[Show abstract][Hide abstract] ABSTRACT: Previously, we associated two mutations with the diseases X-linked congenital spinal muscular atrophy (SMAX2) and a syndromic form of X-linked mental retardation (MRXS10). These mutations are synonymous, meaning that they do not influence the amino acid sequence of the respective protein. They are localized in exon 15 of the gene ubiquitin activating enzyme 1 (UBA1) and exon 5 of the hydroxyacyl-coenzyme A dehydrogenase-II gene (HADH2). As quantitative PCR (qPCR) revealed, these mutations caused a strong decrease of the mRNA expression of the both genes. Herewith, we show that each mutation affects the functionality of exonic splicing enhancers (ESE) critical for proper splice site usage. Firstly, the wild type exonicUBA1 and HADH2 sequences appeared to promote pre-mRNA splicing within an ESE dependent reporter, whereas both mutant sequences exhibited an impaired recognition of the respective 5'splice site (5'ss). Secondly, RNA pull-down assays revealed reduced binding of the splicing regulatory SR-proteins SRSF2 and SRSF6 to the mutant RNA sequences of UBA1 and HADH2. Remarkably, we found that the mutant HADH2 pre-mRNA interacts with hnRNP F/H known to repress splicing from exonic positions. Therefore, we propose that functional ESEs in UBA1 and HADH2 are disabled by synonymous mutations and, in case of HADH2, an hnRNP F/H dependent exonic splicing silencer (ESS) is created. Our findings emphasize the substantial influence of synonymous mutations on gene expression and stress their impact on comprehensive genetic diagnostics and counseling.
[Show abstract][Hide abstract] ABSTRACT: Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.
[Show abstract][Hide abstract] ABSTRACT: Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia-like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.
[Show abstract][Hide abstract] ABSTRACT: In breast cancer, metastases are relatively widely distributed, with the most common sites being bone, regional lymph nodes, lung, liver, and brain. The detailed mechanism of organ-specific metastasis is poorly understood. In this study, we initiated a search for genes that are implicated in brain or bone metastasis of primary human breast cancer. We generated gene expression profiles of 18 brain and eight bone metastases derived from primary breast tumors. We identified 73 genes differentially expressed between brain and bone metastases. Visualization of the differential gene expression profiles by correspondence and cluster analyses shows that the metastases clearly separate into two distinct groups as an exact reflection of their site of metastasis. Moreover, the analysis of this gene set in primary breast tumors relapsing to either bone or brain allowed accurate categorization of the tumors according to their metastatic site. The identified genes may prove to be excellent markers to predict the site of metastasis in breast cancer patients and could lead to tailor-made therapy to an individual patient.
[Show abstract][Hide abstract] ABSTRACT: EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real-time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down-regulation in 59% and 61% of the analyzed tissues, respectively. This down-regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57-62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down-regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease-free (p = 0.037) and overall survival (p = 0.032), particularly in those node-positive patients who received adjuvant anthracycline-based chemotherapy, but not in those treated by either cyclophosphamide-methotrexate-5-fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer.
Full-text · Article · Apr 2009 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3-Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G-->T, p.Met539Ile; c.1639 A-->G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C-->T substitution (c.1731 C-->T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 x 10(-10), p = 0.001815). We also demonstrated that the synonymous C-->T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C-->T transitions might have the potential to affect gene expression.
Full-text · Article · Jan 2008 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: Analysis of allelic imbalance is of great importance for understanding tumorigenesis and the clinical management of malignant disease. Fluorescent-based capillary electrophoresis (CE) of highly polymorphic short tandem repeats (STRs) has become the main method used to detect the loss/gain of alleles. However, there is continued interest in the development of techniques that require no fluorescence and allow the rapid analysis of individual samples. One promising alternative is ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), which is widely available because of its use in denaturing HPLC. Its applicability in combination with ultraviolet (UV) absorbance detection to the efficient separation of di- and tetranucleotide repeats on the short arm of chromosome 11 was tested using 25 matched pairs of normal and ovarian cancer tissues. Loss of heterozygosity (LOH) could be readily identified for all 13 loci tested, based on changes in the ratios between either the alleles or homo- and heteroduplex signals. However, discrimination between noninformative homo- or hemizygous and heterozygous samples was difficult or impossible when HPLC failed to resolve the alleles. Hyphenation of HPLC with electrospray ionization (ESI) quadrupole ion trap (IT) mass spectrometry (MS) not only allowed the identification of coeluting alleles, but also the reliable detection of a 40% reduction of one allele. The size range of DNA fragments amenable to mass spectrometric analysis was effectively tripled to >300 bp by the use of a linear IT and a Taq DNA polymerase cocktail lacking detergents that otherwise adversely affect ESI.
