PMC5938003_01
Pathological view of the colorectal cancers, pedigree, and schematic diagram of a novel MLH1 mutation.. a MLH1 immunostaining of the rectal cancer specimen demonstrated normal expression within the inflammatory cells and interstitial cells, but null expression in the cancer cells, which were found partially floating in a mucinous lake (x200). b Pathology of the ascending colon cancer showed moderate to poor differentiation within the mucosa (H&E, x100). c The patient's parents divorced when he was young, and his father's subsequent health condition was unknown. No maternal relatives were cancer sufferers. P proband. d A novel heterozygous mutation was detected by whole-exome sequencing in the MLH1 intron 6 splice site [NM_000249.3:c.545+2T>C (IVS6+2T>C); localization was Chr3:37050398] and confirmed by the Sanger method
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Fig1
His maternal relatives had no cancer history (Fig. 1c). A novel heterozygous mutation was detected in the splice donor site of MLH1 intron 6 [NM_000249.3:c.545+2T>C (IVS6+2T>C): the localization was Chr3:37050398(GRCh37)] (Fig. 1d).
PMC5938003_02
Pathological view of the colorectal cancers, pedigree, and schematic diagram of a novel MLH1 mutation.. a MLH1 immunostaining of the rectal cancer specimen demonstrated normal expression within the inflammatory cells and interstitial cells, but null expression in the cancer cells, which were found partially floating in a mucinous lake (x200). b Pathology of the ascending colon cancer showed moderate to poor differentiation within the mucosa (H&E, x100). c The patient's parents divorced when he was young, and his father's subsequent health condition was unknown. No maternal relatives were cancer sufferers. P proband. d A novel heterozygous mutation was detected by whole-exome sequencing in the MLH1 intron 6 splice site [NM_000249.3:c.545+2T>C (IVS6+2T>C); localization was Chr3:37050398] and confirmed by the Sanger method
41439_2018_2_Fig1_HTML.jpg
PMC5938003_02_41439_2018_2_Fig1_HTML.jpg
Fig1
His maternal relatives had no cancer history (Fig. 1c). A novel heterozygous mutation was detected in the splice donor site of MLH1 intron 6 [NM_000249.3:c.545+2T>C (IVS6+2T>C): the localization was Chr3:37050398(GRCh37)] (Fig. 1d).
Results of RT-PCR analysis.. a The diagram shows alternatively spliced exons of MLH1. The intron 6 splicing variant was validated by designing primers b and c within intron 6 (Intron 6: 2915 bp). A white box represents an exon, and a black box shows an intron. b Normal mRNA was also detected (PCR product by primer a and d, 157 bp), so this mutation is heterozygous. c Abnormal mRNA including intron 6 was detected (PCR product by primer a and b, 373 bp). d Abnormal mRNA including intron 6 (primer c and d, 359 bp) and introns 6 and 7 (PCR product by primer c and d, 507 bp) were detected
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Fig2
We designed a primer on intron 6 and performed reverse transcription-polymerase chain reaction (RT-PCR) to confirm the morbidity significance of this variant (Fig. 2a). This RT-PCR produced a normal sized product and abnormal variants that bound intron 6 and intron 7 (Figs. 2b-d).