Branchio-otic syndrome caused by a genomic rearrangement: clinical findings and molecular cytogenetic studies in a patient with a pericentric inversion of chromosome 8.

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype.

A family with an inverted tandem duplication 5q22.1q23.2.

Here, we report a 3-year-old boy with short stature, developmental delay and mild facial dysmorphic signs. Karyotype analysis and array-CGH revealed a pure duplication 5q22.1q23.2 with a length of 14.25 Mb. As demonstrated by multicolor-fluorescence in situ hybridization, the duplicated segment was orientated in an inverted tandem manner. One of the 2 older half-brothers of the index patient was intellectually disabled and showed short stature as well. The mother of the siblings was only 149 cm in height. The affected half-brother as well as the mother of the siblings were tested positive for the same duplication. Duplications of the long arm of chromosome 5 are rare. There are 16 reported cases of different 5q segments with a pure duplication and no additional chromosomal imbalance. In order to refine the 5q-duplication phenotype, reported cases were recently classified in 3 groups on the basis of clinical findings and the involved chromosome segments. However, our case does not fit in any of these groups but is placed in the interjacent chromosomal area between 2 of these groups. Overall, this is the second reported family with a duplication of 5q22.1q23.2 and both families share phenotypic features like short stature, facial dysmorphic signs and speech delay. The reported family provides further information for delineating phenotype-genotype correlations of pure duplications of the 5q region.

Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants?

Array comparative genomic hybridization (array CGH) is now widely adopted as a first-tier clinical diagnostic test in individuals with unexplained developmental delay/intellectual disability (DD/ID) and congenital anomalies. Our study aimed at enlarging the phenotypic spectrum associated with clinically relevant copy number variants (CNVs) as well as delineating clinical criteria, which may help separating patients with pathogenic CNVs from those without pathogenic CNVs. We performed a retrospective review of clinical and array CGH data of 342 children with unexplained DD/ID. The phenotypic features of patients with clinically significant CNV were compared with those without pathogenic CNVs. Array CGH detected pathogenic CNVs in 13.2% of the patients. Congenital anomalies, especially heart defects, as well as primary microcephaly, short stature and failure to thrive were clearly more frequent in children with pathogenic CNVs compared with children with normal array CGH results. Thus, we assume that in patients with unexplained DD/ID, array CGH will more probably detect a significant CNV if any of these features is part of the patient's phenotype.

CHD7 mutations causing CHARGE syndrome are predominantly of paternal origin.

CHARGE (coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, ear anomalies and deafness) syndrome is a congenital malformation syndrome caused by mutations in the CHD7 gene in approximately 2/3 of cases. In the vast majority of cases, CHARGE syndrome is sporadic. There are only a few reports of parent-to-child transmission and somatic or gonadal mosaicism. To determine the parental origin of CHD7 mutations in sporadic CHARGE syndrome, we screened 30 families for informative exonic or intronic polymorphisms located near the detected CHD7 mutation. An informative polymorphism could be identified in 13 out of 30 families. Linkage analysis was performed between the CHD7 mutation and the polymorphism in the child. In 12 out of 13 families, the mutation affected the paternal allele (92.3%). In our cohort, the mean paternal age at birth was 32.92 years. Comparing the age of fathers of an affected CHARGE patient with the paternal age of the German population in general, we could not observe any paternal age effect. Taken together, we show in this study that de novo CHD7 mutations occur predominantly in the male germ line.

Targeted disruption of the mouse Npal3 gene leads to deficits in behavior, increased IgE levels, and impaired lung function.

The non-imprinted in Prader-Willi/Angelman syndrome (NIPA) proteins are highly conserved receptors or transporters. Translocation of NIPA genes were found in patients with Prader-Willi syndrome, and loss-of-function of the NIPA1 gene was identified in hereditary spastic paraplegia. The family of NIPA-like domain containing (NPAL) proteins is closely related to the NIPA proteins, but to date nothing is known about their function. Here, we could demonstrate that both human NPAL3 and mouse NPAL3 are ubiquitously expressed and encode highly conserved proteins. To further elucidate the function of the Npal3 gene, knockout (Npal3(-/-)) mice were generated. Intensive phenotypic analyses revealed that disruption of the Npal3 gene results in a pleiotropic phenotype. The function of the nervous system was impaired in both mutant males and females which could be demonstrated in behavioral tests. In addition, in NPAL3 mutants the number of NK cells was decreased and changes in IgM, IgG(2), and IgA were observed, indicating that the immune system is also affected. Interestingly, increased IgE levels as well as impaired lung functions were observed in mutant males but not in mutant females. It should be noted that the human Npal3 gene is located at 1p36.12-->p35.1, and atopic diseases were previously linked to this genomic region. Thus, the Npal3(-/-) mice could serve as a valuable model system for studying atopic diseases.

