Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes

Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long‐range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long‐range product spanning exons 11–15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty‐seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long‐range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2. Hum Mutat 31:588–593, 2010. © 2010 Wiley‐Liss, Inc.     

Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3′ deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11–15, due to extensive gene conversion. A newly designed multiplex ligation‐dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9–15, 11–15, 13–15, 14–15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations. Hum Mutat 32:1063–1071, 2011. © 2011 Wiley‐Liss, Inc.     

Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of lynch syndrome patients

Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair‐deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene‐specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3′ end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2‐specific PCR primers and MLPA probes, designed on PSVs, in the 3′ duplicated region is unreliable, and present an RNA‐based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV‐based mutation detection methods. Hum Mutat 31:578–587, 2010. © 2010 Wiley‐Liss, Inc.     

Reclassification of a frequent African‐origin variant from PMS2 to the pseudogene PMS2CL

Genomic analysis has become a mainstay in the investigation of cancer patients, especially for those suspected of harboring a heritable cancer predisposition syndrome. With ubiquitous short‐read next‐generation sequencing (NGS) technologies, these analyses can be complicated by the inappropriate alignment of variants to homologous genomic regions or pseudogenes. Using distinct primer sets specific to the gene and pseudogene, a nonspecific primer set, and a highly gene‐specific long‐range polymerase chain reaction primer set, we have shown that in at least a subset of patients, the common African PMS2 variant NM_000535.5:c.2182_2184delACTinsG, classified as pathogenic in ClinVar, has been incorrectly assigned to PMS2 from its well‐documented pseudogene, PMS2CL. This result is not only important for patients but also highlights a weakness in short‐read NGS technologies and the racial inequity in genomic analysis.     

The changing landscape of Lynch syndrome due to PMS2 mutations

DNA repair pathways are essential for cellular survival as our DNA is constantly under assault from both exogenous and endogenous DNA damaging agents. Five major mammalian DNA repair pathways exist within a cell to maintain genomic integrity. Of these, the DNA mismatch repair (MMR) pathway is highly conserved among species and is well documented in bacteria. In humans, the importance of MMR is underscored by the discovery that a single mutation in any 1 of 4 genes within the MMR pathway (MLH1, MSH2, MSH6 and PMS2) results in Lynch syndrome (LS). LS is a autosomal dominant condition that predisposes individuals to a higher incidence of many malignancies including colorectal, endometrial, ovarian, and gastric cancers. In this review, we discuss the role of PMS2 in the MMR pathway, the evolving testing criteria used to identify variants in the PMS2 gene, the LS phenotype as well as the autosomal recessive condition called constitutional mismatch repair deficiency syndrome, and current methods used to elucidate the clinical impact of PMS2 mutations.     

Inactivation of DNA Mismatch Repair by Variants of Uncertain Significance in the PMS2 Gene

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell‐free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS.     

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome

Monoallelic PMS2 germline mutations cause 5%–15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA‐ and RNA‐based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety‐one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.     

Identification of Lynch syndrome mutations in the MLH1–PMS2 interface that disturb dimerization and mismatch repair

Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome that predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C‐terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1–PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1–CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased coexpression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations. Hum Mutat 31:975–982, 2010. © 2010 Wiley‐Liss, Inc.     

PMS2 expression decrease causes severe problems in mismatch repair

PMS2 is one of the four susceptibility genes in Lynch syndrome (LS), the most common cancer syndrome in the world. Inherited mutations in DNA mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, account for approximately 90% of LS, while a relatively small number of LS families segregate a PMS2 mutation. This and the low cancer penetrance in PMS2 families suggest that PMS2 is only a moderate or low‐risk susceptibility gene. We have previously shown that even a partial expression decrease in MLH1, MSH2, or MSH6 suggests that heterozygous LS mutation carriers have MMR malfunction in constitutive tissues. Whether and how PMS2 expression decrease affects the repair capability is not known. Here, we show that PMS2 knockdown cells retaining 19%, 33%, or 53% of PMS2 expression all have significantly reduced MMR efficiency. Surprisingly, the cells retaining expression levels comparable to PMS2 mutation carriers indicate the lowest repair efficiency.     

