Key role of phosphorylation sites in ATPase domain and Linker region of MLH1 for DNA binding and functionality of MutLα.

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.

A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation.

DNA mismatch repair (MMR) is essential for correction of DNA replication errors. Germline mutations of the human MMR gene MLH1 are the major cause of Lynch syndrome, a heritable cancer predisposition. In the MLH1 protein, a non-conserved, intrinsically disordered region connects two conserved, catalytically active structured domains of MLH1. This region has as yet been regarded as a flexible spacer, and missense alterations in this region have been considered non-pathogenic. However, we have identified and investigated a small motif (ConMot) in this linker which is conserved in eukaryotes. Deletion of the ConMot or scrambling of the motif abolished mismatch repair activity. A mutation from a cancer family within the motif (p.Arg385Pro) also inactivated MMR, suggesting that ConMot alterations can be causative for Lynch syndrome. Intriguingly, the mismatch repair defect of the ConMot variants could be restored by addition of a ConMot peptide containing the deleted sequence. This is the first instance of a DNA mismatch repair defect conferred by a mutation that can be overcome by addition of a small molecule. Based on the experimental data and AlphaFold2 predictions, we suggest that the ConMot may bind close to the C-terminal MLH1-PMS2 endonuclease and modulate its activation during the MMR process.

Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer.

Lynch syndrome is a heritable condition caused by a heterozygous germline inactivating mutation of the DNA mismatch repair (MMR) genes, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants, for which the clinical significance is unclear in many cases. We have identified three MLH1 missense alterations (p.(Glu736Lys), p.(Pro640Thr) and p.(Leu73Pro)) in six individuals from large Tunisian families. For none of these alterations, a classification of pathogenicity was available, consequently diagnosis, predictive testing and targeted surveillance in affected families was impossible. We therefore performed functional laboratory testing using a system testing stability as well as catalytic activity that includes clinically validated reference variants. Both p.(Leu73Pro) and p.(Pro640Thr) were found to be non-functional due to severe defects in protein stability and catalytic activity. In contrast, p.(Glu736Lys) was comparable to the wildtype protein and therefore considered a neutral substitution. Analysis of residue conservation and of the structural roles of the substituted residues corroborated these findings. In conjunction with the available clinical data, two variants fulfil classification criteria for class 4 "likely pathogenic". The findings of this work clarify the mechanism of pathogenicity of two unclear MLH1 variants and enables predictive testing and targeted surveillance in members of carrier families worldwide.

High Expression of Casein Kinase 2 Alpha Is Responsible for Enhanced Phosphorylation of DNA Mismatch Repair Protein MLH1 and Increased Tumor Mutation Rates in Colorectal Cancer.

DNA mismatch repair (MMR) deficiency plays an essential role in the development of colorectal cancer (CRC). We recently demonstrated in vitro that the serine/threonine casein kinase 2 alpha (CK2α) causes phosphorylation of the MMR protein MLH1 at position serine 477, which significantly inhibits the MMR. In the present study, CK2α-dependent MLH1 phosphorylation was analyzed in vivo. Using a cohort of 165 patients, we identified 88 CRCs showing significantly increased nuclear/cytoplasmic CK2α expression, 28 tumors with high nuclear CK2α expression and 49 cases showing a general low CK2α expression. Patients with high nuclear/cytoplasmic CK2α expression demonstrated significantly reduced 5-year survival outcome. By immunoprecipitation and Western blot analysis, we showed that high nuclear/cytoplasmic CK2α expression significantly correlates with increased MLH1 phosphorylation and enriched somatic tumor mutation rates. The CK2α mRNA levels tended to be enhanced in high nuclear/cytoplasmic and high nuclear CK2α-expressing tumors. Furthermore, we identified various SNPs in the promotor region of CK2α, which might cause differential CK2α expression. In summary, we demonstrated that high nuclear/cytoplasmic CK2α expression in CRCs correlates with enhanced MLH1 phosphorylation in vivo and seems to be causative for increased mutation rates, presumably induced by reduced MMR. These observations could provide important new therapeutic targets.

