Genomic rearrangements account for more than one-third of the BRCA1 mutations in northern Italian breast/ovarian cancer families

The recent identification of major genomic rearrangements in breast and breast/ovarian cancer families has widened the mutational spectrum of the BRCA1 gene, thus increasing the number of informative patients who can benefit from molecular screening. Numerous types of alterations have been identified in different populations with variable frequencies, probably due to both ethnic diversity and the technical approach employed. In fact, although several methods have been successfully used to detect large genomic deletions and insertions, most are laborious, time-consuming, and of variable sensitivity. In order to estimate the contribution of BRCA1 genomic rearrangements to breast/ovarian cancer predisposition in Italian families, we applied, for the first time as a diagnostic tool, the recently described multiplex ligation-dependent probe amplification (MLPA) methodology. Among the 37 hereditary breast/ovarian cancer (HBOC) families selected, all had a high prior probability of BRCA1 mutation, and 15 were previously shown to carry a mutation in either the BRCA2 (five families) or BRCA1 gene (10 families, including one genomic rearrangement). The application of BRCA1-MLPA to the remaining 22 uninformative families allowed the identification of five additional genomic rearrangements. Moreover, we observed that loss of constitutive heterozygosity of polymorphic markers in linkage disequilibrium is predictive of such BRCA1 alterations. By means of this approach, we demonstrate that BRCA1 genomic deletions account for more than one-third (6/15) of the pathogenic BRCA1 mutations in our series. We therefore propose to systematically include MLPA in the BRCA1 mutational analysis of breast/ovarian cancer families.     

SIX1 mutation screening in 247 branchio-oto-renal syndrome families: a recurrent missense mutation associated with BOR

Branchio-oto-renal syndrome (BOR) is a clinically heterogeneous autosomal dominant form of syndromic hearing loss characterized by variable hearing impairment, malformations of the pinnae, the presence of branchial arch remnants, and various renal abnormalities. Both EYA1 and SIX1 are expressed in developing otic, branchial and renal tissue. Consistent with this expression pattern, mutations in both genes cause BOR syndrome. Mutations in EYA1 are found in approximately 40% of patients with the BOR phenotype, however, the role of SIX1 is much lower. To date only three different SIX1 mutations have been described in BOR patients. The current screen of 247 BOR families detected five novel SIX1 mutations (c.50T>A, c.218A>C, c.317T>G, c.329G>A, c.334C>T) and one previously reported mutation (c.328C>T) seen in 5 unrelated families. All mutations are within the protein-binding Six domain. Phenotypic variability was high in these BOR families. Seven of the eight known SIX1 mutations are missense and the one in frame deletion is predicted to be functionally similar. The wide phenotypic variability precludes making genotype-phenotype correlations at this time.     

EYA1 nonsense mutation in a Japanese branchio-oto-renal syndrome family

Advances in molecular genetics have recently revealed that mutations in the EYA1 gene are responsible for branchio-oto-renal (BOR) syndrome in European and other populations. This is the first report confirming that an EYA1 gene mutation is also disease-causing in an Asian population. We have described one Japanese BOR syndrome family showing a novel mutation in exon 7 of the EYA1 gene. There was extensive variation of clinical phenotypes within this family. When the physician is confronted with a BOR family showing a wide variation in clinical expression, molecular genetic testing helps to achieve accurate diagnosis. Received: February 23, 1999 / Accepted: April 2, 1999     

Exclusion of the branchio-oto-renal syndrome locus (EYA1) from patients with branchio-oculo-facial syndrome

In addition to craniofacial, auricular, ophthalmologic, and oral anomalies, the distinctive phenotype of the branchio-oculo-facial (BOF) syndrome (MIM 113620) includes skin defects in the neck or infra/supra-auricular region. These unusual areas of thin, erythematous wrinkled skin differ from the discrete cervical pits, cysts, and fistulas of the branchio-oto-renal (BOR) syndrome (MIM 113650). Although the BOF and BOR syndromes are sufficiently distinctive that they should not be confused, both can be associated with nasolacrimal duct stenosis, deafness, prehelical pits, malformed pinna, and renal anomalies. Furthermore, a reported father and son [Legius et al., 1990, Clin Genet 37:347-500] had features of both conditions. It was not clear whether they had an atypical presentation of either BOR or BOF syndrome, or represented a private syndrome. In light of these issues, we selected the BOR locus (EYA1) as a possible gene mutation for the BOF syndrome. In five BOF patients, there were no mutations detected in the EYA1 gene, suggesting that it is not allelic to the BOR syndrome.     