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study‐based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long‐range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation‐dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2‐suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first‐line method for searching germline mutations in PMS2.     

Recurrent and founder mutations in the PMS2 gene

Germline mutations in PMS2 are associated with Lynch syndrome (LS), the most common known cause of hereditary colorectal cancer. Mutation detection in PMS2 has been difficult due to the presence of several pseudogenes, but a custom‐designed long‐range PCR strategy now allows adequate mutation detection. Many mutations are unique. However, some mutations are observed repeatedly across individuals not known to be related due to the mutation being either recurrent, arising multiple times de novo at hot spots for mutations, or of founder origin, having occurred once in an ancestor. Previously, we observed 36 distinct mutations in a sample of 61 independently ascertained Caucasian probands of mixed European background with PMS2 mutations. Eleven of these mutations were detected in more than one individual not known to be related and of these, six were detected more than twice. These six mutations accounted for 31 (51%) ostensibly unrelated probands. Here, we performed genotyping and haplotype analysis in four mutations observed in multiple probands and found two (c.137G>T and exon 10 deletion) to be founder mutations and one (c.903G>T) a probable founder. One (c.1A>G) could not be evaluated for founder mutation status. We discuss possible explanations for the frequent occurrence of founder mutations in PMS2.     

Insertion of an SVA element, a nonautonomous retrotransposon, in PMS2 intron 7 as a novel cause of Lynch syndrome

Heterozygous germline mutations in the mismatch repair gene PMS2 predispose carriers for Lynch syndrome, an autosomal dominant predisposition to cancer. Here, we present a LINE-1-mediated retrotranspositional insertion in PMS2 as a novel mutation type for Lynch syndrome. This insertion, detected with Southern blot analysis in the genomic DNA of the patient, is characterized as a 2.2 kb long 5' truncated SVA_F element. The insertion is not detectable by current diagnostic testing limited to MLPA and direct Sanger sequencing on genomic DNA. The molecular nature of this insertion could only be resolved in RNA from cultured lymphocytes in which nonsense-mediated RNA decay was inhibited. Our report illustrates the technical problems encountered in the detection of this mutation type. Especially large heterozygous insertions will remain unnoticed because of preferential amplification of the smaller wild-type allele in genomic DNA, and are probably underreported in the mutation spectra of autosomal dominant disorders.     

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

Lynch syndrome (LS) is an autosomal dominant inherited disorder that is associated with an increased predisposition to certain cancers caused by loss‐of‐function mutations in one of four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2). The diagnosis of LS is often challenged by the identification of missense mutations where the functional effects are not known. These are termed variants of uncertain significance (VUSs) and account for 20%–30% of noncoding and missense mutations. VUSs cause ambiguity during clinical diagnosis and hinder implementation of appropriate medical management. In the current study, we focus on the functional and biological consequences of two nonsynonymous VUSs in PMS2. These variants, c.620G>A and c.123_131delGTTAGTAGA, result in the alteration of glycine 207 to glutamate (p.Gly207Glu) and the deletion of amino acid residues 42–44 (p.Leu42_Glu44del), respectively. While the PMS2 p.Gly207Glu variant retains in vitro MMR and ATPase activities, PMS2 p.Leu42_Glu44del appears to lack such capabilities. Structural and biophysical characterization using circular dichroism, small‐angle X‐ray scattering, and X‐ray crystallography of the N‐terminal domain of the PMS2 variants indicate that the p.Gly207Glu variant is properly folded similar to the wild‐type enzyme, whereas p.Leu42_Glu44del is disordered and prone to aggregation.     