Proven germline mosaicism in a father of two children with CHARGE syndrome.

CHARGE syndrome is an autosomal dominant malformation syndrome caused by mutations in the CHD7 gene. The majority of cases are sporadic and only few familial cases have been reported. In these families, mosaicism in one parent, as well as parent- to-child transmission of a CHD7 mutation, has been described. In some further cases, germline mosaicism has been suggested. Here, we report the first case in which germline mosaicism could be demonstrated in a father of two affected children with CHARGE syndrome. The truncating mutation c.7302dupA in exon 34 of the CHD7 gene was found in both affected children but was not detected in parental lymphocytes. However, in DNA extracted from the father's spermatozoa, the c.7302dupA mutation could be identified. Furthermore, mutation analysis of DNA isolated from 59 single spermatozoa revealed that the c.7302dupA mutation occurs in 16 spermatozoa, confirming germline mosaicism in the father of the affected children. This result has a high impact for genetic counselling of the family and for their recurrence risk in further pregnancies.

PHF5A represents a bridge protein between splicing proteins and ATP-dependent helicases and is differentially expressed during mouse spermatogenesis.

PHF5A is a highly conserved protein from yeast to man, and based on studies in yeast, it was suggested that the homologous protein RDS3P in S. cerevisiae takes part in the organization of U2 snRNP particles. By using the yeast two-hybrid assay we could demonstrate that PHF5A interacted both with ATP-dependent helicases EP400 and DDX1 and with arginine-serine (RS)-rich domains of splicing factors U2AF1 and SFRS5 in mouse. Furthermore, domain interaction studies revealed that PHF5A interaction with EP400 and DDX1 is restricted to the N-terminal part of PHF5A, whereas the C-terminal region of PHF5A was found to be responsible for the association with U2AF1 and SFRS5. By using the yeast three-hybrid assay, we could further show that both EP400 and DDX1 interacted only indirectly with U2AF1 and SFRS5 proteins via the bridge protein PHF5A. The subcellular localization of a PHF5A-GFP fusion protein was predominantly observed in the nucleus and, in addition, PHF5A co-localized with both U2AF1 and SFRS5 proteins in nuclear speckles of NIH3T3 cells. Moreover, expression analyses demonstrated that PHF5A and U2AF1 gene expression coincided in spermatocytes during murine spermatogenesis and interaction between these proteins was also detectable in the spermatocyte-specific cell line GC-4spc by using in vivo co-immunoprecipitation studies. Taken together, our results indicate that PHF5A resembles a protein which interacts with splicing factors U2AF1 and SFRS5 and helicases EP400 and DDX1 and functions as a bridge protein between these proteins.

The putative peroxisomal gene Pxt1 is exclusively expressed in the testis.

Genes reported to be crucial for spermatogenesis are often exclusively expressed in the testis. We have identified a novel male germ cell-specific expressed gene named peroxisomal testis specific 1 (Pxt1) with expression starting at the spermatocyte stage during mouse spermatogenesis. The putative amino acid sequence encoded by the cDNA of the Pxt1 gene contains a conserved Asn-His-Leu (NHL)-motif at its C-terminal end, which is characteristic for peroxisomal proteins. Pxt1-EGFP fusion protein is co-localized with known peroxisomal marker proteins in transfected NIH3T3 cells. In addition, we could demonstrate that the peroxisomal targeting signal NHL is functional and responsible for the correct subcellular localization of the Pxt1-EGFP fusion protein. In male germ cells peroxisomes were reported only in spermatogonia. The Pxt1 gene is so far the first gene coding for a putative peroxisomal protein which is expressed in later steps of spermatogenesis, namely in pachytene spermatocytes.

Onconase, an anti-tumor ribonuclease suppresses intracellular oxidative stress.

Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.

