A Probabilistic Model for Cushing's Syndrome Screening in At-Risk Populations: A Prospective Multicenter Study.

CONTEXT: Cushing's syndrome (CS) is challenging to diagnose. Increased prevalence of CS in specific patient populations has been reported, but routine screening for CS remains questionable. To decrease the diagnostic delay and improve disease outcomes, simple new screening methods for CS in at-risk populations are needed. OBJECTIVE: To develop and validate a simple scoring system to predict CS based on clinical signs and an easy-to-use biochemical test. DESIGN: Observational, prospective, multicenter. SETTING: Referral hospital. PATIENTS: A cohort of 353 patients attending endocrinology units for outpatient visits. INTERVENTIONS: All patients were evaluated with late-night salivary cortisol (LNSC) and a low-dose dexamethasone suppression test for CS. MAIN OUTCOME MEASURES: Diagnosis or exclusion of CS. RESULTS: Twenty-six cases of CS were diagnosed in the cohort. A risk scoring system was developed by logistic regression analysis, and cutoff values were derived from a receiver operating characteristic curve. This risk score included clinical signs and symptoms (muscular atrophy, osteoporosis, and dorsocervical fat pad) and LNSC levels. The estimated area under the receiver operating characteristic curve was 0.93, with a sensitivity of 96.2% and specificity of 82.9%. CONCLUSIONS: We developed a risk score to predict CS in an at-risk population. This score may help to identify at-risk patients in non-endocrinological settings such as primary care, but external validation is warranted.

Characterization of four novel BRCA2 large genomic rearrangements in Spanish breast/ovarian cancer families: review of the literature, and reevaluation of the genetic mechanisms involved in their origin.

Large genomic rearrangements (LGRs) at the BRCA2 locus explain a non-negligible proportion of hereditary breast and ovarian cancer (HBOC) syndromes. The multiplex ligation and probe amplification (MLPA) assay has permitted in recent years to identify several families carrying LGRs at this locus, but very few such alterations have been fully characterized at the molecular level. Yet, molecular characterization is essential to identify recurrent alterations, to analyze the genetic mechanisms underlying such alterations, or to investigate potential genotype/phenotype relationships. We have used MLPA to identify BRCA2 LGRs in 7 out of 813 Spanish HBOC families previously tested negative for BRCA1 and BRCA2 small genomic alterations (substitutions and indels) and BRCA1 LGRs. We used a combination of long-range PCR, restriction mapping, and cDNA analysis to characterize the alterations at the molecular level. We found that Del Exon1-Exon2, Del Exon12-Exon16 and Del Exon22-Exon24 explain one family each, while Del Exon2 appears to be a Spanish founder mutation explaining four independent families. Finally, we have combined our data with a comprehensive review of the literature to reevaluate the genetic mechanisms underlying LGRs at the BRCA2 locus. Our study substantially increases the spectrum of BRCA2 LGRs fully characterized at the molecular level. Further on, we provide data to suggest that non-allelic homologous recombination has been overestimated as a mechanism underlying these alterations, while the opposite might be true for microhomology-mediated events.

Association of MUTYH and MSH6 germline mutations in colorectal cancer patients.

Colorectal cancer (CRC) risk associated with germline monoallelic MUTYH mutations remains controversial, although a slightly increased risk for this disease has been suggested. MUTYH and MSH6 proteins act in cooperation during the DNA repair process. Based on this interaction, it was hypothesized that the combination of heterozygote germline mutations in both genes could result in an increased CRC risk. To further clarify the interaction between MUTYH and MSH6, we analyzed the prevalence of MSH6 mutations in a cohort of CRC patients and controls previously tested for MUTYH mutations: CRC patients with and without a monoallelic MUTYH mutation (group I, n = 26; group II, n = 50, respectively), and healthy carriers with a monoallelic MUTYH mutation (group III, n = 21). In group I, we found three patients (11.5%) with MSH6 mutations, a missense mutation (p.R635G), a change in the 3'UTR region (c.*4098A > C) and a nonsense mutation (p.Q982X). In group II and III, no mutations were detected. In CRC patients, MSH6 mutations were more frequently found in MUTYH mutation carriers than in noncarriers (11.5% vs. 0%, P = 0.037). CRC patients carrying monoallelic MUTYH mutations harbor more frequently concomitant MSH6 mutations than patients without them, thus suggesting that both genes could act cooperatively and confer together an increased CRC risk.

The average cumulative risks of breast and ovarian cancer for carriers of mutations in BRCA1 and BRCA2 attending genetic counseling units in Spain.

