HNPCC Syndrome Research Articles

Identification of Germline Variants in Patients with Hereditary Cancer Syndromes in Northeast Mexico.

Hereditary cancer syndromes (HCS) are genetic diseases with an increased risk of developing cancer. This research describes the implementation of a cancer prevention model, genetic counseling, and germline variants testing in an oncologic center in Mexico. A total of 315 patients received genetic counseling, genetic testing was offered, and 205 individuals were tested for HCS. In 6 years, 131 (63.90%) probands and 74 (36.09%) relatives were tested. Among the probands, we found that 85 (63.9%) had at least one germline variant. We identified founder mutations in BRCA1 and a novel variant in APC that led to the creation of an in-house detection process for the whole family. The most frequent syndrome was hereditary breast and ovarian cancer syndrome (HBOC) (41 cases with BRCA1 germline variants in most of the cases), followed by eight cases of hereditary non-polyposic cancer syndrome (HNPCC or Lynch syndrome) (with MLH1 as the primarily responsible gene), and other high cancer risk syndromes. Genetic counseling in HCS is still a global challenge. Multigene panels are an essential tool to detect the variants frequency. Our program has a high detection rate of probands with HCS and pathogenic variants (40%), compared with other reports that detect 10% in other populations.

Les cancers gastriques héréditaires: rôle du pathologiste en 2020

Gastric cancer is the third most common cancer worldwide. The majority of gastric cancers are sporadic but familial clustering is seen in more than 10% of cases. This manuscript is divided into two parts. The first part is dedicated to the non-syndromic hereditary gastric cancer, particularly the hereditary diffuse gastric cancer (HDGC) and other gastric polyposes including the recently described GAPPS (Gastric adenocarcinoma and proximal polyposis of the stomach). The second part concerns the syndromic gastric cancer, namely the HNPCC syndrome (Hereditary Non Polyposis Colorectal Cancer) occurring as part of a genetic predisposition syndrome to cancer. Recent advances in oncogenetics and next generation sequencing technology have enabled the identification of new entities. This enhancement in knowledge regarding inherited syndromes predisposing to gastric cancer has consequently improved the management of patients and their families. In this context, pathologists play a major role in identifying particular morphologic entities prompting genetic investigation. The aim of this manuscript is to provide an update on the current knowledge about hereditary gastric cancer.

384P - Prevalence of abnormal microsatellite instability test among ovary and endometrial cancer patients

BackgroundMicrosatellite instability (MSI)test has been found and developed with relationship of HNPCC or Lynch syndrome. Its role has been extended from screening marker for Lynch syndrome detection to response marker for immune checkpoint therapy. We investigated to know prevalence of abnormal microsatellite instability (MSI-L/H) among endometrial cancer ovary cancer. MethodsWe identified 51 cases of MSI samples from ovary and endometrial cancer visiting hereditary gynecologic cancer center. ResultsWe found 34 ovary cancer,13 endometrial cancer and 4 colon cancer patients. Median age was 56.6(18∼78). About thirty percent have family cancer history among patient relatives (14/45(31.1%)). Early stage cancer (I∼II) (4/25,16.0%) have higher incidence than late stage cancer (III∼IV) (2/28,7.1%). Abnormal MSI have three serous type and endometrioid type pathology. Among abnormal MSI, we found two MSI-L and four MSI-H (6/51(11.8%)). We found one MSI-H ovary cancer,3 MSI-H endometrial cancer patients (1/34(2.9%),3/13(23.1%)). ConclusionsThere are about 10 percent of abnormal MSI in this study group. Prospective large clinical study is needed for hereditary cancer prevention and precision medicine. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

The Prevalence and Associated Factors of Microsatellite Instability in Ovarian Epithelial Cancers Detected by Molecular Genetic Studies in a Sample of Iranian Women

