Gene-specific ACMG/AMP classification criteria for germline APC variants: Recommendations from the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel.

PURPOSE: The Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) was established by the International Society for Gastrointestinal Hereditary Tumours and the Clinical Genome Resource, who set out to develop recommendations for the interpretation of germline APC variants underlying Familial Adenomatous Polyposis, the most frequent hereditary polyposis syndrome. METHODS: Through a rigorous process of database analysis, literature review, and expert elicitation, the APC VCEP derived gene-specific modifications to the ACMG/AMP (American College of Medical Genetics and Genomics and Association for Molecular Pathology) variant classification guidelines and validated such criteria through the pilot classification of 58 variants. RESULTS: The APC-specific criteria represented gene- and disease-informed specifications, including a quantitative approach to allele frequency thresholds, a stepwise decision tool for truncating variants, and semiquantitative evaluations of experimental and clinical data. Using the APC-specific criteria, 47% (27/58) of pilot variants were reclassified including 14 previous variants of uncertain significance (VUS). CONCLUSION: The APC-specific ACMG/AMP criteria preserved the classification of well-characterized variants on ClinVar while substantially reducing the number of VUS by 56% (14/25). Moving forward, the APC VCEP will continue to interpret prioritized lists of VUS, the results of which will represent the most authoritative variant classification for widespread clinical use.

Merged testing for colorectal cancer syndromes and re-evaluation of genetic variants improve diagnostic yield: Results from a nationwide prospective cohort.

Approximately 5% of patients with colorectal cancer (CRC) have a Mendelian predisposition for the disease. Identification of the disease-causing genetic variant enables carrier testing and tailored cancer prevention within affected families. To determine the panorama and genetic variation of Mendelian CRC syndromes among referrals at the cancer genetics clinics in Sweden, 850 patients clinically selected for CRC genetic investigation were included in a prospective study that tested for all major hereditary polyposis and nonpolyposis CRC conditions. Genetically defined syndromes were diagnosed in 11% of the patients. Lynch syndrome was predominant (n = 73) followed by familial adenomatous polyposis (n = 12) and MUTYH-associated polyposis (n = 8); the latter of which two patients presented with CRC before polyposis was evident. One patient with a history of adolescent-onset CRC and polyposis had biallelic disease-causing variants diagnostic for constitutional mismatch repair deficiency syndrome. Post-study review of detected variants of unknown clinical significance (n = 129) resulted in the reclassification of variants as likely benign (n = 59) or as diagnostic for Lynch syndrome (n = 2). Our results reveal the panorama of Mendelian CRC syndromes at the cancer genetics clinics in Sweden and show that unified testing for polyposis and nonpolyposis CRC conditions as well as regular reexamination of sequence data improve the diagnostic yield.

Identification of known and novel familial cancer genes in Swedish colorectal cancer families.

Identifying new candidate colorectal cancer (CRC) genes and mutations are important for clinical cancer prevention as well as in cancer care. Genetic counseling is already implemented for known high-risk variants; however, the majority of CRC are of unknown causes. In our study, 110 CRC patients in 55 Swedish families with a strong history of CRC but unknown genetic causes were analyzed with the aim of identifying novel candidate CRC predisposing genes. Exome sequencing was used to identify rare and high-impact variants enriched in the families. No clear pathogenic variants were found in known CRC predisposing genes; however, potential pathogenic variants in novel CRC predisposing genes were identified. Over 3000 variants with minor allele frequency (MAF) <0.01 and Combined Annotation Dependent Depletion (CADD) > 20 were seen aggregating in the CRC families. Of those, 27 variants with MAF < 0.001 and CADD>25 were considered high-risk mutations. Interestingly, more than half of the high-risk variants were detected in three families, suggesting cumulating contribution of several variants to CRC. In summary, our study shows that despite a strong history of CRC within families, identifying pathogenic variants is challenging. In a small number of families, few rare mutations were shared by affected family members. This could indicate that in the absence of known CRC predisposing genes, a cumulating contribution of mutations leads to CRC observed in these families.

Biallelic germline nonsense variant of MLH3 underlies polyposis predisposition.