PURPOSE: It is not clear that the published estimates of the breast and ovarian cancer penetrances of mutations in BRCA1 and BRCA2 can be used in genetic counseling in countries such as Spain, where the incidence of breast cancer in the general population is considerably lower, the prevalence of BRCA2 mutations seems to be higher, and a distinct spectrum of recurrent mutations exists for both genes. We aimed to estimate these penetrances for women attending genetic counseling units in Spain. EXPERIMENTAL DESIGN: We collected phenotype and genotype data on 155 BRCA1 and 164 BRCA2 mutation carrier families from 12 centers across the country. Average age-specific cumulative risks of breast cancer and ovarian cancer were estimated using a modified segregation analysis method. RESULTS: The estimated average cumulative risk of breast cancer to age 70 years was estimated to be 52% [95% confidence interval (95% CI), 26-69%] for BRCA1 mutation carriers and 47% (95% CI, 29-60%) for BRCA2 mutation carriers. The corresponding estimates for ovarian cancer were 22% (95% CI, 0-40%) and 18% (95% CI, 0-35%), respectively. There was some evidence (two-sided P = 0.09) that 330A>G (R71G) in BRCA1 may have lower breast cancer penetrance. CONCLUSIONS: These results are consistent with those from a recent meta-analysis of practically all previous penetrance studies, suggesting that women with BRCA1 and BRCA2 mutations attending genetic counseling services in Spain have similar risks of breast and ovarian cancer to those published for other Caucasian populations. Carriers should be fully informed of their mutation- and age-specific risks to make appropriate decisions regarding prophylactic interventions such as oophorectomy.

Molecular analysis of colorectal cancer tumors from patients with mismatch repair proficient hereditary nonpolyposis colorectal cancer suggests novel carcinogenic pathways.

PURPOSE: A subset of colorectal cancers (CRC) arises in families that, despite fulfilling clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC), do not show evidence of a mismatch repair (MMR) deficiency. The main objective of this study was to characterize these tumors at the molecular level. EXPERIMENTAL DESIGN: After comprehensive germ line mutation scanning, microsatellite analysis, and MMR protein expressions, we selected a well-defined cohort of 57 colorectal tumors with no evidence of MMR defects. In this group of tumors, we analyzed KRAS, BRAF, and APC somatic mutations, as well as methylguanine methyltransferase (MGMT) and beta-catenin expression. We correlated these alterations with clinicopathologic data and explored the relationship between KRAS G > A transitions and lack of MGMT expression. RESULTS: The mutation profile at the RAS/RAF/MAPK pathway mimics sporadic microsatellite-stable CRCs. We found an average age of diagnosis 10 years older in KRAS-mutated patients (P = 0.001). In addition, we show that KRAS G > A transitions are actively selected by tumors, regardless of MGMT status. Similarities with HNPCC high-microsatellite instability tumors are observed when APC data are analyzed. The APC mutation rate was low and small insertions/deletions accounted for 70% of the alterations. In addition, we found a low frequency of beta-catenin nuclear staining. Finally, we did not find evidence of tumors arising in individuals from the same family sharing molecular features. CONCLUSIONS: We show evidence that CRC tumors arising in HNPCC families without MMR alterations have distinctive molecular features. Overall, our work shows that systematic analysis of somatic alterations in a well-defined subset of CRCs is a good approach to provide new insights into the mechanisms of colorectal carcinogenesis.

Molecular haplotyping of tandem single nucleotide polymorphisms by allele-specific PCR.

BARD1 Val507Met (1592A>G) is an interesting marker for association studies on cancer risk. However, studies are scarce in the literature, probably reflecting the methodological problem imposed by the fact that next to the 1592A>G stands the 1591C>T single nucleotide polymorphism (SNP). We have designed an allele-specific PCR method capable of molecular haplotyping tandem SNPs. In the tandem SNPs haplotyping assay (tSNPh), four reverse primers are designed to be perfect matches of each potential haplotype. The forward primer is labeled with a fluorochrome. PCR products are analyzed by capillary electrophoresis. Haplotyping is performed by size calling. To ascertain the accuracy and reproducibility of the assay, we measured the level of concordance with sequencing data in 124 samples. In vitro-generated templates have been used for further testing. We developed a novel and reliable assay that permits typing two SNPs directly adjacent to each other, avoiding mutual interferences. The method is amenable to automation and high throughput. We expect that this assay will contribute to clarifying the role of BARD1 in cancer susceptibility. In addition, we suggest that tandem SNPs are potentially interesting polymorphic markers in which molecular haplotyping can be performed easily.

Screening for large rearrangements of the BRCA2 gene in Spanish families with breast/ovarian cancer.

