Using the in vitro MMR assay to recognize reduced MMR gene expression levels

Assessing how reduced expression levels of the mismatch repair genes MLH1, MSH2 and MSH6 affect repair efficiency

Minttu Kansikas, Mariann Kasela, Jukka Kantelinen and Minna Nyström

Lynch syndrome (LS), the most common familial colon cancer, is associated with mismatch repair (MMR) malfunction. As mutation carriers inherit one normal and one defected MMR gene allele, cancer risk can be considered as limited amount of normal MMR gene product. How reductions in different MMR gene expressions affect MMR capability is, however, not known. The in vitro MMR assay is a method for the pathogenicity assessment of MMR gene variants causing functional or expressional defects and thus also suitable to evaluate the effects of reduced expression of normal mRNA. Here, the assay was applied to quantify repair efficiencies of human cells retaining varying expression levels (25%/50%/75%) of the main LS susceptibility genes MLH1,MSH2 or MSH6. Compared to the shRNA knockdown control, already a 50% reduction in mRNA levels could be detected as decreased MMR function although without statistical significance in MLH1. In MSH2 and MLH1, total loss of MMR was achieved with 25% expression, while in MSH6 and MSH2 the repair capability decreased significantly already with 75% expression. Our results provide a preliminary indication of relative expressions required for wild-type function and suggest that the in vitro MMR assay could be used to recognize expression levels indicative of LS.

Human Mutation, 2014.

A Novel Pathogenic 132 kb Deletion in the APC Promoter 1B Recognized as a Rare Cause for Mutation-negative FAP

Promoter-Specific Alterations of APC are a Rare Cause for Mutation-Negative Familial Adenomatous Polyposis

Walter Pavicic, Taina T. Nieminen, Annette Gylling, Juha-Pekka Pursiheimo, Asta Laiho, Attila Gyenesel, Heikku J. Järvinen and Päivi Peltomäki

In familial adenomatous polyposis (FAP), 20% of classical and 70% of attenuated/atypical (AFAP) cases remain mutation-negative after routine testing; yet, allelic expression imbalance may suggest an APC alteration. Our aim was to determine the proportion of families attributable to genetic or epigenetic changes in the APC promoter region. We studied 51 unrelated families/cases (26 with classical FAP and 25 with AFAP) with no point mutations in the exons and exon/intron borders and no rearrangements by multiplex ligation-dependent probe amplification (MLPA, P043-B1). Promoter-specific events of APC were addressed by targeted re-sequencing, MLPA (P043-C1), methylation-specific MLPA, and Sanger sequencing of promoter regions. A novel 132-kb deletion encompassing the APC promoter 1B and upstream sequence occurred in a classical FAP family with allele-specific APC expression. No promoter-specific point mutations or hypermethylation were present in any family. In conclusion, promoter-specific alterations are a rare cause for mutation-negative FAP (1/51, 2%). The frequency and clinical correlations of promoter 1B deletions are poorly defined. This investigation provides frequencies of 1/26 (4%) for classical FAP, 0/25 (0%) for AFAP, and 1/7 (14%) for families with allele-specific expression of APC. Clinically, promoter 1B deletions may associate with classical FAP without extracolonic manifestations.

Genes, Chromosomes and Cancer, 2014.


New publication in Gastroenterology

Germline mutation of RPS20, a ribosomal protein gene, predisposes to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency

Taina T. Nieminen, Marie-Françoise O’Donohue, Yunpeng Wu, Hannes Lohi, Stephen W. Scherer, Andrew D. Paterson, Pekka Ellonen, Wael M. Abdel-Rahman, Satu Valo,Jukka-Pekka Mecklin, Heikki J. Järvinen, Pierre-Emmanuel Gleizes, and Päivi Peltomäki

The predisposing genes for FCCX, hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects, are largely unknown. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of a four-generation FCCX family resulted in the identification of a truncating germline mutation in RPS20, a novel colon cancer predisposition gene encoding a component (S20) of the small ribosomal subunit. The mutation was associated with a defect in pre-rRNA maturation. Our findings show that a mutant ribosomal protein gene can underlie hereditary predisposition to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted.

Gastroenterology, 2014.