SPTAN1 Expression Predicts Treatment and Survival Outcomes in Colorectal Cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality. In a cohort of 189 patients with CRC, we recently showed that expression of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1) was lower in advanced metastatic tumours. The aim of the present study was to clarify the association of intratumoural SPTAN1 expression levels with treatment and survival outcomes in patients with CRC. The analysis was based on histologic assessment of SPTAN1 protein levels in our own CRC cohort, and transcriptome data of 573 CRC cases from The Cancer Genome Atlas (TCGA). We first establish that high intratumoural levels of SPTAN1 protein and mRNA associate with favourable survival outcomes in patients with CRC. Next, a response prediction signature applied to the TCGA data reveals a possible link between high SPTAN1 transcript levels and improved patient responses to FOLFOX chemotherapy. Complementary in vitro experiments confirm that SPTAN1 knockdown strains of the colon cancer cell lines HT-29, HCT116 mlh1-2 and Caco-2 are less responsive to FOLFOX chemotherapy compared with SPTAN1-proficient control strains. Taken together, we identify SPTAN1 as a novel prognostic biomarker in CRC and show that SPTAN1 expression levels may predict patient responses to chemotherapy. These investigations illustrate how an affordable, histology-based diagnostic test could directly impact therapeutic decision-making at the bedside.

Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations.

Pathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.

Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region.

Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) mediate proliferation, metabolism, differentiation and anti-apoptotic signals. AKT isoforms are activated downstream of PI3-kinase and also by PI3-kinase independent mechanisms. Mutations in the lipid phosphatase PTEN and PI3-kinase that increase PIP3 levels increase AKT signaling in a large proportion of human cancers. AKT and other AGC kinases possess a regulatory mechanism that relies on a conserved hydrophobic motif (HM) C-terminal to the catalytic core. In AKT, the HM is contiguous to the serine 473 and two other newly discovered (serine 477 and tyrosine 479) regulatory phosphorylation sites. In AKT genes, this regulatory HM region is encoded in the final exon. We identified a splice variant of AKT2 (AKT2-13a), which contains an alternative final exon and lacks the HM regulatory site. We validated the presence of mRNA for this AKT2-13a splice variant in different tissues, and the presence of AKT2-13a protein in extracts from HEK293 cells. When overexpressed in HEK293 cells, AKT2-13a is phosphorylated at the activation loop and at the zipper/turn motif phosphorylation sites but has reduced specific activity. Analysis of the human transcriptome corresponding to other AGC kinases revealed that all three AKT isoforms express alternative transcripts lacking the HM regulatory motif, which was not the case for SGK1-3, S6K1-2, and classical, novel and atypical PKC isoforms. The transcripts of splice variants of Akt1-3 excluding the HM regulatory region could lead to expression of deregulated forms of AKT.

Expression and secretion of the pro­inflammatory cytokine IL­8 is increased in colorectal cancer cells following the knockdown of non­erythroid spectrin αII.

Non­erythroid spectrin αII (SPTAN1) expression is decreased in ~40% of cases of MLH1­deficient colorectal cancer (CRC). SPTAN1 knockdown reduces cell viability, cellular mobility and cell­cell contact formation, indicating that the SPTAN1 plays an important role in tumour growth, attachment and in regulating the tumour microenvironment. Changes in the tumour microenvironment can affect the immune response. Therefore, in the present study, proteome arrays were used to analyse the expression of 119 different chemokines and soluble receptors in CRC cell lines in which mutL homologue 1 (MLH1) or SPTAN1 were knocked down. The levels of interleukin (IL)­8 were significantly increased in the cells in which SPTAN1 was knocked down, both at the mRNA and protein level. ELISA demonstrated that the cells in which SPTAN1 was knocked down secreted increased quantities of IL­8, and chemotaxis assays revealed the enhanced trafficking of neutrophils, which was induced by media containing higher levels of IL­8. The IL­8 receptors, CRCX1 and CRCX2, were expressed in all the cell lines examined; however, their expression was not directly associated with IL­8 expression. The results of the present study thus demonstrated that CRC cells in which SPTAN1 was knocked down secreted significantly higher levels of IL­8, which in­turn increased the migration of neutrophilic granulocytes. As MLH1­deficient CRC exhibits an increased infiltration of cytotoxic T­cells and is associated with a decreased SPTAN1 expression, it can thus be hypothesized that CRC with a low SPTAN1 expression may release increased quantities of IL­8, resulting in increased immune cell infiltration.