[Show abstract][Hide abstract] ABSTRACT: Recently, we defined a new syndromic form of X-linked mental retardation in a 4-generation family with a unique clinical phenotype characterized by mild mental retardation, choreoathetosis, and abnormal behavior (MRXS10). Linkage analysis in this family revealed a candidate region of 13.4 Mb between markers DXS1201 and DXS991 on Xp11; therefore, mutation analysis was performed by direct sequencing in most of the 135 annotated genes located in the region. The gene (HADH2) encoding L-3-hydroxyacyl-CoA dehydrogenase II displayed a sequence alteration (c.574 C-->A; p.R192R) in all patients and carrier females that was absent in unaffected male family members and could not be found in 2,500 control X chromosomes, including in those of 500 healthy males. The silent C-->A substitution is located in exon 5 and was shown by western blot to reduce the amount of HADH2 protein by 60%-70% in the patient. Quantitative in vivo and in vitro expression studies revealed a ratio of splicing transcript amounts different from those normally seen in controls. Apparently, the reduced expression of the wild-type fragment, which results in the decreased protein expression, rather than the increased amount of aberrant splicing fragments of the HADH2 gene, is pathogenic. Our data therefore strongly suggest that reduced expression of the HADH2 protein causes MRXS10, a phenotype different from that caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, which is a neurodegenerative disorder caused by missense mutations in this multifunctional protein.
Full-text · Article · Mar 2007 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: X-linked mental retardation has been traditionally divided into syndromic (S-XLMR) and non-syndromic forms (NS-XLMR), although the borderlines between these phenotypes begin to vanish and mutations in a single gene, for example PQBP1, can cause S-XLMR as well as NS-XLMR. Here, we report two maternal cousins with an apparently X-linked phenotype of mental retardation (MR), microphthalmia, choroid coloboma, microcephaly, renal hypoplasia, and spastic paraplegia. By multipoint linkage analysis with markers spanning the entire X-chromosome we mapped the disease locus to a 28-Mb interval between Xp11.4 and Xq12, including the BCOR gene. A missense mutation in BCOR was described in a family with Lenz microphthalmia syndrome, a phenotype showing substantial overlapping features with that described in the two cousins. However, no mutation in the BCOR gene was found in both patients. Subsequent mutation analysis of PQBP1, located within the delineated linkage interval in Xp11.23, revealed a 2-bp deletion, c.461_462delAG, that cosegregated with the disease. Notably, the same mutation is associated with the Hamel cerebropalatocardiac syndrome, another form of S-XLMR. Haplotype analysis suggests a germline mosaicism of the 2-bp deletion in the maternal grandmother of both affected individuals. In summary, our findings demonstrate for the first time that mutations in PQBP1 are associated with an S-XLMR phenotype including microphthalmia, thereby further extending the clinical spectrum of phenotypes associated with PQBP1 mutations.
Full-text · Article · Feb 2007 · European Journal of HumanGenetics
[Show abstract][Hide abstract] ABSTRACT: To define non-bacterial osteitis (NBO) as a clinical entity possibly associated with autoimmune manifestations. Patients with sterile osteitis were analysed to develop diagnostic criteria.
A total of 89 patients with non-bacterial inflammatory bone lesions were observed for a median of 49 months. History, diagnostic imaging, laboratory and histological data were obtained. Mutation analysis in the genes PSTPIP1 and PSTPIP2 was performed.
