Parallel deletion and duplication at 7q11.23 in a silent carrier for two reciprocal syndromic disorders.

Partial deletions at chromosome 7q11.23 are causative for the autosomal-dominant Williams-Beuren syndrome (WBS), whereas the partial duplication of this region leads to the 7q11.23 duplication syndrome. Both syndromes are highly penetrant and occur with a frequency of 1:7500-10,000 (WBS) and 1:13,000-20,000 (7q11.23 duplication syndrome). They are associated with multiple organ defects, intellectual disability, and typical facial dysmorphisms showing broad phenotypic variability. The 7q11.23 region is susceptible to chromosomal rearrangements due to flanking segmental duplications and regions of long repetitive DNA segments. Here, we report on a family with two children affected by WBS and clinically unaffected parents. Interestingly, metaphase fluorescence in situ hybridization (FISH) revealed a deletion on 7q11.23 in the father. Intensive genetic testing, using interphase FISH, whole genome sequencing and optical genome mapping led to the confirmation of a 1.5 Mb deletion at one 7q11.23 allele and the identification of a reciprocal 1.8 Mb duplication at the other allele. This finding is highly important regarding genetic counseling in this family. The father is a silent carrier for two syndromic disorders, thus his risk to transmit a disease-causing allele is 100%. To the best of our knowledge we, here, report on the first case in which the phenotype of a microdeletion/microduplication syndrome was compensated by its reciprocal counterpart.

Cancer incidence and spectrum among children with genetically confirmed Beckwith-Wiedemann spectrum in Germany: a retrospective cohort study.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a cancer predisposition syndrome caused by defects on chromosome 11p15.5. The quantitative cancer risks in BWS patients depend on the underlying (epi)genotype but have not yet been assessed in a population-based manner. METHODS: We identified a group of 321 individuals with a molecularly confirmed diagnosis of BWS and analysed the cancer incidence up to age 15 years and cancer spectrum by matching their data with the German Childhood Cancer Registry. RESULTS: We observed 13 cases of cancer in the entire BWS cohort vs 0.4 expected. This corresponds to a 33-fold increased risk (standardised incidence ratio (SIR) = 32.6; 95% confidence interval = 17.3-55.7). The specific cancers included hepatoblastoma (n = 6); nephroblastoma (n = 4); astrocytoma (n = 1); neuroblastoma (n = 1) and adrenocortical carcinoma (n = 1). The cancer SIR was highest in patients with a paternal uniparental disomy of 11p15.5 (UPDpat). A high cancer risk remained when cases of cancer diagnosed prior to the BWS diagnosis were excluded. CONCLUSIONS: This study confirms an increased cancer risk in children with BWS. Our findings suggest that the highest cancer risk is associated with UPDpat. We were unable to confirm an excessive cancer risk in patients with IC1 gain of methylation (IC1-GOM) and this finding requires further investigation.

Clinical and molecular characterization of the BRCA2 p.Asn3124Ile variant reveals substantial evidence for pathogenic significance.

Variants of uncertain clinical significance (VUS) in the high-penetrance breast cancer susceptibility genes BRCA1 and BRCA2 represent a major obstacle in genetic counseling of high-risk breast cancer families. We analyzed a missense VUS located in BRCA2 (p.Asn3124Ile; HGVS: BRCA2 c.9371A > T) present in seven independent high-risk breast cancer families that were counseled and genetically tested in South-West Germany. The VUS was identified by DNA sequencing. We analyzed co-occurrence with deleterious BRCA1/2 mutations, segregation, evolutionary conservation, in silico impact prediction, and prevalence in the general population. All carriers of the VUS suffered from breast or ovarian cancer. In two families, an additional high burden of other cancers such as pancreatic, prostate, and gastric cancers was reported, one further family included two cases of male breast cancer. The VUS did not co-occur with deleterious BRCA1/2 mutations and segregated in two affected individuals of one family. In contrast to the 7/1,347 (0,5 %) tested high-risk BC families without clearly pathogenic mutations in BRCA1/2, none of 3,126 healthy population controls sharing the same ethnic and geographical background were found to carry this VUS (p = 0.0002). In-silico prediction revealed strong evolutionary conservation of the asparagine residue, residing in the C-terminal oligonucleotide-binding-fold-3 region, and a most likely damaging impact of this exchange on the protein structure. The BRCA2 p.Asn3124Ile (BRCA2 c.9371A > T) variant is a rare mutation with a damaging effect on the BRCA2 protein that is strongly associated with familial breast and ovarian cancer risk, indicating its most likely pathogenic nature and clinical relevance.

A palindrome-mediated recurrent translocation with 3:1 meiotic nondisjunction: the t(8;22)(q24.13;q11.21).

Palindrome-mediated genomic instability has been associated with chromosomal translocations, including the recurrent t(11;22)(q23;q11). We report a syndrome characterized by extremity anomalies, mild dysmorphia, and intellectual impairment caused by 3:1 meiotic segregation of a previously unrecognized recurrent palindrome-mediated rearrangement, the t(8;22)(q24.13;q11.21). There are at least ten prior reports of this translocation, and nearly identical PATRR8 and PATRR22 breakpoints were validated in several of these published cases. PCR analysis of sperm DNA from healthy males indicates that the t(8;22) arises de novo during gametogenesis in some, but not all, individuals. Furthermore, demonstration that de novo PATRR8-to-PATRR11 translocations occur in sperm suggests that palindrome-mediated translocation is a universal mechanism producing chromosomal rearrangements.