PURPOSE: Some 10% of familial adenomatous polyposis (FAP) and 80% of attenuated polyposis (AFAP) cases remain molecularly unexplained. We scrutinized such cases by exome-wide and targeted methods to search for novel susceptibility genes. METHODS: Exome sequencing was conducted on 40 unexplained (mainly sporadic) cases with FAP or AFAP from Finland. The DNA mismatch repair (MMR) gene MLH3 (MutL Homolog 3) was pinpointed and prompted a subsequent screen of ~1000 Swedish patients referred to clinical panel sequencing for colon tumor susceptibility. RESULTS: Three homozygous carriers of a truncating variant in MLH3, c.3563C>G, p.Ser1188Ter, were identified among the index cases from the Finnish series. An additional biallelic carrier of the same variant was present in the Swedish series. All four patients shared a 0.8-Mb core haplotype around MLH3, suggesting a founder variant. Colorectal polyps from variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats, in agreement with previous findings of a minor role of MLH3 in MMR. Multiple loci were affected by loss of heterozygosity, suggesting chromosomal instability. CONCLUSION: Our results show that a biallelic nonsense variant of MLH3 underlies a novel syndrome with susceptibility to classical or attenuated adenomatous polyposis and possibly extracolonic tumors, including breast cancer.

GREM1 and POLE variants in hereditary colorectal cancer syndromes.

Hereditary factors are thought to play a role in at least one third of patients with colorectal cancer (CRC) but only a limited proportion of these have mutations in known high-penetrant genes. In a relatively large part of patients with a few or multiple colorectal polyps the underlying genetic cause of the disease is still unknown. Using exome sequencing in combination with linkage analyses together with detection of copy-number variations (CNV), we have identified a duplication in the regulatory region of the GREM1 gene in a family with an attenuated/atypical polyposis syndrome. In addition, 107 patients with colorectal cancer and/or polyposis were analyzed for mutations in the candidate genes identified. We also performed screening of the exonuclease domain of the POLE gene in a subset of these patients. The duplication of 16 kb in the regulatory region of GREM1 was found to be disease-causing in the family. Functional analyses revealed a higher expression of the GREM1 gene in colorectal tissue in duplication carriers. Screening of the exonuclease domain of POLE in additional CRC patients identified a probable causative novel variant c.1274A>G, p.Lys425Arg. In conclusion a high penetrant duplication in the regulatory region of GREM1, predisposing to CRC, was identified in a family with attenuated/atypical polyposis. A POLE variant was identified in a patient with early onset CRC and a microsatellite stable (MSS) tumor. Mutations leading to increased expression of genes can constitute disease-causing mutations in hereditary CRC syndromes.

Systematic large-scale application of ClinGen InSiGHT APC -specific ACMG/AMP variant classification criteria substantially alleviates the burden of variants of uncertain significance in ClinVar and LOVD databases.

Background: Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUS), APC-specific ACMG/AMP variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP). Methods: A streamlined algorithm using the APC -specific criteria was developed and applied to assess all APC variants in ClinVar and the InSiGHT international reference APC LOVD variant database. Results: A total of 10,228 unique APC variants were analysed. Among the ClinVar and LOVD variants with an initial classification of (Likely) Benign or (Likely) Pathogenic, 94% and 96% remained in their original categories, respectively. In contrast, 41% ClinVar and 61% LOVD VUS were reclassified into clinically actionable classes, the vast majority as (Likely) Benign. The total number of VUS was reduced by 37%. In 21 out of 36 (58%) promising APC variants that remained VUS despite evidence for pathogenicity, a data mining-driven work-up allowed their reclassification as (Likely) Pathogenic. Conclusions: The application of APC -specific criteria substantially reduced the number of VUS in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalisable model for other gene-/disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUS that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.

Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors-a nationwide, prospective Swedish study.

Background: Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors. Methods: gWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients. Findings: The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35). Interpretation: Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients. Funding: The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.

BRCA1 gene mutations may explain more than 80% of excess number of ovarian cancer cases after breast cancer - a population based study from the Western Sweden Health Care region.

