A novel frameshift mutation (+G) at codons 15/16 in a beta0 thalassaemia gene results in a significant reduction of beta globin mRNA values

Abstract

Aims: To identify a novel beta globin gene mutation found in a Chinese family, and also to assess its functional consequences.

Methods: Haematological analysis was performed on all family members. The 23 common mutations of beta thalassaemia found in Chinese populations were detected by means of a reverse dot blot method. Direct DNA sequencing of polymerase chain reaction (PCR) amplified complete beta globin gene was carried out to identify the novel mutation. A real time, one step reverse transcription PCR assay was used to measure beta globin mRNA in the reticulocytes of heterozygous patients.

Results: A novel frameshift mutation-an insertion of G between codons 15 and 16 in a homonucleotide run of four guanines-was determined, which generates a new premature chain terminator at the 22nd codon. Relative quantitative analysis of the beta globin mRNA in heterozygous subjects demonstrated a 39.83% reduction compared normal controls.

Conclusions: The significantly lower amounts of beta globin mRNA found in mutation carriers is probably caused by the rapid nonsense mediated degradation of the mutant mRNA. These data, combined with haematological analysis, suggest that this novel mutation of CDs 15/16 (+G) results in a beta(0) thalassaemia phenotype.

Figure 1

(A) Mutant sequence of the sense strand. The inserted base (G) and the generated premature termination codon of TGA are marked in red and underlined. This one base insertion mutation results in numerous ambiguities (peaks overlapping, indicated by N) immediately after it occurs in the sequencing chromatogram of a heterozygote. (B) Mutant sequence of the antisense strand.

Figure 2

Reverse dot blot assay for detecting the novel CDs15/16(+G) mutation. The normal probes are at the top and the mutant probes are at the bottom of each filter strip. The mutations represented by each probe are listed above, and the subjects tested are shown on the right.

Figure 3

(A) Comparison of the threshold cycle value (C_T) of (a) two heterozygous subjects with the novel CDs15/16(+G) frameshift mutation, (b) five patients with β⁺ thalassaemia trait, and (c) eight normal individuals. The proband with β thalassemia major (−28/CDs15/16) was excluded from the study of β globin mRNA values for heterozygous patients. All statistical analyses were performed using the SPSS10.0 software package for mean, standard deviation, and ANOVA with the Student-Newman-Keuls test. Significant differences were found (p < 0.05) between the three groups by pairwise multiple comparison and as a whole. (B) Relative quantitative analysis of β globin mRNA. (a) Normal individuals, (b) patients with β⁺ thalassemia trait, and (c) heterozygotes with the CDs15/16 mutation show 100%, 73.56%, and 60.17% overall β globin mRNA values, respectively.