Background: Microsatellite instability, the main genetic element in HNPCC syndrome, is associated with a number of cancers, including ovarian epithelial carcinomas. These cancers have distinct characteristics compared to non-MSI related ones. Objectives: The present study aims at determining the prevalence of microsatellite instability in ovarian carcinomas and their associated factors in Iranian patients. Methods: Paraffin-embedded blocks, belonging to 37 patients with definite diagnosis of ovarian epithelial cancers, were retrieved from the archives. After DNA extraction from tumor tissue and PCR reaction, the results were assessed in accordance with melting curve analysis. Subsequently, the relationship among microsatellite status and tumor histology, grade, stage, and size were investigated statistically. Results: The predominant histological type was serous histology. Four out of 37 carcinomas were microsatellite unstable (10.8%) and only 1 was MSI-high type (2.1%). The MSI was more frequent among younger patients with unilateral, non-serous histology, non-high grade, and stage I tumors without omental involvement. After statistical analysis, the only significant relationship was found between histological type (non-serous) and microsatellite status. Conclusions: Microsatellite stable and unstable ovarian cancers may have different associations with various factors in a sample of Iranian women. The identification of these characteristics may help narrow down indications to test this prognostic and predictive genetic error.

Abstract 3847: MSI status frequency, MSI-target genes mutation profile and ancestry proportions in Brazilian colorectal carcinoma patients

Abstract Introduction: Colorectal cancer (CRC) is the fourth in incidence and in mortality worldwide for both sexes. There are three pathways in the pathogenesis of CRC: chromosome instability (CIN), microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). These pathways are closely related and some tumors may harbor alterations in multiple pathways. The MSI is the less common and is more frequently related with HNPCC Syndrome. In Brazil, CRC incidence is increasing and the frequency of MSI and its biological and clinical impact are largely unexplored. Furthermore, the literature has reported the influence of ancestry factors on the incidence and prognosis in this tumor type. Goals: to assess the frequency of MSI status in Brazil CRC tumors; to analyze the mutation profile of MSI target genes in MSI-positive tumors and to determine the patients ancestry by molecular markers. Materials and Methods: 1013 CRC patients were enrolled in the study. The MSI evaluation was performed using a multiplex PCR comprising 5 markers (NR27, NR21, NR24, BAT25, and BAT26). The MSI-H tumors were also assessed for mutations in a panel of 25 MSI-target genes, and their genetic ancestry will be evaluated using a panel of 46 AIMs (Ancestry-Informative Markers). Results: MSS status was observed in 86.0%, MSI-L in 4.0% and MSI-H in 10.0% of cases. The analysis of 25 MSI-target genes in 101 MSI-H cases showed distinct mutations frequencies, with the highest mutated genes been ATM (83.0%), EGFR (80.0%) and MRE11 (78.0%) and the lowest mutated genes been BLM (3.0%), WISP3 (2.0%) and TRBP2 (1.0%). No alterations were observed in BRCA1, BRCA2, XRCC2, PTEN and XPO5. We showed that the average ancestry proportions for MSS+MSI-L group were 74.1% of European, 12.3% of African, 6.6% of Native Americans, and 7.0% of East Asian, while for MSI-H group were 73.7% of European, 14.3% of African, 6.8% of Native Americans, and 5.2% of East Asian. So, no statistical difference was observed between distinct MSI status groups. Conclusion: The MSI frequencies identified in Brazilian CRC patients are in agreement with the international literature. We identify altered MSI-target genes in MSI-H tumors samples with potential clinical impact. For while, the ancestry proportions not showed an important factor for MSI status in Brazilian CRC patients. Citation Format: Gustavo N. Berardinelli, Cristovam Scapulatempo-Neto, Ronilson Duraes, Denise Peixoto-Guimaraes, Armando Melani, Rui Pereira, Rui Reis. MSI status frequency, MSI-target genes mutation profile and ancestry proportions in Brazilian colorectal carcinoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3847. doi:10.1158/1538-7445.AM2015-3847

Épidémiologie et facteurs de risque des tumeurs de la voie excrétrice urinaire supérieure : revue de la littérature pour le rapport annuel de l’Association française d’urologie