Early age of onset and broad cancer spectrum persist in MSH6- and PMS2-associated Lynch syndrome

PurposeThis study aimed to characterize MSH6/PMS2-associated mismatch repair–deficient (MMR-D)/microsatellite instability-high (MSI-H) tumors, given revised guidelines suggesting more modest phenotypes.MethodsPatients who consented to Institutional Review Board–approved protocols of tumor/germline sequencing or Lynch syndrome registry at a single institution from February 2005 to January 2021 with germline, heterozygous MSH6/PMS2 pathogenic/likely pathogenic variants were identified. Clinical data were abstracted and correlated with MMR/microsatellite instability status using nonparametric tests.ResultsWe identified 243 patients (133 sequencing, 110 registry) with germline MSH6/PMS2 pathogenic/likely pathogenic variants; 186 (77%) had >1 cancer. Of 261 pooled tumors, colorectal cancer (CRC) and endometrial cancer (EC) comprised 55% and 43% of cancers in MSH6 and PMS2, respectively; 192 tumors underwent molecular assessments and 122 (64%) were MMR-D/MSI-H (77 in MSH6, 45 in PMS2). MMR-D/MSI-H cancers included CRC (n = 56), EC (n = 35), small bowel cancer (n = 6), ovarian cancer (n = 6), urothelial cancer (n = 5), pancreas/biliary cancer (n = 4), gastric/esophageal cancer (n = 3), nonmelanoma skin tumors (n = 3), prostate cancer (n = 2), breast cancer (n = 1), and central nervous system/brain cancer (n = 1). Among MMR-D/MSI-H CRC and EC, median age of diagnosis was 51.5 (range = 27-80) and 55 (range = 39-74) years, respectively; 9 of 56 (16%) MMR-D/MSI-H CRCs were diagnosed at age <35 years.ConclusionMSH6/PMS2 heterozygotes remain at risk for a broad spectrum of cancers, with 16% of MMR-D/MSI-H CRCs presenting before upper threshold of initiation of colonoscopy per guidelines.     

Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives.     

Functional PMS2 hybrid alleles containing a pseudogene‐specific missense variant trace back to a single ancient intrachromosomal recombination event

Sequence exchange between PMS2 and its pseudogene PMS2CL, embedded in an inverted duplication on chromosome 7p22, has been reported to be an ongoing process that leads to functional PMS2 hybrid alleles containing PMS2‐ and PMS2CL‐specific sequence variants at the 5′‐and the 3′‐end, respectively. The frequency of PMS2 hybrid alleles, their biological significance, and the mechanisms underlying their formation are largely unknown. Here we show that overall hybrid alleles account for one‐third of 384 PMS2 alleles analyzed in individuals of different ethnic backgrounds. Depending on the population, 14–60% of hybrid alleles carry PMS2CL‐specific sequences in exons 13–15, the remainder only in exon 15. We show that exons 13–15 hybrid alleles, named H1 hybrid alleles, constitute different haplotypes but trace back to a single ancient intrachromosomal recombination event with crossover. Taking advantage of an ancestral sequence variant specific for all H1 alleles we developed a simple gDNA‐based polymerase chain reaction (PCR) assay that can be used to identify H1‐allele carriers with high sensitivity and specificity (100 and 99%, respectively). Because H1 hybrid alleles harbor missense variant p.N775S of so far unknown functional significance, we assessed the H1‐carrier frequency in 164 colorectal cancer patients. So far, we found no indication that the variant plays a major role with regard to cancer susceptibility. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Extensive gene conversion at the PMS2 DNA mismatch repair locus

Mutations of the PMS2 DNA repair gene predispose to a characteristic range of malignancies, with either childhood onset (when both alleles are mutated) or a partially penetrant adult onset (if heterozygous). These mutations have been difficult to detect, due to interference from a family of pseudogenes located on chromosome 7. One of these, the PMS2CL pseudogene, lies within a 100-kb inverted duplication (inv dup), 700 kb centromeric to PMS2 itself on 7p22. Here, we show that the reference genomic sequences cannot be relied upon to distinguish PMS2 from PMS2CL, because of sequence transfer between the two loci. The 7p22 inv dup occurred prior to the divergence of modern ape species (15 million years ago [Mya]), but has undergone extensive sequence homogenization. This process appears to be ongoing, since there is considerable allelic diversity within the duplicated region, much of it derived from sequence exchange between PMS2 and PMS2CL. This sequence diversity can result in both false-positive and false-negative mutation analysis at this locus. Great caution is still needed in the design and interpretation of PMS2 mutation screens.     

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

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.