Germ-line mutations in BRCA1 and BRCA2 are responsible for about 30-60% of the hereditary breast and ovarian cancer (HBOC). A large number of point mutations have been described in both genes. However, large deletions and duplications that disrupt one or more exons are overlooked by point mutation detection approaches. Over the past years several rearrangements have been identified in BRCA1, while few studies have been designed to screen this type of mutations in BRCA2. Our aim was to estimate the prevalence of large genomic rearrangements in the BRCA2 gene in Spanish breast/ovarian cancer families. The multiplex ligation-dependent probe amplification (MLPA) was employed to search gross deletions or duplications of BRCA2 in 335 Spanish moderate to high-risk breast/ovarian cancer families previously screened negative for point mutations by conventional methods. Four different and novel large genomic alterations were consistently identified by MLPA in five families, respectively: deletions of exon 2, exons 10-12 and exons 15-16 and duplication of exon 20 (in two families). RT-PCR experiments confirmed the deletion of exons 15-16. All patients harbouring a genomic rearrangement were members of high-risk families, with three or more breast/ovarian cancer cases or the presence of breast cancer in males. We provide evidence that the BRCA2 rearrangements seem to account for a relatively small proportion of familial breast cancer cases in Spanish population. The screening for these alterations as part of the comprehensive genetic testing can be recommended, especially in multiple case breast/ovarian families and families with male breast cancer cases.

Genomic rearrangements at the BRCA1 locus in Spanish families with breast/ovarian cancer.

BACKGROUND: Large genomic rearrangements (LGRs) account for a substantial proportion of the BRCA1 disease-causing changes, or variations, identified in families with hereditary breast/ovarian cancer [HB(O)C]. Great differences in the spectrum and prevalence of BRCA1 LGR have been observed among populations. Here we report the first comprehensive analysis of BRCA1 LGRs conducted in Spain. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to screen for BRCA1 LGRs in the index case individuals of 384 HB(O)C families who previously tested negative for BRCA1 and BRCA2 point variations, small insertions, and deletions. An alternative set of MLPA probes, long-range PCR, and real-time PCR were used to confirm positive results. RESULTS: We have identified 8 different BRCA1 rearrangements (del exon 1-24, del exon 8-13, del exon 11-15, del exon 14, dup exon 19-20, dup exon 20, exon 21-22 amplification, and del exon 23-24). With the exception of del exon 8-13, they are novel alterations. Overall, BRCA1 LGRs explain 1.4% of the Spanish HB(O)C families, and they account for 8.2% of all BRCA1 pathogenic variations identified in our study population. BRCA1 genetic variants affecting hybridization of commercially available MLPA probes are very rare in our population. CONCLUSIONS: Screening for BRCA1 LGRs should be mandatory in Spanish HB(O)C families. A high proportion of country-specific rearrangements are scattered along the gene. MLPA is a robust method to screen for LGRs in our population. MLPA analysis of positive samples with an alternative set of probes, together with long-range PCR and real-time PCR, is a feasible approach to confirm results in cases in which LGR breakpoints have not been characterized.

Increased frequency of disease-causing MYH mutations in colon cancer families.

The genetic factors that cause clustering of colorectal cancers (CRCs) other than mutations in the mismatch repair (MMR) genes are not well understood. Clustering in families who lack MMR gene mutations may be attributable to low-penetrance mutations. Hypothetically, mono-allelic MYH mutations could contribute to the risk of CRC in these families. Using Fisher's exact test and logistic regression, we compared the frequency of the known disease-causing MYH mutations Y165C, G382D and 466delE in 137 probands (117 cases with CRC and 20 cases diagnosed on the basis of adenomatous polyps only) from families with three or more CRCs but negative for mutations in the MMR genes and in 967 healthy controls with comparable ethnic backgrounds. Of 137 cases, 6 (4.4%) carried mono-allelic MYH mutations compared with 16 of 967 (1.6%) controls. In addition, three bi-allelic MYH mutation carriers, who eventually developed MYH-associated polyposis, were also identified in families with pedigree structures consistent with dominant inheritance of CRC susceptibility. By Fisher's exact tests, there was a statistically different frequency of cases with any MYH mutation (mono- or bi-allelic carriers; P-value = 0.002) and of cases with mono-allelic MYH mutation (P = 0.04) compared with the controls. Using a logistic regression model, the unadjusted odds ratio associated with any MYH mutation was 4.14 (P-value < 0.001); for mono-allelic carriers, it was 2.79 (P-value = 0.04). Adjusting for ethnic backgrounds, gender and age, the odds ratio associated with any disease-causing MYH mutation was 3.23 (P-value = 0.01); for mono-allelic carriers, it was 1.99 (P-value = 0.20). Overall, the results support previous studies suggesting that mono-allelic mutations of MYH constitute low-penetrance CRC-causing alleles. These data further support a model in which low-penetrance alleles are enriched in MMR gene mutation-negative CRC families.

Lack of germ-line mutations at the specific BRCA1-IRIS coding sequence in 114 Spanish high-risk breast/ovarian families.