To describe the epidemiology, the risk and genetic factors involved in carcinogenesis pathways of upper urinary tumors UTUCs. A systematic review of the scientific literature was performed from the database Medline (National Library of Medicine, PubMed) and websites of the HAS and the ANSM using the following keywords: epidemiology; risk factor; tobacco; aristolochic acid; urothelial carcinoma; ureter; renal pelvis. The search was focused on the characteristics, the mode of action, the efficiency and the side effects of the various drugs concerned. The estimated UTUC incidence is 1.2 cases/100,000 inhabitant per year in Europe. The incidence of renal pelvis tumor has been stable for 30years, while the frequency of ureteric locations has increased over time. Locally advanced stage and high grade are more frequent at the time of diagnosis. The median age for diagnosis is 70-years-old. Male-to-female ratio is nearly 2. Main carcinogenic factors are tobacco consumption and occupational exposure. There are specific risk factors for UTUC such acid aristolochic (balkan's nephropathy and Chinese herbs nephropathy). Familial cases are distinct from sporadic cases. UTUCs belong to the HNPCC syndrome and they rank third in its tumor spectrum. UTUCs are scarce tumors with specific epidemiologic characteristics. UTUCs share common risk factors with other urothelial carcinomas such as bladder tumors but have also specific risk factors that clinicians should know.

Relationship between DNA Mismatch Repair Deficiency and Endometrial Cancer

Some cases of endometrial cancer are associated with a familial tumor and are referred to as hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Lynch syndrome is thought to be induced by germline mutation of the DNA mismatch repair (MMR) gene. An aberration in the MMR gene prevents accurate repair of base mismatches produced during DNA replication. This phenomenon can lead to an increased frequency of errors in target genes involved in carcinogenesis, resulting in cancerization of the cell. On the other hand, aberrant DNA methylation is thought to play a key role in sporadic endometrial carcinogenesis. Hypermethylation of unmethylated CpG islands in the promoter regions of cancer-related genes associated with DNA repair leads to the cell becoming cancerous. Thus, both genetic and epigenetic changes are intricately involved in the process through which cells become cancerous. In this review, we introduce the latest findings on the DNA mismatch repair pathway in endometrial cancer.

SP1-80 Program evaluation familial cancer genetic counselling in the community of Valencia. Spain (2005–2010)

BackgroundIn 2005 the Health Department of the Valencia (Spain), implemented a program of familial cancer. The objective of this program is to reduce cancer incidence and mortality in those with...

Le syndrome HNPCC (hereditary non polyposis colorectal cancer) ou syndrome de Lynch : un syndrome en rapport avec une défaillance du système de réparation de l’ADN

The HNPCC syndrome (hereditary non polyposis colon cancer) or Lynch syndrome stands for an autosomic dominant condition leading to the most prevalent hereditary colo-rectal cancers (CCR). MMR (mismatch repair)’s genes are involved in carcinogenesis as they play a role in ADNA mismatch repair. Microsatellite instability (MSI+ phenotype) induced by germline mutations is characteristic of such tumors and is necessary to assert the diagnosis. The HNPCC syndrome is associated with a significant increased risk of CCR altogether with endometrium, upper urinary tract and small bowel carcinomas as well as ovarian, biliary system and gastric cancers although of lesser extent. It is of importance to diagnose HNPCC syndrome prior to the treatment starts because it may influence patient's (as well as her/his relatives) disease management (type of surgery, surveillance and screening exams). New French recommendations, developed in 2009, about prophylactic colo-rectal and gynecologic surgeries and monitoring update latest ones published on 2004.

Hereditary non polyposis colorectal carcinoma (HNPCC syndrome)

Review on Hereditary non polyposis colorectal carcinoma (HNPCC syndrome), with data on clinics, and the genes involved.

