PMC5011803_01
Patient's pedigree. The arrow indicates the early-onset ovarian cancer proband. Filled symbols indicate subjects affected by cancer; square = male; circle = female. The numbers after cancer sites indicate the age at diagnosis. The age of death is reported if known. Abbreviations: D = deceased
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Fig1
The heterozygous state for the mutations (c.1706A > T) and (c.1711G > T) at exon 11 of MSH2 gene. a MSH2 genomic sequence analysis (fragment including exon 11) showing the two mutations identified in our index case. The arrow indicates the position of two mutations. b MSH2 cDNA sequence analysis (fragment including exons 9-15) showing the two mutations at mRNA level. The arrow indicates the lowering peak of mutant allele
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Fig2
MSH2 mRNA transcript including exon 9-15 revealed the presence of only the wild type allele (Fig. 2). Moreover, electrophoresis of this transcript showed an abnormal fragment, suggestive of an aberrant splicing event.
Characterization of aberrant splicing fragment. a 8 % polyacrilamide gel electrophoresis of MSH2 cDNA RT-PCR analysis of fragment in a normal control (1) and in the index case (2). SM size marker XIV Roche. The arrow indicates the abnormal band lower than the wild type band (1172 bp) of about 1000 bp. b MSH2 cDNA sequence analysis of abnormal band showing the skipping of exon 11. The arrow indicates the frameshift (between 11 and 12 exons) due to contamination with wild type allele
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Fig3
Direct sequencing of this aberrantly spliced transcript revealed the loss in frame of the entire exon 11; this transcript fragment resulted in a new exon connection between 10 and 12 exons (Fig. 3).
MSH2, MSH6 and MLH1 immunohistochemistry (IHC) results in the ovary tumor section of patient carrier of c.1706A > T and c.1711G > T variants in MSH2 exon 11. a Absence of MSH2 protein in the tumor cells; b weak presence of MSH6 protein in the tumor cells; c normal IHC for MLH1 protein in the tumor cells. Filled arrow heads point to IHC- tumor cells, open arrows heads point to IHC+ tumor cells. Blue nuclear staining of lymphocytes indicates positive internal control
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Fig4