Journal: Molecular carcinogenesis

Article Title: 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

doi: 10.1002/mc.22116

Figure Lengend Snippet: 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.

Article Snippet: Two downstream ATG sites in exon 4 at c.323 and c.367 were selected for functional analysis to determine their potential use as alternate translation initiation sites. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 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.

Techniques: Binding Assay, Synthesized

Journal: Molecular carcinogenesis

Article Title: 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

doi: 10.1002/mc.22116

Figure Lengend Snippet: 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.

Article Snippet: Two downstream ATG sites in exon 4 at c.323 and c.367 were selected for functional analysis to determine their potential use as alternate translation initiation sites. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 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.

Techniques: Construct, Sequencing, Variant Assay, Produced