Validation of an in Vitro Mismatch Repair Assay Used in the Functional Characterization of Mismatch Repair Variants.

A significant proportion of DNA-mismatch repair (MMR) variants are classified as of unknown significance, precluding diagnosis. The in vitro MMR assay is used to assess their MMR capability, likely the most important function of an MMR protein. However, the robustness of the assay, crucial for its use in the clinical setting, has been rarely evaluated. The aim of the present work was to validate an in vitro MMR assay approach to the functional characterization of MMR variants, as a first step to meeting quality standards of diagnostic laboratories. The MMR assay was optimized by testing a variety of reagents and experimental conditions. Reference materials and standard operating procedures were established. To determine the intra- and interexperimental variability of the assay and its reproducibility among centers, independent transfections of six previously characterized MLH1 variants were performed in two independent laboratories. Reagents and conditions optimal for performing the in vitro MMR assay were determined. The validated assay demonstrated no significant intra- or interexperimental variability and good reproducibility between centers. We set up a robust in vitro MMR assay that can provide relevant in vitro functional evidence for MMR variant pathogenicity assessment, eventually improving the molecular diagnosis of hereditary cancer syndromes associated with MMR deficiency.

Downregulation of SPTAN1 is related to MLH1 deficiency and metastasis in colorectal cancer.

INTRODUCTION: Colorectal cancers (CRCs) deficient in the DNA mismatch repair protein MutL homolog 1 (MLH1) display distinct clinicopathological features and require a different therapeutic approach compared to CRCs with MLH1 proficiency. However, the molecular basis of this fundamental difference remains elusive. Here, we report that MLH1-deficient CRCs exhibit reduced levels of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1), and that tumor progression and metastasis of CRCs correlate with SPTAN1 levels. METHODS AND RESULTS: To investigate the link between MLH1 and SPTAN1 in cancer progression, a cohort of 189 patients with CRC was analyzed by immunohistochemistry. Compared with the surrounding normal mucosa, SPTAN1 expression was reduced in MLH1-deficient CRCs, whereas MLH1-proficient CRCs showed a significant upregulation of SPTAN1. Overall, we identified a strong correlation between MLH1 status and SPTAN1 expression. When comparing TNM classification and SPTAN1 levels, we found higher SPTAN1 levels in stage I CRCs, while stages II to IV showed a gradual reduction of SPTAN1 expression. In addition, SPTAN1 expression was lower in metastatic compared with non-metastatic CRCs. Knockdown of SPTAN1 in CRC cell lines demonstrated decreased cell viability, impaired cellular mobility and reduced cell-cell contact formation, indicating that SPTAN1 plays an important role in cell growth and cell attachment. The observed weakened cell-cell contact of SPTAN1 knockdown cells might indicate that tumor cells expressing low levels of SPTAN1 detach from their primary tumor and metastasize more easily. CONCLUSION: Taken together, we demonstrate that MLH1 deficiency, low SPTAN1 expression, and tumor progression and metastasis are in close relation. We conclude that SPTAN1 is a candidate molecule explaining the tumor progression and metastasis of MLH1-deficient CRCs. The detailed analysis of SPTAN1 is now mandatory to substantiate its relevance and its potential value as a candidate protein for targeted therapy, and as a predictive marker of cancer aggressiveness.

Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants.

The human DNA repair gene MUTYH, whose mutational loss causes a colorectal polyposis and cancer predisposition, contains three alternative first exons. In order to analyze alternative transcription and the effect of genetic alterations found in humans, we established a cell-based minigene experimental model supporting transcription and splicing and thoroughly verified its functionality. We identified highly conserved promoter areas and inactivated them in the minigene, and also introduced six human variants. Moreover, the potential contribution of CpG island methylation and specific transcription factors on MUTYH transcription was addressed. The findings allowed to attribute regulatory roles to three conserved motifs in the promoter: an M4 motif, a transcription factor IIB recognition element, and a GC box. Moreover, the data showed that three patient variants compromised MUTYH expression and therefore have the potential to cause pathogenic effects. We did not find evidence for a biologically relevant contribution of CpG island methylation or a direct transcriptional activation by DNA damage. Besides insight into the regulation of MUTYH transcription, the work therefore provides a functional MUTYH minigene experimental system suitable as a diagnostic tool for analyzing patient variants, and a functional map of the promotor that also can facilitate pathogenicity classifications of human variants.