AIM: In a previous cohort study, we showed that there was a significant variation in the frequency of ovarian cancer after having breast cancer in Sweden, with the highest risk occuring in the Western region. The present study aimed to evaluate whether the high prevalence of the founder mutation BRCA1 3171ins5 may explain the excess number of ovarian cancer. METHOD: Among more than 26 000 women with breast cancer in the Western Swedish Health Care Region, 159 cases were subsequently diagnosed with ovarian cancer, whereas the expected number was 96. Archived tissue material was analysed for six common Scandinavian BRCA1 and BRCA2 gene mutations. RESULTS: The excess number of cases was 63 (95% CI 47-77), based on person-years at risk and national incidence rates of ovarian cancer. A BRCA1 gene mutation was detected in 33 cases corresponding to 52% of the excess number. The founder mutation, BRCA1 3171ins5, was detected in 44% of the excess number. The identified mutations decreased from 45% in women less than 50 years of age at follow-up to 14% at 60+ years at follow-up. There was no obvious decrease in mutation frequency by excess numbers with age. Age at follow-up and first-degree relatives with breast and/or ovarian cancer were the best predictors of a mutation in this material. CONCLUSION: The founder mutation, BRCA1 3171ins5, explains the excess of ovarian cancer after breast cancer in the region. From the relative frequency of the studied mutations found at the cancer genetic counselling clinic, it is estimated that BRCA1 gene mutations are associated with about 80-85% of the excess cases. This means that a negative screening for these mutations in similar cases may have a predictive value and could strongly reduce the risk of ovarian cancer in relatives.

Parallel sequencing used in detection of mosaic mutations: comparison with four diagnostic DNA screening techniques.

We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments.

Update on genetic predisposition to colorectal cancer and polyposis.

The present article summarizes recent developments in the characterization of genetic predisposition to colorectal cancer (CRC). The main themes covered include new hereditary CRC and polyposis syndromes, non-CRC hereditary cancer genes found mutated in CRC patients, strategies used to identify novel causal genes, and review of candidate genes that have been proposed to predispose to CRC and/or colonic polyposis. We provide an overview of newly described genes and syndromes associated with predisposition to CRC and polyposis, including: polymerase proofreading-associated polyposis, NTHL1-associated polyposis, mismatch repair gene biallelic inactivation-related adenomatous polyposis (including MSH3- and MLH3-associated polyposes), GREM1-associated mixed polyposis, RNF43-associated serrated polyposis, and RPS20 mutations as a rare cause of hereditary nonpolyposis CRC. The implementation of next generation sequencing approaches for genetic testing has exposed the presence of pathogenic germline variants in genes associated with hereditary cancer syndromes not traditionally linked to CRC, which may have an impact on genetic testing, counseling and surveillance. The identification of new hereditary CRC and polyposis genes has not deemed an easy endeavor, even though known CRC-related genes explain a small proportion of the estimated familial risk. Whole-genome sequencing may offer a technology for increasing this proportion, particularly if applied on pedigree data allowing linkage type of analysis. The final section critically surveys the large number of candidate genes that have been recently proposed for CRC predisposition.

Clinical characterization and the mutation spectrum in Swedish adenomatous polyposis families.

BACKGROUND: The dominantly inherited condition familial adenomatous polyposis (FAP) is caused by germline mutations in the APC gene. Finding the causative mutations has great implications for the families. Correlating the genotypes to the phenotypes could help to improve the diagnosis and follow-up of patients. METHODS: Mutation screening of APC and the clinical characterization of 96 unrelated FAP patients from the Swedish Polyposis Registry was performed. In addition to generally used mutation screening methods, analyses of splicing-affecting mutations and investigations of the presence of low-frequency mutation alleles, indicating mosaics, have been performed, as well as quantitative real-time polymerase chain reaction to detect lowered expression of APC. RESULTS: Sixty-one different APC mutations in 81 of the 96 families were identified and 27 of those are novel. We have previously shown that 6 of the 96 patients carried biallelic MUTYH mutations. The 9 mutation-negative cases all display an attenuated or atypical phenotype. Probands with a genotype (codon 1250-1464) predicting a severe phenotype had a median age at diagnosis of 21.8 (range, 11-49) years compared with 34.4 (range, 14-57) years among those with mutations outside this region (P < 0.017). Dense polyposis (> 1000) occurred in 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal cancer among probands was 25% at a mean age of 37.5 years and 29% at a mean age of 46.6 years. CONCLUSION: Using a variety of mutation-detection techniques, we have achieved a 100% detection frequency in classical FAP. Probands with APC mutations outside codon 1250-1464, although exhibiting a less-severe phenotype, are at high risk of having a colorectal cancer at diagnosis indicating that age at diagnosis is as important as the severity of the disease for colorectal cancer morbidity.