A new BRCA1 locus product called BRCA1-IRIS has been identified recently. High-risk breast/ovarian families have not been screened for germ-line mutations at the specific BRCA1-IRIS coding sequence, as it was considered merely as part of BRCA1 intron 11. Here we report the first comprehensive screening of germ-line mutations in a cohort of 116 index cases from high-risk breast/ovarian families in which no germ-line mutation was identified in BRCA1 or BRCA2. We did not find germ-line mutations at the specific BRCA1-IRIS coding sequence in any sample. The only heterozygous patter identified by DGGE was caused by a C to A substitution in the non-coding 3' sequence, 123 bases downstream of the BRCA1-IRIS stop codon (IVS11+268C/A). The data indicates that it is probably a neutral change not associated with cancer risk. Our analysis suggests that the role of germ-line mutations at the specific BRCA1-IRIS sequence in breast cancer susceptibility, if any, is marginal and do not explain a significant fraction of high-risk breast/ovarian families, at least in the population analyzed.

Low prevalence of germline hMSH6 mutations in colorectal cancer families from Spain.

AIM: To investigate the prevalence and penetrance of hMSH6 mutations in Spanish HNPCC families that was negative for mutation in hMLH1 or hMSH2. METHODS: We used PCR-based DGGE assay and direct sequencing to screen for hMSH6 gene in 91 HNPCC families. RESULTS: we have identified 10 families with germ-line mutations in the DNA sequence. These mutations included two intronic variation, three missense mutation, one nonsense mutation, and four silent mutations. Among the 10 germ-line mutations identified in the Spanish cohort, 8 were novel, perhaps, suggesting different mutational spectra in the Spanish population. Detailed pedigrees were constructed for the three families with a possible pathogenic hMSH6 mutation. The two silent mutations H388H and L758L, detected in a person affected of colorectal cancer at age 29, produce loss of the wild-type allele in the tumor sample. Immunohistochemical analysis showed that expression of MSH6 protein was lost only in the tumors from the carriers of V878A and Q263X mutations. CONCLUSION: Altogether, our results indicate that disease-causing germ-line mutations of hMSH6 are very less frequent in Spanish HNPCC families.

The CHEK2 1100delC allele is not relevant for risk assessment in HNPCC and HBCC Spanish families.

The frame-shift mutation 1100delC in the cell-cycle-checkpoint kinase 2 gene (CHEK2) has been reported to be a low penetrance breast cancer gene in Northern European populations. However, the variant may be relevant for breast cancer risk in other populations, due to its low prevalence. Recent studies have proposed a role for the mutation in colorectal cancer, finding a strong association between the CHEK21100delC mutation and hereditary breast and colorectal cancer (HBCC). A previous study suggested that the CHEK21100delC variant was not of clinical relevance in Spanish breast/ovarian cancer families. Here, we demonstrate that this genetic variant is not of clinical relevance for HNPCC and HBCC Spanish families.

Association between BRCA1 mutations and ratio of female to male births in offspring of families with breast cancer, ovarian cancer, or both.

CONTEXT: Defects in X-chromosome inactivation distort sex ratio in mice. The BRCA1 gene is also involved in X-chromosome inactivation, suggesting the possibility that some sex-ratio distortion may be associated with BRCA1-related human cancer syndromes. OBJECTIVE: To determine whether BRCA1 mutations are associated with distortion of the sex ratio of births in families with breast cancer, ovarian cancer, or both. DESIGN AND SETTING: Analysis of germline mutations in participants from Spain who had been screened for BRCA between 1998 and 2002. PARTICIPANTS: Sixty-eight families with at least 3 breast cancer cases or ovarian cancer cases, or both types of cancer in 2 generations (germline mutations: BRCA1, n = 17; BRCA2, n = 15; and BRCA unrelated, n = 36). An average of 4 relatives per family were tested for the corresponding BRCA mutation. MAIN OUTCOME MEASURE: Male and female births registered in breast and/or ovarian pedigrees tested for the presence of BRCA1 and BRCA2 germline mutations. RESULTS: Of BRCA1-related breast and/or ovarian cancer pedigrees, there was a 2-fold excess of female births (218 female vs 109 male births). Of BRCA2-related or BRCA-unrelated breast and/or ovarian cancer pedigrees, there was not an excess of female births (175 female/150 male and 344 female/315 male, respectively). Of 327 BRCA1 births, 218 (67%) were female births compared with 54% among BRCA2 pedigrees (175/327; P<.001) and 52% among BRCA-unrelated pedigrees (344/659; P<.001). Female births increased in the offspring of BRCA1 carriers compared with BRCA2 carriers (67% vs 52%; P =.004). CONCLUSION: In these families with breast and/or ovarian cancer, mutations in BRCA1 but not BRCA2 were associated with a sex ratio skewed against male births.