Type-dependent integration frequency of human papillomavirus genomes in cervical lesions.

Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.

Molecular genetic analysis of NBS1 in German melanoma patients.

The aim of this study was to investigate the role of NBS1 in the pathogenesis of malignant melanoma of the skin. To exclude the common 657del5 founder mutation, a total of 376 melanoma patients from Southern Germany were analyzed for sequence alterations in exon 6 of NBS1 by direct sequencing. Analyses revealed one 657del5 mutation and three nonsynonymous sequence variations in exon 6 of NBS1 (V210F, R215W, and F222L). Analysis of an additional sample of 629 melanoma patients and 604 controls revealed no F222L mutation, indicating that this newly identified sequence alteration is not a common polymorphism. In a case-control association study including 632 melanoma patients and 615 cancer-free control participants from Southern Germany, three publicly known single nucleotide polymorphisms located in the NBS1 gene region were analyzed. No significant associations between single nucleotide polymorphisms (rs9995, rs867185 and rs1063045) or referring calculated haplotypes and melanoma risk were identified. These results suggest that NBS1 does not play a major role in predisposition to melanoma in the Southern German population but that alterations of this gene might contribute to the risk of this cancer.

Trisomy 8q and partial trisomy 22 in a 43-year-old man with moderate intellectual disability, epilepsy and large cell non-Hodgkin lymphoma.

Partial trisomies are chromosome abnormalities resulting in a broad range of malformations depending on the size and location of the chromosomal rearrangement. Whereas diagnosis of these syndromes is usually made in early childhood, few descriptions exist about the clinical picture in adulthood. We report on a patient diagnosed at the age of 43 years with a 47,XY,+der(22)t(8;22)(q24.13;q11.21) karyotype and predominant clinical features of trisomy 8q. To our knowledge, this is the oldest patient described with a partial trisomy 8. The patient presented with moderate intellectual disability, a past history of epilepsy and facial anomalies. In addition, a large cell non-Hodgkin lymphoma was diagnosed in adulthood. Detailed breakpoint mapping by single nucleotide polymorphism (SNP) arrays showed that the derivative chromosome contains a full-length copy of the C-MYC oncogene. Given that trisomy 8q is the most frequent secondary chromosomal abnormality in hematological diseases, the possibility of a genetic predisposition for these disorders in patients with 8q duplication is raised.

Clonal history of papillomavirus-induced dysplasia in the female lower genital tract.

BACKGROUND: Dysplastic lesions of the vagina or the vulva often occur in women who have a previous history of cervical dysplasia. Most lesions in the female lower genital tract are induced by infections with high-risk oncogenic human papillomaviruses (HR-HPVs), including HPV16 and HPV18. HR-HPV genomes frequently integrate into host cell chromosomes at random sites. We analyzed viral integration sites in multiple metachronous lesions of the lower genital tract from women previously treated for HR-HPV-positive cervical dysplasia or cancer to determine whether the metachronous lesions emerged from a single common preexisting dysplastic cell clone or as consequence of independent HR-HPV infection events in the female lower genital tract. METHODS: From among 1500 patients with anogenital lesions, seven patients with high-grade vaginal or vulvar lesions and with a previous history of cervical disease (five with prior high-grade cervical dysplasia and two with a history of cervical cancer) were included in this study. Integration sites of HPV16 or HPV18 in vaginal or vulvar lesions were mapped by an adaptor ligation polymerase chain reaction (PCR) method. The sequence information was used to design an integrate-specific PCR assay that was applied to DNA extracted from archival paraffin-embedded material derived from biopsy samples of cervical lesions. RESULTS: Identical HPV DNA integration loci were found in vaginal or vulvar and cervical samples of all lesions available for four of the five patients with a prior history of high-grade cervical dysplasia and for both patients with a history of cervical cancer. CONCLUSIONS: These data indicate that high-grade dysplastic lesions in the female lower genital tract may emerge primarily as monoclonal lesions from a transformed cell population derived from the uterine cervix.

Induction of antibodies to the PreS region of surface antigens of woodchuck hepatitis virus (WHV) in chronic carrier woodchucks by immunizations with WHV surface antigens.