Patients had an onset of disease at a median age of 10 yrs [interquartile range (IQR) 7.5-12] and suffered a median period of 21 (IQR 9-52) months with a median of three foci per patient. Twenty percent of all the patients demonstrated associated autoimmune disorders, particularly of the skin and bowel. The majority of bone lesions were located in the vertebrae and metaphyses. Slight-to-moderate elevation of inflammation values were found in all the patients and antinuclear antibodies were elevated in 30%. Non-steroidal anti-inflammatory drugs (NSAIDs) were effective in 85% of the patients. HLA-B27 and Human Leukocyte Antigen-DR (HLA-DR)-classification did not differ from the general population. Autoimmune diseases in 40% of all the families, multiply affected family members, linkage to 18q21 and mouse models strongly indicate a genetic basis for NBO. We observed three different courses of disease regarding the duration of complaints, rate of complications and associated autoimmune manifestations leading to a new classification of NBO.
Clinical analysis of our cohort leads us to define NBO as a distinct disease entity with three clinical presentations: acute NBO, chronic recurrent multifocal osteomyelitis or persistent chronic NBO. Diagnostic criteria were proposed to differentiate NBO from diseases with similar clinical presentation.
[Show abstract][Hide abstract] ABSTRACT: Careful manual annotation of the human reference sequence provides a solid basis for the identification of disease-associated genes. Toward this end, we focused on a medically relevant 2.6-Mb region of the human chromosome Xp11.4 between markers DXS9851 and DXS9751 and identified 16 transcription units according to the Vertebrate Genome Annotation (Vega) rules. In order to validate these annotations, we performed a comprehensive RT-PCR expression analysis and a human-mouse comparison. This revealed, despite the high overall genomic conservation of the region, remarkable differences of the gene content between human and mouse. Whereas 12 of 16 annotations were confirmed by RT-PCR in human tissues, for only seven genes mouse orthologs could be identified and found to be expressed. This indicates that a comprehensive and experimentally supported annotation effort of the human genome simultaneously highlights regions with striking differences in gene organization to other species and may indicate evolutionary events specific to the human lineage demanding further functional analyses.
No preview · Article · Jan 2006 · Mammalian Genome
[Show abstract][Hide abstract] ABSTRACT: The renin-angiotensin system (RAS) is essential for blood pressure control and water-electrolyte balance. Until the discovery of the renin receptor, renin was believed to be mainly a circulating enzyme with a unique function, the cleavage of angiotensinogen. We report a unique mutation in the renin receptor gene (ATP6AP2) present in patients with X-linked mental retardation and epilepsy (OMIM no. 300423), but absent in 1200 control X-chromosomes. A silent mutation (c.321C>T, p.D107D) residing in a putative exonic splicing enhancer site resulted in inefficient inclusion of exon 4 in 50% of renin receptor mRNA, as demonstrated by quantitative RT-PCR. Analysis of membrane associated-receptor molecular forms showed the presence of full-length and truncated proteins in the patient. Functional analysis demonstrated that the mutated receptor could bind renin and increase renin catalytic activity, similar to the wild-type receptor, but resulted in a modest and reproducible impairment of ERK1/2 activation. Thus, our findings confirm the importance of the RAS in cognitive processes and indicate a novel specific role for the renin receptor in cognitive functions and brain development.
Full-text · Article · May 2005 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
[Show abstract][Hide abstract] ABSTRACT: Mental retardation is the most frequent cause of serious handicap in children and young adults. The underlying causes of this heterogeneous condition are both acquired and genetically based. A recently performed refinement of the linkage interval in a large Belgian family with mild to severe non-syndromic X linked mental retardation, classified as MRX9, revealed a candidate region of 11.3 Mb between markers DXS228 and DXS1204 on the short arm of the X chromosome. In order to identify the underlying disease gene in the MRX9 family, we established a gene catalogue for the candidate region and performed comprehensive mutation analysis by direct sequencing. A human homologue of the bacterial 23S rRNA methyltransferase Fstj, the FTSJ1 gene, is located within this region and displayed a sequence alteration in the conserved acceptor splice site of intron 3 (IVS3-2A>G) in all tested patients and carrier females of this family. In contrast, it was absent in all unaffected male family members tested. The mutation results in skipping of exon 4 and introduces a premature stop codon in exon 5, probably leading to a severely truncated protein. Our finding indicates that a protein, possibly associated with ribosomal stability, can be linked to X linked mental retardation (XLMR).
Full-text · Article · Sep 2004 · Journal of Medical Genetics