[Molecular principles of alternative treatment approaches for hormone-refractory prostate cancer]. / Molekulare Grundlagen alternativer Therapieansätze für das hormonrefraktäre Prostatakarzinom.

Prostate cancer is more frequently diagnosed in men from Western countries than from Asian societies. Therefore, nutritional factors such as phyto-oestrogens from soya are considered to cause this prostate cancer prevention effect. As there is no curative therapy for hormone-refractory prostate cancer, new strategies are in demand which might include phyto-oestrogens or inhibitors of histone deacetylases. Both approaches have in common the potential to reduce the aberrant androgen receptor and IGF receptor signalling. Furthermore, invasiveness and acquired survival strategies of tumours can be diminished. Reduced tumour cell proliferation and PSA secretion coincide with altered gene expression in the aforementioned processes. In addition, selective knock-down of genes by RNA interference afforded functional analyses regarding impact and succession of expression events involved in the beneficial effects caused by phyto-oestrogens and histone deacetylase inhibitors.

The genes for human brain factor 1 and 2, members of the fork head gene family, are clustered on chromosome 14q.

Brain factor-1 (BF-1) is a member of the fork head gene family which shows expression restricted to the neurons of the developing telencephalon in rodents and man. We have isolated a second human gene (HBF-2), which is also strongly expressed in embryonic brain and has very high homology to both the rat and human brain factor-1 genes and the retroviral oncogene qin. The HBF-2 cDNA was isolated from a human fetal brain expression library and contains a putative open reading frame of 479 amino acids. The HBF-2 gene is strongly expressed in fetal brain and also with lower levels of expression in several adult tissues. At the genomic level the gene for HBF-1 contains an 500 bp intron situated between the DNA binding domain II and the fork head domain while that of HBF-2 is intronless. The two genes are clustered on human chromosome 14q11-13.

Potential triple helix-mediated inhibition of IGF-I gene expression significantly reduces tumorigenicity of glioblastoma in an animal model.

Oligonucleotide-directed triple helix formation is a powerful approach to block transcription of specific genes. Although the oligonucleotide triplex approach is efficient for inhibiting gene expression in cultured cells, suppression is transient. We developed an approach which inhibits insulin-like growth factor-I (IGF-I) expression following stable transfection of C6 rat glioblastoma cells with a plasmid from which an RNA is transcribed that codes for the third strand of a potential triple helix. We tested the ability of this expression vector to inhibit IGF-I gene expression in vitro as well as tumorigenesis in an animal. A dramatic reduction of IGF-I RNA and protein levels in cultured cells occurred following transfection of rat C6 cells with a eukaryotic expression plasmid encoding the oligopurine variant of the triple helix but not the oligopyrimidine or a control sequence. The cells transfected with the oligopurine variant displayed morphological changes, upregulation of major histocompatibility complex I, and increased expression of protease nexin I. Dramatic inhibition of tumor growth occurred in nude mice following injection of transfected C6 cells. To our knowledge, this is the first example of tumor growth inhibition in an animal model employing a triple helix approach.

Supernumerary small marker chromosome (SMC) and uniparental disomy 22 in a child with confined placental mosaicism of trisomy 22: trisomy rescue due to marker chromosome formation.

Trisomy rescue is one of various proposed mechanisms in formation of supernumerary small marker chromosomes (SMC) and uniparental disomy (UPD). In the present report a small de novo marker chromosome derived from chromosome 14 or 22 was diagnosed at prenatal diagnosis due to maternal age. Follow up investigations at birth revealed mosaicism 47,XX,+mar/46,XX. Using FISH, the marker was positive for the probe D14/22Z1, but negative for the probes midi 54 and D22Z4. Using three informative markers both chromosomes 22 were shown to be inherited from the mother (UPDmat). The results are consistent with nondisjunction at maternal meiosis I. The girl is 18 months old now and phenotypically normal. Cardiac and abdominal malformations were excluded by sonographic examinations. Motor and mental development is according to or ahead of developmental milestones (free walking with 10 months, first words at 12 months). The case confirms that maternal UPD 22 most likely is not associated with clinical abnormalities. According to FISH results, UPD 22, and 47,XX,+22 in the placenta, we conclude that the SMC was derived from alpha satellite sequences of chromosome 22. This case for the first time gives evidence that early postzygotic reduction of a chromosome to a small marker chromosome is a real existing mechanism to rescue a conceptus with trisomy.