53. Gastrointestinal tract findings in patients with muir torre syndrome (MTS)

<h3>Background</h3> MTS is a condition characterised by the association of cutaneous tumours (in particular sebaceous neoplasms) and visceral tumours. It is now considered a variant of HNPCC syndrome and the underlying defect is an autosomal dominant inherited deficiency in mismatch repair (MMR) leading to micro-satellite instability (MSI) with subsequent predisposition to neoplasia. The association of sebaceous neoplasms with MTS is strong enough to warrant testing of all such tumours for MMR by way of immunohistochemistry (IHC) for MLH-1, MSH-2, MSH-6 and PMS-2 proteins. A loss of at least one of these proteins is considered to represent MTS and ideally these patients should be screened for visceral malignancy as per HNPCC guidelines. The findings in gastrointestinal tract (GIT) pathology in patients with MMR loss in at least one cutaneous sebaceous tumour have not yet been reported. <h3>Methods</h3> A retrospective search (2003-2009) was done for patients with a clinical history of MTS and/or showing loss of at least one MMR by IHC in at least one cutaneous sebaceous neoplasm and yielded a database of 107 patients. GIT findings, if any, within 2 years prior and subsequent to the diagnosis of their cutaneous tumour were recorded and assessed for MMR deficiency by way of IHC. <h3>Results</h3> 15 patients had documented GIT pathology. 12 had a sebaceous tumour with documented MMR loss and 3 had a clinical history of MTS. There were 7 males and 8 females with a mean age of 59 years at diagnosis. 12/15 patients had ≥1 adenomatous polyps (24 colorectal, 2 duodenal). There were 6 colorectal carcinomas in 5 patients. 9 hyperplastic polyps/sessile serrated polyps were documented in 5 patients. At time of abstract submission IHC for MMR had been performed on 8 patients. Loss of at least one MMR marker was observed in 10/13 colorectal adenomas, 2/2 duodenal adenomas and 5/5 colorectal carcinomas. All hyperplastic polyps (4/9) showed preserved staining. In all cases tested to date, the MMR loss in the GIT lesion matched that documented in the skin tumours. <h3>Conclusion</h3> At least 10% of MTS patients will have significant GIT pathology and most neoplasms show MMR loss. GIT screening is recommended in all patients with MMR deficient sebaceous skin tumours.

En bloc right hemicolectomy and pancreaticoduodenectomy for HNPCC with simultaneous mutations of MSH-2 and MSH-6

Dear Editor: Hereditary nonpolyposis colon cancer (HNPCC or Lynch syndrome) is a multisystemic disorder, affecting the colon and rectum in up to 70% of cases. It is characterized by autosomal dominant inheritance and the development of multiple neoplasms including colorectal, endometrial, ovarian, pancreatic, small bowel, and urinary tract tumors. HNPCC is the leading cause of hereditary colorectal cancers (CRC) with an incidence of 2–5%. The genetic defects associated with this disorder arise from errors in the mismatch repair genes (MMR). Currently, five different genetic mutations have been identified (hMLH1, hMSH2, hMSH3, hMSH6, hPMS1) that lead to accelerated carcinogenic transformation, some of which show distinct phenotypic features like inflammatory component or expansive growth. Herein, we describe the first case report of an HNPCC patient with simultaneous loss of expression of MSH-2 and MSH-6 genes in the setting of a T4N0 colon cancer invading the duodenum and the head of the pancreas. A 38-year-old man presented to the emergency department of a tertiary referral center complaining of right-sided abdominal pain, nausea, vomiting, skinny stools, and weight loss (20 lbs) for the past 6 weeks prior to admission. He had no significant past medical history. However, he reported that his father died at the age of 50 of colon cancer. Physical exam was significant for mild discomfort in the right upper quadrant. His stool guaiac test was positive for occult blood, but no frank blood was seen and no mass was palpated on rectal digital exam. Laboratory tests were significant for mild transaminitis and a CT scan of the abdomen and pelvis revealed a 7.6-cm mass arising from the hepatic flexure of the right colon and extending into the second and third portion of the duodenum and pancreatic head. An upper and lower GI endoscopy with biopsy of the mass showed moderately differentiated adenocarcinoma of the colon. A subsequent PET scan showed no metastatic focus of fludeoxyglucose uptake. The patient underwent an exploratory laparotomy with an extended right hemicolectomy and pancreaticoduodenectomy. No contraindication to resection was encountered in the exploratory phase. The patient had an uneventful postoperative course and was discharged home on POD#14. Upon follow-up in the clinic 2 weeks later, he was eating a regular diet, having normal bowel movements, and was back to regular activities. The surgical specimen consisted of the resected colon, duodenum, and the head of the pancreas. Microscopic examination revealed a high grade (G3), poorly differentiated adenocarcinoma of the hepatic flexure, with mild to moderate lymphocytic invasion, infiltrating the full thickness of the colonic wall into the duodenum. The authors declare no conflict of interest and/or relationships with people or businesses that could influence or bias the outcome of this paper.