Diagnostic yield and clinical utility of a comprehensive gene panel for hereditary tumor syndromes.

BACKGROUND: In a considerable number of patients with a suspected hereditary tumor syndrome (HTS), no underlying germline mutation is detected in the most likely affected genes. The present study aimed to establish and validate a large gene panel for HTS, and determine its diagnostic yield and clinical utility. METHODS: The study cohort comprised 173 patients with suspected, but unexplained, HTS (group U) and 64 HTS patients with a broad spectrum of known germline mutations (group K). All patients in group U presented with early age at onset, multiple tumors, and/or a familial clustering of various tumor types; no germline mutation had been identified during routine diagnostics. Sequencing of leukocyte DNA was performed for the 94 HTS genes of the Illumina TruSight™Cancer Panel and 54 additional HTS genes. RESULTS: The sensitivity of the panel to identify known germline variants was 99.6%. In addition to known mutations, a total of 192 rare, potentially pathogenic germline variants in 86 genes were identified. Neither the proportion of rare variants per patient (group K: 0.9 variants; group U: 0.8 variants) nor the proportion of variants in the most frequently mutated, moderately penetrant genes CHEK2 and ATM showed significant inter-group difference. Four of the five patients from group U with a truncating CHEK2 mutation had thyroid cancer, pointing to a broader tumor spectrum in patients with pathogenic CHEK2 variants. In 22% of patients from group K, a further potential causative variant was identified. Here, the most interesting finding was an NF1 nonsense mutation in a child with a known TP53 frameshift mutation. In 17% of patients from group U, potential causative variants were identified. In three of these patients (2%), mutations in PMS2, PTEN, or POLD1 were considered to be causative. In both groups, incidental findings with presumptive predictive value were generated. CONCLUSIONS: The gene panel identified the genetic cause in some prescreened, unexplained HTS patients and generated incidental findings. Some patients harbored predicted pathogenic mutations in more than one established HTS gene, rendering interpretation of the respective alterations challenging. Established moderate risk genes showed an almost equal distribution among patients with known and unexplained disease.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.

Evaluation of MLH1 variants of unclear significance.

Inactivating mutations in the MLH1 gene cause the cancer predisposition Lynch syndrome, but for small coding genetic variants it is mostly unclear if they are inactivating or not. Nine such MLH1 variants have been identified in South American colorectal cancer (CRC) patients (p.Tyr97Asp, p.His112Gln, p.Pro141Ala, p.Arg265Pro, p.Asn338Ser, p.Ile501del, p.Arg575Lys, p.Lys618del, p.Leu676Pro), and evidence of pathogenicity or neutrality was not available for the majority of these variants. We therefore performed biochemical laboratory testing of the variant proteins and compared the results to protein in silico predictions on structure and conservation. Additionally, we collected all available clinical information of the families to come to a conclusion concerning their pathogenic potential and facilitate clinical diagnosis in the affected families. We provide evidence that four of the alterations are causative for Lynch syndrome, four are likely neutral and one shows compromised activity which can currently not be classified with respect to its pathogenic potential. The work demonstrates that biochemical testing, corroborated by congruent evolutionary and structural information, can serve to reliably classify uncertain variants when other data are insufficient.

Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1L749P and MLH1Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα.

Elucidating the clinical significance of two PMS2 missense variants coexisting in a family fulfilling hereditary cancer criteria.

