A Point Mutation Creating a 3' Splice Site in C8A Is a Predominant Cause of C8α-γ Deficiency in African Americans

Abstract

C8α-γ deficiency was examined in four unrelated African Americans. Two individuals were compound heterozygotes for a previously reported point mutation in exon 9. mRNA from the remaining six C8A alleles contained a 10 nt insertion between nt 992 and 993 corresponding to the junction between exons 6 and 7. This suggested that C8α-γ deficiency in these individuals was caused by a splicing defect. Genomic sequencing revealed a G→A point mutation in intron 6, upstream of the exon 7 acceptor site. This mutation converts a GG to an AG, generates a consensus 3' splice site that shifts the reading frame, and creates a premature stop codon downstream. To verify that the point mutation caused a splicing defect, we tested wild-type and mutant mRNA substrates, containing 333 nt of the C8α intron 6/exon 7 boundary, in an in vitro splicing assay. This assay generated spliced RNA containing the 10 bp insertion observed in the C8α mRNA of affected patients. In addition, in mutant RNA substrates, the new 3' splice site was preferentially recognized compared with wild-type. Preferential selection of the mutant splice site likely reflects its positioning adjacent to a polypyrimidine tract that is stronger than that adjacent to the wild-type site. In summary, we have identified a G→A mutation in intron 6 of C8A as a predominant cause of C8α-γ deficiency in African Americans. This mutation creates a new and preferred 3' splice site, results in a 10 nt insertion in mRNA, shifts the reading frame, and produces a premature stop codon downstream.

FIGURE 1.

Immunoblots comparing normal and C8 α-γ deficient sera. Western blots of C8 or patient sera were probed using rabbit polyclonal antibodies raised against C8 or C8 α-γ and developed with alkaline phosphatase labelled goat anti-rabbit IgG. Left Panel: non-reducing conditions. The position of the C8α−γ and β subunits is shown in the far-left lane. Right Panel: reducing conditions. The position of the individual C8α and C8γ polypeptide chains is shown in the far-right lane. In contrast to their presence in normal human sera (NHS), neither the C8α-γ subunit, nor the individual α and γ polypeptides was evident in the sera from C8α-γ deficient patients (Pt). Each patient’s serum was examined at least twice. In reducing gels, the bands (*) at ~55 and 25 kDa are consistent with the heavy and light chains of IgG.

FIGURE 2.

C8α-γ-deficient Patients’ C8α cDNA contains a 10 base pair insertion. C8α cDNA, generated from C8α-γ-deficient individuals’ mRNA was sequenced directly. Comparing this sequence to that reported previously (11, 15) revealed a 10 bp insertion between nt 992 and 993. Each patient’s cDNA was sequenced at least twice.

FIGURE 3.

Comparison of genomic sequences from normal (Nl) and C8α-γ deficient (Def) persons. a) DNA was extracted from normal and patient fibroblasts, amplified by PCR, and sequenced. Sequencing revealed a G→A mutation (arrows) 12 bp upstream from the start of exon 7, at nt 993. This mutation creates a new consensus 3′ SS (box) in the intronic sequence in the DNA from deficient individuals that is upstream of the usual 3′ SS (box) in DNA from normal individuals. Amplified genomic DNA from each patient was sequenced at least twice. b) The G→A mutation creates a new consensus 3′ SS upstream of the intron 6/exon 7 boundary in DNA from the normal individual. The vertical arrow indicates the G→A point mutation, which is noted by a lower case “a.” The newly created 3′ SS signal sequence and the one found in normal individuals are designated by the broad vertical bars. A bracket encompasses the 10 nt of intronic sequence that becomes included in exon 7 when the newly created 3′ SS is used. c) The newly created, putative 3′ SS has enhanced polypyrimidine tract strength. The DNA sequence upstream of the 3′ SS of C8A exon 7 selected in individuals who are deficient (upper sequence) or normal (lower sequence) are aligned to facilitate comparison of their polypyrimidine tracts. The point mutation in deficient individuals is in lower case; 3′ SS consensus is in bold; and pyrimidines in the polypyrimidine tract are indicated by black circles below the nucleotide. The DNA sequence upstream of the 3′ SS of C8A exon 7 in normal individuals contains fewer pyrimidines (6, vs 8, respectively) and fewer of these occur as contiguous nucleotides (4 of 6 vs 7 of 8, respectively) than the analogous sequence in the DNA from deficient persons (see the discussion section for the relevance of these changes).

FIGURE 4.

The newly created SS is highly preferred. (A) Schematic of the RNA splicing substrates, which contain the heterologous HIV 116 nt 59 exon (box 1), 169 nt intervening sequence followed by 106 nt from the 39 end of C8A intron 6, and 177 nt from the 59 end of C8A exon 7 (box 2). (B) DNA m.w. standards (Std) and their respective lengths (in bp) are shown in the left lane. Intermediates and products of splicing reactions containing either normal (Nl) or deficient (Def) RNA substrates are shown in the right two lanes, respectively. The vertical white line denotes that the standard and the experimental lanes were separated in the original gel and later assembled to create a composite figure from the corresponding autoradiograph. The diagram at the right depicts spliced products and intermediates [compare with (A)]. Substrates derived from a C8α-γ-deficient patient sequence are consistent with the G→A mutation at the 212 position, creating a new 39 SS. The mutant transcript produced a slightly smaller excised lariat (~396 versus ~406 nt) and a slightly larger spliced product (~308 versus ~298 nt) compared with the normal intron and spliced products. Lariat products migrate with slower mobility than linear RNA of the same size. Splicing reactions were performed once as described in the Materials and Methods.