Analysis of mismatch repair gene mutations in Turkish HNPCC patients

Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by the inheritance of a mutant allele of a DNA mismatch repair gene. We aimed to investigate types and frequencies of mismatch repair (MMR) gene mutations in Turkish patients with HNPCC and to identify specific biomarkers for early diagnosis of their non-symptomatic kindred's. The molecular characteristics of 28 Turkish colorectal cancer patients at high-risk for HNPCC were investigated by analysis of microsatellite instability (MSI), immunohistochemistry and methylation-specific PCR in order to select tumors for mutation analysis. Ten cases (35.7%) were classified as MSI (+). Lack of expression of the main MMR proteins was observed in MSI (+) tumors. Hypermethylation of the MLH1 promoter region was observed in one tumor. Nine Lynch syndrome cases showed novel germ-line alterations of the MMR gene: two frame-shifts (MLH1 c.1843dupC and MLH1 c.1743delG) and three missense mutations (MLH1 c.293G>C, MLH1 c.954_955delinsTA and MSH2 c.2210G>A). Unclassified variants were evaluated as likely to be pathogenic by using the in-silico analyses. In addition, the MSH2 c.2210G>A alteration could be considered as a founder mutation for the Turkish population due to its identification in five different Lynch syndrome families and absence in control group. The present study adds new information about MMR gene mutation types and their role in Lynch syndrome. This is the first detailed research on Turkish Lynch syndrome families.

Search and repair of bona fide base‐pair mismatches in DNA

The DNA mismatch repair system (MMR) helps maintain genome integrity by correcting base‐pair mismatches and insertion/deletion loops in DNA. MMR proteins are thus critical for limiting mutagenesis, and their malfunction is linked to sporadic cancers and the HNPCC syndrome. MutS proteins perform the challenging task of finding a few errors against a background of millions of correct base pairs in DNA, and initiate repair. In order to understand the mechanism by which MutS proteins locate these errors, we measured the movement of S. cerevisiae MutS (Msh2‐6), between near Watson‐Crick (2‐Aminopurine:T) and mismatched (G:T) base‐pair sites on DNA. We found that Msh2‐6 binds both sites at equally high rates (2x107 M−1s−1), but it leaves the matched site ~50‐fold faster than the mismatched site (t1/2 ~ 0.4 versus 18 s, respv). These data suggest that while Msh2‐6 makes similar initial contacts with various base pairs, it recognizes mismatches by forming stable contacts specifically at these sites. Notably, Msh2‐6 leaves the matched site and arrives at the mismatched site without dissociating from DNA, which suggests that the protein slides on DNA, probing base pairs via transient contacts until it is trapped at a bona fide mismatch. A sliding mechanism likely enables MutS proteins to scan long stretches of DNA rapidly, thereby ensuring efficient repair of infrequent aberrations in the double helix. Research support: NSF.