Identification of three novel mutations in human EYA1 protein associated with branchio-oto-renal syndrome

The Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial clefts, preauricular sinuses, hearing loss, and renal anomalies. Recent studies have shown that mutations in EYA1 are associated with BOR. However, the underlying molecular mechanisms by which mutations in the EYA1 gene cause BOR syndrome are unknown. We have investigated 12 unrelated Caucasian families for mutations by heteroduplex analysis and direct sequencing of products from the polymerase chain reaction. In this study, we identified two novel frameshift deletions and a single base substitution that introduces a stop codon mutation in the C-terminal region of the EYA1 gene. No obvious relationships were observed between the nature of the mutations and the variable clinical features associated with BOR syndrome. Hum Mutat 11:443–449, 1998. © 1998 Wiley-Liss, Inc.     

Screening for large rearrangements of the BRCA1 gene in German breast or ovarian cancer families using semi-quantitative multiplex PCR method

Since the identification of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2, a large number of different germline mutations in both genes have been found by conventional PCR-based mutation detection methods. Complex germline rearrangements such as those reported in the BRCA1 gene are often not detectable by these standard diagnostic techniques. To detect large deletions or duplications encompassing one or more exons of the BRCA1 gene and in order to estimate the frequency of BRCA1 rearrangements in German breast or ovarian cancer families, a semi-quantitative multiplex PCR method was developed and applied to DNA samples of patients from families negatively tested for disease causing mutations in the BRCA1 and BRCA2 coding regions by direct sequencing. Out of 59 families analysed, one family was found to carry a rearrangement in the BRCA1 gene (duplication of exon 13). The results indicate that the semi-quantitative multiplex PCR method is useful for the detection of large rearrangements in the BRCA1 gene and therefore represents an additional valuable tool for mutation analysis of BRCA1 and BRCA2.     

Branchio-oto-renal syndrome: detection of EYA1 and SIX1 mutations in five out of six Danish families by combining linkage, MLPA and sequencing analyses

The branchio-oto-renal (BOR) syndrome is an autosomal-dominant disorder characterized by hearing loss, branchial and renal anomalies. BOR is genetically heterogeneous and caused by mutations in EYA1 (8q13.3), SIX1 (14q23.1), SIX5 (19q13.3) and in an unidentified gene on 1q31. We examined six Danish families with BOR syndrome by assessing linkage to BOR loci, by performing EYA1 multiplex ligation-dependent probe amplification (MLPA) analysis for deletions and duplications and by sequencing of EYA1, SIX1 and SIX5. We identified four EYA1 mutations (c.920delG, IVS10-1G>A, IVS12+4A>G and p.Y591X) and one SIX1 mutation (p.W122R), providing a molecular diagnosis in five out of the six families (83%). The present, yet preliminary, observation that renal and temporal bone malformations are less frequent in SIX1-related disease suggests a slightly different clinical profile compared to EYA1-related disease. Unidentified mutations impairing mRNA expression or further genetic heterogeneity may explain the lack of mutation finding in one family despite LOD score indications of EYA1 involvement.     

Exclusion of large deletions and other rearrangements in BRCA1 and BRCA2 in Finnish breast and ovarian cancer families

In the Finnish population, identified mutations in BRCA1 and BRCA2 account for a less than expected proportion of hereditary breast and ovarian cancer. All previous studies performed in our country have concentrated on finding germ-line mutations in the coding and splice-site regions of these two genes. Therefore, we wanted to use a different methodological approach and search for large genomic rearrangements, to exclude the possibility of biased BRCA1 and BRCA2 mutation spectra due to known limitations of the previously used PCR-based detection methods. Our results support earlier notions that other genes than BRCA1 and BRCA2 will explain a majority of the still unexplained cases of hereditary susceptibility to breast and ovarian cancer.     

Real-time PCR-based gene dosage assay for detecting BRCA1 rearrangements in breast-ovarian cancer families

BRCA1 and BRCA2 germline mutations, mainly point mutations and other small alterations, are responsible for most hereditary cases of breast-ovarian cancer. However, the observed frequency of BRCA1 alterations is lower than that predicted by linkage analysis. Several large BRCA1 rearrangements have been identified with a variety of technical approaches in some families. We have developed a gene dosage assay based on real-time quantitative PCR and used it to extensively analyze 91 French families of breast-ovarian cancer in which no BRCA1 or BRCA2 point mutations was identified. This gene dosage method calculates the copy number of each BRCA1 exon to readily detect one, two, and three or more copies of BRCA1 target exons. In the series of 91 families at high risk of carrying BRCA1 mutations, we detected seven large rearrangements of the BRCA1 gene by using this real-time PCR approach. This simple, rapid, and semiautomated real-time quantitative polymerase chain reaction (PCR) assay is a promising alternative technique to Southern blot, bar code analysis on combed DNA, quantitative multiplex PCR of short fluorescent fragments, and cDNA length analysis for the detection of large rearrangements. Therefore, this technique should be considered as a powerful diagnostic method for breast/ovarian cancer susceptibility in clinical and research genetic surveys.