutation, respectively.

Figure 3

Pedigree analysis of a Thai family with the − 530 ^Gγ (A>G) HPFH. The arrow indicates the proband who was homozygous for the − 530 ^Gγ (A>G) whereas his parents and his sister were heterozygotes. The hematological parameters, Hb analysis, globin genotypes as well as β-globin gene haplotypes segregated in the family are presented.