No germline mutations in supposed tumour suppressor genes SAFB1 and SAFB2 in familial breast cancer with linkage to 19p.

BACKGROUND: The scaffold attachment factor B1 and B2 genes, SAFB1/SAFB2 (both located on chromosome 19p13.3) have recently been suggested as tumour suppressor genes involved in breast cancer development. The assumption was based on functional properties of the two genes and loss of heterozygosity of intragenic markers in breast tumours further strengthened the postulated hypothesis. In addition, linkage studies in Swedish breast cancer families also indicate the presence of a susceptibility gene for breast cancer at the 19p locus. Somatic mutations in SAFB1/SAFB2 have been detected in breast tumours, but to our knowledge no studies on germline mutations have been reported. In this study we investigated the possible involvement of SAFB1/SAFB2 on familiar breast cancer by inherited mutations in either of the two genes. RESULTS: Mutation analysis in families showing linkage to the SAFB1/2 locus was performed by DNA sequencing. The complete coding sequence of the two genes SAFB1 and SAFB2 was analyzed in germline DNA from 31 affected women. No missense or frameshift mutations were detected. One polymorphism was found in SAFB1 and eight polymorphisms were detected in SAFB2. MLPA-anlysis showed that both alleles of the two genes were preserved which excludes gene inactivation by large deletions. CONCLUSION: SAFB1 and SAFB2 are not likely to be causative of the hereditary breast cancer syndrome in west Swedish breast cancer families.

Occurrence of both breast and ovarian cancer in a woman is a marker for the BRCA gene mutations: a population-based study from western Sweden.

AIM: This study aimed to analyze whether the occurrence of both breast and ovarian cancer in a woman serves as a marker for BRCA gene mutations. MATERIAL AND METHODS: This population-based study included 256 women in western Sweden who developed both invasive breast and ovarian tumors between 1958 and 1999. Archival paraffin tissue blocks of their tumors were retrieved for DNA-extraction to analyze the founder mutation, BRCA1 c.3171_3175dup (c.3171ins5), which is most common in this geographic area and four other common Scandinavian BRCA1 gene mutations and one BRCA2 mutation. Together, account these mutations for approximately 75% of the BRCA1/2 gene mutations in the clinical unit. RESULTS: Ninteen percent (95% confidence interval (CI) 14-24%) of the women carried one of the analyzed BRCA1 gene mutations but none of the women were positive for the analyzed BRCA2 mutation. One-third of the women with both tumors before age 60 were mutation carriers. BRCA1 c.3171_3175dup (c.3171ins5) constituted 84% of all identified mutations. Although the majority of breast cancers were invasive ductal and atypical medullary types, a variety of other breast malignancies were seen among mutation carriers. Serous ovarian carcinomas predominated among ovarian tumors. A variety of other ovarian tumors, including three granulosa-theca cell tumors, were also observed among mutation carriers. CONCLUSIONS: The occurrence of both breast and ovarian cancer in a woman is associated with a high likelihood of a constitutional BRCA1 mutation. These women and their families might therefore be considered for mutation screening after appropriate genetic counselling.

Expanding the genotype-phenotype spectrum in hereditary colorectal cancer by gene panel testing.

Hereditary syndromes causing colorectal cancer include both polyposis and non-polyposis syndromes. Overlapping phenotypes between the syndromes have been recognized and this make targeted molecular testing for single genes less favorable, instead there is a gaining interest for multi-gene panel-based approaches detecting both SNVs, indels and CNVs in the same assay. We applied a panel including 19 CRC susceptibility genes to 91 individuals of six phenotypic subgroups. Targeted NGS-based sequencing of the whole gene regions including introns of the 19 genes was used. The individuals had a family history of CRC or had a phenotype consistent with a known CRC syndrome. The purpose of the study was to demonstrate the diagnostic difficulties linked to genotype-phenotype diversity and the benefits of using a gene panel. Pathogenicity classification was carried out on 46 detected variants. In total we detected sixteen pathogenic or likely pathogenic variants and 30 variants of unknown clinical significance. Four of the pathogenic or likely pathogenic variants were found in BMPR1A in patients with unexplained familial adenomatous polyposis or atypical adenomatous polyposis, which extends the genotype-phenotype spectrum for this gene. Nine patients had more than one variant remaining after the filtration, including three with truncating mutations in BMPR1A, PMS2 and AXIN2. CNVs were found in three patients, in upstream regions of SMAD4, MSH3 and CTNNB1, and one additional individual harbored a 24.2 kb duplication in CDH1 intron1.

Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by decreased levels of survival motor neuron protein (SMN). In the majority of cases, this decrease is due to absence of the SMN1 gene. Multiplex ligation-dependent probe amplification (MLPA) is a modern quantitative molecular method. Applied in SMA cases, it improves diagnostics by simultaneously identifying the number of copies of several target sequences in the SMN1 gene and in nearby genes. Using MLPA in clinical diagnostics, we have identified a previously unreported, partial deletion of SMN1 (exons 1-6) in two apparently unrelated Swedish families. This mutation would not have been detected by conventional diagnostic methods. This paper illustrates the broad clinical and genetic spectrum of SMA and includes reports of MLPA results and clinical descriptions of a patient with homozygous absence of SMN1 and only one SMN2 (prenatal onset SMA type 1), an asymptomatic woman with five SMN2 (lacking SMN1) and representative patients with SMA types 1, 2 and 3.

Mismatch repair gene mutation spectrum in the Swedish Lynch syndrome population.

Lynch syndrome caused by constitutional mismatch­repair defects is one of the most common hereditary cancer syndromes with a high risk for colorectal, endometrial, ovarian and urothelial cancer. Lynch syndrome is caused by mutations in the mismatch repair (MMR) genes i.e., MLH1, MSH2, MSH6 and PMS2. After 20 years of genetic counseling and genetic testing for Lynch syndrome, we have compiled the mutation spectrum in Sweden with the aim to provide a population-based perspective on the contribution from the different MMR genes, the various types of mutations and the influence from founder mutations. Mutation data were collected on a national basis from all laboratories involved in genetic testing. Mutation analyses were performed using mainly Sanger sequencing and multiplex ligation-dependent probe amplification. A total of 201 unique disease-predisposing MMR gene mutations were identified in 369 Lynch syndrome families. These mutations affected MLH1 in 40%, MSH2 in 36%, MSH6 in 18% and PMS2 in 6% of the families. A large variety of mutations were identified with splice site mutations being the most common mutation type in MLH1 and frameshift mutations predominating in MSH2 and MSH6. Large deletions of one or several exons accounted for 21% of the mutations in MLH1 and MSH2 and 22% in PMS2, but were rare (4%) in MSH6. In 66% of the Lynch syndrome families the variants identified were private and the effect from founder mutations was limited and predominantly related to a Finnish founder mutation that accounted for 15% of the families with mutations in MLH1. In conclusion, the Swedish Lynch syndrome mutation spectrum is diverse with private MMR gene mutations in two-thirds of the families, has a significant contribution from internationally recognized mutations and a limited effect from founder mutations.

Nuclear accumulation of full-length and truncated adenomatous polyposis coli protein in tumor cells depends on proliferation.

The adenomatous polyposis coli (APC) tumor suppressor is a nucleocytoplasmic protein. The nuclear accumulation of APC was recently found to vary depending on cell density, suggesting that putative APC function(s) in the nucleus is controlled by the establishment of cell contacts. We report here that the density-dependent redistribution of APC between nucleus and cytoplasm prevails in 6/6 thyroid and colorectal carcinoma cell lines. Moreover, mutated APC lacking known nuclear localization sequences had the similar distribution pattern as the full-length protein. APC invariably accumulated in the nuclei of Ki-67 expressing cells, but was largely cytoplasmic when cell cycle exit was induced by serum starvation or at high cell density. APC colocalized with beta-catenin in the nucleus only in one cell line (SW480). Also, APC maintained a predominantly nuclear position in early confluent states when cytoplasmic beta-catenin was recruited to newly formed adherens-like junctions. The results indicate that nuclear targeting of APC is driven by cell cycle entry rather than altered cell-cell contact. The ability of C-terminally truncated APC to accumulate in the nucleus suggests that nuclear import signals other than NLS1(APC) and NLS2(APC) are functionally important. Residual function(s) of N-terminal APC fragments in tumor cells carrying APC mutations might be beneficial to tumor growth and survival.

A high frequency of germline BRCA1/2 mutations in western Sweden detected with complementary screening techniques.

