Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2: Use of potential alternative start sites and implications for predicting variant pathogenicity

Abstract

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However, few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site.

Keywords: cancer syndrome genes; in vitro assay; unclassified variant.

Figure 1. Alternative translation initiation codons in MLH1 and BRCA2

A) Translation from all but one of these potential start codons (c.103) is predicted to result in an out-of-frame protein product. Two of the ATP-binding and hydrolysis domain motifs are shown (bases 91–129 and 187–204). Two further ATP-binding and hydrolysis motifs lie at bases 289–321 and 436–441. B) Alternative translation start sites following the aberrant splicing event as a result of BRCA2:c.67+3A>G are highlighted at position c.323 and c.367. The N-terminal transactivation domain that spans bases 67–315 is lost as a result of translation initiation at c.323 and c.367. In the event that the ATG at c.323 is recognized as the translation initiation signal, an out of frame protein would be synthesized terminating 12 amino acids after initiation. Initiation at c.367 would result in an in-frame protein product, lacking the N-terminal transactivation domain.

Figure 2. BRCA2:c.67+3A>G splicing products detected by RT-PCR

M – 100bp DNA marker (New England Biolabs); Lane 1 variant carrier; Lanes 2–5, normal unaffected female controls; lane 6, no-template control. Controls revealed only expected product sizes of 654bp for full length transcript, and 406bp for common exon 3 deletion isoform previously reported in healthy controls. The variant carrier in Lane 1 shows an aberrant product of 548bp, that was confirmed by sequencing to be an exon 2 deletion. The loss of exon 2 includes the position of the ATG start site, complicating prediction of the molecular consequences of this aberration.

Figure 3. Relative GFP fluorescent level from alternative downstream translation initiation codons in MLH1 and BRCA2.

Schematic representation of each construct is shown. The GFP sequence is in-frame with the most 3′ potential start site. Error bars are based on the standard error between repeat experiments. A) Relative GFP fluorescent level of predicted potential alternate start sites with and without the presence of the variant MLH1c.1A>G(p.Met1Val). The ATG at position c.103 is in-frame with the ATG at c.1, and produced higher GFP protein level than the two initiation sites not in-frame with position c.1 (c.89 and c.122, which are in-frame with each other). B) Effect of exon 2 loss from BRCA2 transcripts on the initiation of translation. An out-of-frame ATG codon at position c.323 in exon 4 is preferred to an in-frame alternative at c.370. Each experiment was conducted in triplicate, and then repeated twice.

Figure 4. In vitro cell-free mismatch repair assay confirms loss of function proteins translated from MLH1c.1A>G p.(Met1?) alleles

A) Alleles generated by PCR including the full-length variant allele and an allele carrying a deletion of 34 amino acids as anticipated from MLH1-GFP fusion construct assays. B) Sequence confirmed alleles. C) ³⁵S-Methionine labelled proteins derived for the cell-free assay. The p.Met1Val allele was unable to produce a detectable protein. D) A protein lacking amino acids 1–34 reduces MMR activity to a level equivalent to known pathogenic missense mutation, MLH1:p.Gly67Arg.