Endometrial Cancer as a Familial Tumor: Pathology and Molecular Carcinogenesis (Review)

Some cases of endometrial cancer are associated with a familial tumor and are referred to as hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Such tumors are thought to be induced by germline mutation of the DNA mismatch repair (MMR) gene, but many aspects of the pathology of familial endometrial cancer are unclear and no effective screening method has been established. However, the pathology of endometrial cancer with familial tumor has been progressively clarified in recent studies. At present, about 0.5% of all cases of endometrial cancers meet the clinical diagnostic criteria for HNPCC. A recent analysis of the three MMR genes (hMLH1, hMSH2 and hMSH6) revealed germline mutations in 18 of 120 cases (15.0%) of endometrial cancer with familial accumulation of cancer or double cancer, with a frameshift mutation of the hMSH6 gene being the most common. Many cases with mutation did not meet the current clinical diagnostic criteria for HNPCC, indicating that familial endometrial cancer is often not diagnosed as HNPCC. The results suggest that the hMSH6 gene mutation may be important in carcinogenesis in endometrial cancer and germline mutations of the MMR gene may be more prevalent in cases associated with familial accumulation of cancer. An international large-scale muticenter study is required to obtain further information about the pathology of endometrial cancer as a familial tumor.

Consideration of hereditary nonpolyposis colorectal cancer in BRCA mutation‐negative familial ovarian cancers

Inherited mutations account for approximately 10% of all epithelial ovarian cancers. Breast cancer (BRCA1 and BRACA2) gene mutations are responsible for up to 85% of inherited breast and/or ovarian cancer. Another condition that has been associated with ovarian cancer is hereditary nonpolyposis colorectal cancer syndrome (HNPCC), which carries a lifetime risk of up to 13% for ovarian cancer. The objective of this study was to determine the incidence of HNPCC-related gene mutations in patients with familial ovarian cancer who previously tested negative for BRCA1 and BRCA2 gene mutations. Seventy-seven probands were identified who had familial ovarian cancer and negative BRCA gene mutation testing. Their pedigrees were analyzed for HNPCC syndrome. DNA samples underwent gene sequencing and Southern blot analysis for mutations in the 3 most common HNPCC-associated genes: mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) with reflex testing for MSH6 if tests for the first 2 genes were negative. None of the probands met Amsterdam criteria for the clinical diagnosis of HNPCC. DNA testing revealed 2 patients (2.6%) with deleterious mutations in the MSH2 gene. An additional 8 patients (10.4%) had substitutions in either the MLH1 gene or the MSH2 gene that were classified as variants of uncertain significance. If Amsterdam criteria were expanded to include ovarian cancer, then 15 of 77 patients (19.5%) would have met these expanded criteria. One deleterious mutation was noted in this group, yielding a mutation incidence of 6.7%. This percentage may be even higher if any of the identified variants of uncertain significance are confirmed to be deleterious. HNPCC should be considered when evaluating patients with suspected hereditary ovarian cancer who have had negative BRCA mutation testing.

Cancer de l’endomètre du syndrome HNPCC

The Hereditary Non-Polyposis Colorectal Cancer syndrome (HNPCC) has initially been described as a predisposition to colorectal cancers (CRC). Subsequently, other cancers, such as endometrial cancers (EC), have been added. The objective of this review was to update data on endometrial cancers of HNPCC syndrome. Endometrial cancers of the HNPCC syndrome are characterized by a younger age at diagnosis (46-48 year old), and a higher cumulative risk along life (30% at 70 years). Complex atypical hyperplasia seems to occur before the cancer, but the transition between precursors and cancer seems to be short. Histology of endometrial cancers of the HNPCC syndrome appears quite similar to that of sporadic cases, except for non-endometrioid lesions which seem more frequent and could occur in younger women. Screening of endometrial cancer in predisposed women should associate annual clinical examination, transvaginal sonography and endometrial sampling. Unfortunately, available data on screening by sonography show that this test seems poorly accurate, with no asymptomatic cancer or hyperplasia recognized and interval cancers between screenings. Endometrial biopsy appears as the most interesting method, since 11 asymptomatic cancers and 14 hyperplasia have been diagnosed in 175 mutation carriers. Diagnostic hysteroscopy seems also interesting, but requires further evaluation. Prophylactic hysterectomy confers a complete protection against endometrial cancer. However, perioperative morbidity (especially in women with history of colorectal surgery) and long-term effects of ovarian suppression should also be considered. Screening of endometrial cancer remains the main objective of the management of those patients. Endometrial biopsy should have a larger place.

Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction

Deletions of one or more exons in the mismatch repair genes MLH1 and MSH2 have been implicated in a significant fraction of hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Multiplex ligation-dependent probe amplification (MLPA) detection of deletions of multiple sequential exons is widely accepted; however, there is concern over the reliability of MLPA results showing single exon deletions. Given the clinical implications of a diagnosis of Lynch syndrome, it is important to use an alternative technique to confirm single exon deletions. To verify single exon deletions, we developed a SYBR Green-based quantitative polymerase chain reaction (PCR) assay. Clinical DNA samples containing deletions in 33 of the 35 exons in MLH1 and MSH2, previously screened by MLPA, were evaluated by quantitative PCR. Gene dosage ratios were determined by both the relative standard curve method and by the 2(-DeltaDeltaC(T)) method. Deleted exons had gene dosage ratios of 0.4 to 0.6, while nondeleted exons exhibited ratios of 0.8-1.3. We found 100% concordance between the quantitative PCR and MLPA results, including confirmation of all single exon deletions. The 2(-DeltaDeltaC(T)) method was as accurate as using standard curves for the calculation of ratios. Single exon deletions in MLH1 and MSH2 can be verified using quantitative PCR. Assays using this method are simple to design and easy to perform, making them ideal for confirmatory testing.

A large fraction of unclassified variants of the mismatch repair genesMLH1andMSH2is associated with splicing defects

Numerous unclassified variants (UVs) have been found in the mismatch repair genes MLH1 and MSH2 involved in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Some of these variants may have an effect on pre-mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). In order to determine the consequences of UVs on splicing, we used a functional assay of exon inclusion. For each variant, mutant and wild-type exons to be tested were PCR-amplified from patient genomic DNA together with approximately 150 bp of flanking sequences and were inserted into a splicing reporter minigene. After transfection into HeLa cells, the effects on splicing were evaluated by RT-PCR analysis and systematic sequencing. A total of 22 UVs out of 85 different variant alleles examined in 82 families affected splicing, including four exonic variants that affected putative splicing regulatory elements. We analyzed short stretches spanning the latter variants by cloning them into the ESE-dependent central exon of a three-exon splicing minigene and we showed in cell transfection experiments that the wild-type sequences indeed contain functional ESEs. We then used this construct to query for ESE elements in the MLH1 or MSH2 regions affected by 14 previously reported exonic splicing mutations and showed that they also contain functional ESEs. These splicing assays represent a valuable tool for the interpretation of UVs and should contribute to the optimization of the molecular diagnosis of the Lynch syndrome and of other genetic diseases.

283 – A study of alternative splicing of the KPNB3 gene in schizophrenia

Deletions of one or more exons in the mismatch repair genes MLH1 and MSH2 have been implicated in a significant fraction of hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Multiplex ligation-dependent probe amplification (MLPA) detection of deletions of multiple sequential exons is widely accepted; however, there is concern over the reliability of MLPA results showing single exon deletions. Given the clinical implications of a diagnosis of Lynch syndrome, it is important to use an alternative technique to confirm single exon deletions. To verify single exon deletions, we developed a SYBR Green-based quantitative polymerase chain reaction (PCR) assay. Clinical DNA samples containing deletions in 33 of the 35 exons in MLH1 and MSH2, previously screened by MLPA, were evaluated by quantitative PCR. Gene dosage ratios were determined by both the relative standard curve method and by the 2−ΔΔCT method. Deleted exons had gene dosage ratios of 0.4 to 0.6, while nondeleted exons exhibited ratios of 0.8–1.3. We found 100% concordance between the quantitative PCR and MLPA results, including confirmation of all single exon deletions. The 2−ΔΔCT method was as accurate as using standard curves for the calculation of ratios. Single exon deletions in MLH1 and MSH2 can be verified using quantitative PCR. Assays using this method are simple to design and easy to perform, making them ideal for confirmatory testing.