RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference

Abstract

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects.

Agenesis of the corpus callosum and gray matter heterotopia in three patients with constitutional mismatch repair deficiency syndrome

“…To confirm the mutation identified in patient 1 at transcript level, a PMS2-specific RT-PCR product containing exons 10-15 (PCR product B in Etzler et al 14 ) was generated from RNA isolated from a cycloheximide treated short-term lymphocytes culture and sequenced with three-internal primers. Only lowquality RNA was available of patient 2.…”

“…Therefore, only a 366-bp RT-PCR fragment from exons 2-5 (fwd primer: 5 0 -ATCCAGCGGCCAGCTAATG-3 0 ; reverse primer: 5 0 -CTTGATTGCCAGCACATGG-3 0 ) was amplified from RNA isolated from the puromycin-treated short-term lymphocytes culture of the patient to exclude a possible splicing effect of the identified MLH1 missense variant in exon 3. The previously described RNA-based mutation analysis protocol 14 was used as primary assay to analyze MLH1 and PMS2 in patient 3.…”

Constitutional mismatch repair-deficiency syndrome

“…[12][13][14][15] However, signs reminiscent of neurofibromatosis type 1 (NF1), in particular café-aulait macules (CALMs), are much more common and were observed in the majority of the reported cases (63/92). There are only 2 patients explicitly reported to lack CALMs or other signs of NF1.…”

“…The identification of a truncating NF1 mutation in the blood of one patient 16 and data supporting the notion that the NF1 gene is a mutational target of MMR deficiency 17 are in line with this assumption. However, extensive mutation analysis in other CMMR-D patients has not confirmed this theory (see 8,12,18 and papers cited therein).…”

Clinical utility gene card for: Lynch syndrome (MLH1, MSH2, MSH6, PMS2, EPCAM) - update 2012

“…3. Germline analysis 7,8 9 Germline MLH1 promoter methylation characterisation by MSP, bisulphite pyrosequencing, or MLPA (useful for diagnostic purpose, not for predictive testing).…”