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.2149G>A (p.V717M) and c.2444C>T (p.S815L), were identified in trans in one individual diagnosed with early-onset colorectal cancer (CRC) who belonged to a family fulfilling clinical criteria for hereditary cancer. Clinico-pathological data, multifactorial likelihood calculations and functional analyses were used to refine their clinical significance. Likelihood analysis based on cosegregation and tumor data classified the c.2444C>T variant as pathogenic, which was supported by impaired MMR activity associated with diminished protein expression in functional assays. Conversely, the c.2149G>A variant displayed MMR proficiency and protein stability. These results, in addition to the conserved PMS2 expression in normal tissues and the absence of germline microsatellite instability (gMSI) in the biallelic carrier ruled out a CMMRD diagnosis. The use of comprehensive strategies, including functional and clinico-pathological information, is mandatory to improve the clinical interpretation of naturally occurring MMR variants. This is critical for appropriate clinical management of cancer syndromes associated to MMR gene mutations.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment.

Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas.

In a number of families with colorectal adenomatous polyposis or suspected Lynch syndrome/HNPCC, no germline alteration in the APC, MUTYH, or mismatch repair (MMR) genes are found. Missense mutations in the polymerase genes POLE and POLD1 have recently been identified as rare cause of multiple colorectal adenomas and carcinomas, a condition termed polymerase proofreading-associated polyposis (PPAP). The aim of the present study was to evaluate the clinical relevance and phenotypic spectrum of polymerase germline mutations. Therefore, targeted sequencing of the polymerase genes POLD1, POLD2, POLD3, POLD4, POLE, POLE2, POLE3 and POLE4 was performed in 266 unrelated patients with polyposis or fulfilled Amsterdam criteria. The POLE mutation c.1270C>G;p.Leu424Val was detected in four unrelated patients. The mutation was present in 1.5% (4/266) of all patients, 4% (3/77) of all familial cases and 7% (2/30) of familial polyposis cases. The colorectal phenotype in 14 affected individuals ranged from typical adenomatous polyposis to a HNPCC phenotype, with high intrafamilial variability. Multiple colorectal carcinomas and duodenal adenomas were common, and one case of duodenal carcinoma was reported. Additionally, various extraintestinal lesions were evident. Nine further putative pathogenic variants were identified. The most promising was c.1306C>T;p.Pro436Ser in POLE. In conclusion, a PPAP was identified in a substantial number of polyposis and familial colorectal cancer patients. Screening for polymerase proofreading mutations should therefore be considered, particularly in unexplained familial cases. The present study broadens the phenotypic spectrum of PPAP to duodenal adenomas and carcinomas, and identified novel, potentially pathogenic variants in four polymerase genes.

Functional testing strategy for coding genetic variants of unclear significance in MLH1 in Lynch syndrome diagnosis.

Lynch syndrome is caused by inactivating mutations in the MLH1 gene, but genetic variants of unclear significance frequently preclude diagnosis. Functional testing can reveal variant-conferred defects in gene or protein function. Based on functional defect frequencies and clinical applicability of test systems, we developed a functional testing strategy aimed at efficiently detecting pathogenic defects in coding MLH1 variants. In this strategy, tests of repair activity and expression are prioritized over analyses of subcellular protein localization and messenger RNA (mRNA) formation. This strategy was used for four unclear coding MLH1 variants (p.Asp41His, p.Leu507Phe, p.Gln689Arg, p.Glu605del + p.Val716Met). Expression was analyzed using a transfection system, mismatch repair (MMR) activity by complementation in vitro, mRNA formation by reverse transcriptase-PCR in carrier lymphocyte mRNA, and subcellular localization with dye-labeled fusion constructs. All tests included clinically meaningful controls. The strategy enabled efficient identification of defects in two unclear variants: the p.Asp41His variant showed loss of MMR activity, whereas the compound variant p.Glu605del + p.Val716Met had a defect of expression. This expression defect was significantly stronger than the pathogenic expression reference variant analyzed in parallel, therefore the defect of the compound variant is also pathogenic. Interestingly, the expression defect was caused additively by both of the compound variants, at least one of which is non-pathogenic when occurring by itself. Tests were neutral for p.Leu507Phe and p.Gln689Arg, and the results were consistent with available clinical data. We finally discuss the improved sensitivity and efficiency of the applied strategy and its limitations in analyzing unclear coding MLH1 variants.