Dominant inheritance is presumed in 6-10% of breast and ovarian cancers. Mutations in BRCA1 and BRCA2 genes are the most commonly identified causative genes in such families. The frequency of mutation carriers with breast/ovarian cancer depends on the population studied, and display considerable variation that coincides with ethnic and geographical diversity. Mutation analyses were performed in 143 families registered at the Cancer Genetic Counseling Clinic of western Sweden. In a thorough mutation screening procedure, the entire BRCA1 and BRCA2 genes were analyzed using a combination of complementary mutation detection techniques. Mutations in either BRCA1 or BRCA2 were detected in 36% (52 out of 143) of all screened families. All families were clinically evaluated regarding age at diagnosis, type of cancer and number of cancer cases in the family. Among high-risk families, the mutation detection rate was 39% (46 out of 117). The detection rate observed among families with cases of ovarian cancer (42 out of 62, 68%), was substantially higher than in families with only breast cancer (10 out of 81, 12%). Age at ovarian cancer did not seem to have an effect on the detection rate. The analyses revealed 11 frameshift mutations, 4 nonsense mutations and 2 large deletions. Notably, the BRCA1 c.3171ins5 mutation accounted for 34 of 52 (65%) identified mutations. Seven mutations are novel: BRCA1c.409_410del; c.1912T>G; c.2228_2229del; c.3029delA; c.3433delA, a large deletion covering exons 1-3 of BRCA1and one BRCA2 mutation; BRCA2c.6287_6290del. We have shown that the founder mutation BRCA1 c.3171ins5 has a great influence on western Swedish breast/ovarian cancer families along with a high number of mutations unique for the region. In order to achieve a high mutation detection rate we suggest a combination of several detection techniques.

Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques.

Large deletions in the APC (adenomatous polyposis coli) gene, causing familial adenomatous polyposis (FAP), cannot easily be detected by conventional mutation-detection techniques. Therefore, we have developed two independent quantitative methods for the detection of large deletions, encompassing one or more exons, of APC. Multiplex ligation-dependent probe amplification (MLPA) is performed in one reaction for the initial quantification of all APC exon copy numbers. Subsequently, quantitative real-time PCR (QRT-PCR) is used to verify the results obtained in the MLPA reaction. The identification of a deletion of the whole APC gene in a patient with classical FAP is described. The mutation was detected with the two quantitative methods and further verified on chromosomal level by the use of FISH (fluorescence in situ hybridization) on metaphase spreads. Furthermore, a large deletion covering exons 11-13 of the APC gene was detected in two apparently unrelated families. This deletion was further verified and characterized with long-range PCR. The MLPA test ensures a sensitive high-throughput screening for large deletions of the APC gene and can easily be implemented in the diagnostic testing for FAP.

Genotype-phenotype correlations, and retinal function and structure in von Hippel-Lindau disease.

PURPOSE: To investigate genotype-phenotype correlation and to analyze functional and structural changes in the retina of patients with von Hippel-Lindau (VHL) disease. METHODS: Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined. All patients underwent clinical examination and optical coherence tomography (OCT). Full-field electroretinography (full-field ERG) was performed in twelve patients. RESULTS: Family A, with deletion of exon 3 in the VHL gene, and family B, with the missense mutation p.R79P, exhibited type 1 VHL characterized by the absence of pheochromocytoma and a high incidence of central nervous system hemangioblastomas. One member of family B exhibited Goldenhar syndrome. A novel missense mutation (p.L198P) was identified in the VHL gene in the patient from family C. This p.L198P mutation caused a phenotype with early onset of a neuroendocrine tumor of the pancreas, bilateral pheochromocytomas, and optic nerve hemangioblastoma. Full-field ERG showed significantly prolonged implicit times of the b-wave and maximal combined a-wave in VHL patients, compared to controls. Examination of the retinal structure in all patients with VHL, using OCT, showed a significant decrease in retinal thickness in VHL patients without ocular hemangioblastomas, compared to controls. CONCLUSIONS: Our findings support previously established genotype-phenotype correlations. However, we here describe an unusual phenotype with a novel missense mutation, p.L198P, and report the finding that VHL disease can be associated with Goldenhar syndrome. Electrophysiological and structural findings suggest that VHL disease is a progressive, neurodegenerative disease of the retina.