BACKGROUND/AIMS: One goal of therapeutic vaccinations against chronic hepatitis B virus infection is to stimulate the B-cell responses to viral surface antigens in chronic carriers. Here we investigated the induction of antibody responses to hepadnaviral surface antigens in the woodchuck model, with emphasis on the vaccination of woodchucks chronically infected with woodchuck hepatitis virus (WHV). METHODS: Naive and chronically WHV-infected woodchucks were immunized with plasma-derived WHV surface antigens (p-WHsAg) containing the S and PreS sequences. Antibody responses to WHsAg and the WHV PreS region and viral load in immunized woodchucks were monitored. RESULTS: After repeated immunizations with WHsAg, 17 of 18 chronic WHV carriers developed a persistent antibody response to WHsAg. These antibodies were mainly directed to epitopes within the PreS region and detectable by Western blotting. However, neither WHV DNA nor WHsAg concentrations in these woodchucks changed significantly by immunizations and during the follow up. Sequence analysis of WHV genomes showed that no WHV mutants emerged after the induction of anti-WHs/anti-WHpreS antibodies. No immunopathological changes in livers of immunized animals were recognized thus far. CONCLUSIONS: Our study demonstrated that the immunological unresponsiveness of chronically WHV-infected woodchucks to WHsAg can be partially overcome by repeated immunizations with WHsAg.

Twenty-three novel BRCA1 and BRCA2 sequence alterations in breast and/or ovarian cancer families in Southern Germany.

BRCA1 and BRCA2 germline mutations cause a substantially increased life time risk of both breast and ovarian cancer. Mutational screening of these genes by means of Denaturing High Performance Liquid Chromatography (DHPLC) in breast and/or ovarian cancer-prone families from Southern Germany revealed 15 novel BRCA1 and 8 novel BRCA2 sequence variants. Predictions on the BRCA1/BRCA2 protein functions lead to the identification of 11 novel deleterious cancer predisposing mutations. Mutation types and their functional relevances are discussed. Our data contribute to phenotype-genotype correlation studies and to the characterisation of the mutation spectrum of BRCA1/BRCA2.

Exclusion of BRAFV599E as a melanoma susceptibility mutation.

Very recently, BRAF mutations were found in about 2/3 of malignant melanomas and at lower frequencies in other human cancers. The BRAF gene codes for a protein in the mitogen-activated protein kinase (MAPK) pathway. All mutations identified to date are within the kinase domain, with a single missense mutation (V599E) accounting for 80%. We investigated the hypothesis that this common somatic BRAF mutation (V599E) would contribute to melanoma predisposition in familial and polygenic malignant melanoma if occurring as a germ-line mutation. We performed comprehensive mutational screening of exon 15 of BRAF using DHPLC (denaturing high-performance liquid chromatography) and DNA sequencing techniques. No V599E mutation could be detected in 172 melanoma patients comprising 46 familial cases, 21 multiple melanoma patients and 106 cases with at least one first-degree relative suffering from other cancers. We therefore conclude that the common somatic BRAF mutation V599E does not contribute to polygenic and familial melanoma predisposition.

Identification and characterization of UEV3, a human cDNA with similarities to inactive E2 ubiquitin-conjugating enzymes.

Recent studies have shown that ubiquitination is an essential factor in endosomal sorting and virus assembly. The human TSG101 gene has been demonstrated to belong to a group of genes coding for apparently inactive E2 ubiquitin-conjugating enzymes, which exert regulatory effects on E2 activity in cellular ubiquitination processes. In this study, a novel human cDNA (UEV3) encoding a putative protein of 379 amino acids was isolated from a human placenta library that may represent a partial paralogue of human TSG101. The predicted protein contains an N-terminal domain homologous to the catalytic domain of ubiquitin-conjugating enzymes (Ubc), which is fused to a sequence showing significant homology to members of the lactate dehydrogenase protein family. The UEV3 gene is located on chromosome 11 closely adjacent to TSG101 and LDH-C. Northern blot and UEV3-specific reverse transcription/polymerase chain reaction (RT/PCR) analyses of various colon carcinoma cell lines as well as both normal and tumor samples from colon revealed an expression of the UEV3 cDNA in all tested samples.

Characterization of viral-cellular fusion transcripts in a large series of HPV16 and 18 positive anogenital lesions.

Persistent high risk type human papillomavirus (HR-HPVs) infections induce dysplasia or cancer of the anogenital tract, most notably of the uterine cervix. The viral genome usually persists and replicates as an episomal molecule in early dysplasia, whereas in advanced dysplasia or cervical cancer HPV genomes are frequently integrated into the chromosomal DNA of the host cell. Previous studies suggested that modification of critical cellular sequences by integration of HPV genomes might significantly contribute to the neoplastic transformation of anogenital epithelia (insertional mutagenesis). This prompted us to characterize the integration loci of high risk HPV genomes in a large set of genital lesions. We amplified E6/E7 oncogene transcripts derived from integrated HPV16 and HPV18 genomes and characterized in detail the co-transcribed cellular sequences of 64 primary genital lesions and five cervical cancer cell lines. Database analyses of the cellular parts of these fusion transcripts revealed 51 different integration loci, including 26 transcribed genes (14 known genes, 12 EST sequences with unknown gene function). Seventeen sequences showed similarity to repetitive elements, and 26 sequences did not show any database match other than genomic sequence. Chromosomal integration loci were distributed over almost all human chromosomes. Although we found HPV sequences integrated into cancer related genes and close to fragile sites, no preferential site or integration motif could be identified. These data demonstrate that target directed insertional mutagenesis might occur in few HPV-induced anogenital lesions, however, it is rather the exception than the rule.