Isolation and characterization of differentially expressed genes in invasive and non-invasive immortalized murine male germ cells in vitro.

In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally, after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-1spg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-1spg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin alpha 6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement variant histone 3.3, alpha-catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-1spg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.

Cell cycle effects and control of gene expression by resveratrol in human breast carcinoma cell lines with different metastatic potentials.

Trans-resveratrol, a polyphenol present in red wines and various human foods, is an antioxidant also with reported chemopreventive properties. However, whether resveratrol may exert different effects in malignant cells with a common anatomical origin yet displaying different invasive characteristics is not known. Since invasiveness and metastasis are considered to be the most insidious and life-threatening aspects for all cancers, we compared the ability of resveratrol to control growth and cell cycle transition in the highly invasive MDA-MB-435 with the minimally invasive MCF-7 breast carcinoma cells. The data revealed that resveratrol exerted a greater inhibitory effect on the MDA-MB-435 cells. A diminution of percentage of cells in G1 phase and a corresponding accumulation of cells in S phase of the cell cycle was observed. We also studied the effect of resveratrol on a panel of MDA-MB-435 cells transfected with nm23-H1 and nm23-H2 genes, which have been suggested to play a role in controlling metastasis in breast cancer cells. These cells are designated as Vbeta, 1beta, 1Tbeta, 2beta, and 2Tbeta, respectively. The control Vbeta consists of MDA-MB-435 cells transfected with bacterial beta-glucuronidase. Cells labeled 1beta and 1Tbeta correspond to those carrying beta-glucuronidase and overexpressed wild-type (His118) or mutant (Tyr118, catalytically inactive) nm23-H1 genes. The 2beta and 2Tbeta refer to cells transfected with wild-type and mutant nm23-H2 genes. The responses of these cells to resveratrol were assessed by measuring proliferation, cell cycle phase distribution, and changes in expression of several genes. These studies have shown that resveratrol (25 microM, 3 days) reduced growth of all cell types by 60-80%. Overexpression of both wild-type and catalytically inactive nm23-H1 (1beta, 1Tbeta) but not nm23-H2 (2beta, 2Tbeta) reduced the proportion of cells in G1 phase, compared to the Vbeta control cells. Little changes in expression of PCNA, Rb, p53, and bcl-2 were observed in the five cell types treated with resveratrol, compared to untreated cells. Noted exceptions included reduced expression of Rb protein and increased expression of p53 in 2beta and 2Tbeta cells, and increased expression of bcl-2 in 2beta cells, treated with resveratrol. In contrast, resveratrol upregulated expression of cathepsin D by 50-100% in all cell lines except 1beta. These results suggest that the intrinsic metastatic potential of cancer cells may affect their responses to chemopreventive agents such as resveratrol.

Leupaxin acts as a mediator in prostate carcinoma progression through deregulation of p120catenin expression.

Recently, we could show that the focal adhesion protein leupaxin (LPXN) is expressed in human prostate carcinomas (PCa) and induces invasiveness of androgen-independent PCa cells. In this study we show that LPXN enhanced the progression of existing PCa in vivo by breeding transgenic mice with prostate-specific LPXN expression and TRAMP mice (transgenic adenocarcinoma of mouse prostate). Double transgenic LPXN/TRAMP mice showed a significant increase in poorly differentiated PCa and distant metastases as compared with control TRAMP mice. Additional studies on primary PCa cells generated from both transgenic backgrounds confirmed the connection regarding LPXN overexpression and increased motility and invasiveness of PCa cells. One mediator of LPXN-induced invasion was found to be the cell-cell adhesion protein p120catenin (p120CTN). Both in vitro and in vivo experiments revealed that p120CTN expression negatively correlates with LPXN expression, followed by a redistribution of beta-catenin. Downregulation of LPXN using small interfering RNAs (siRNAs) resulted in a membranous localization of beta-catenin, whereas strong nuclear accumulation of beta-catenin was observed in p120CTN knockdown cells leading to enhanced transcription of the beta-catenin target gene matrix metalloprotease-7. In conclusion, the present results indicate that LPXN enhances the progression of PCa through downregulation of p120CTN expression and that LPXN could function as a marker for aggressive PCa in the future.

A disease causing deletion of 29 base pairs in intron 15 in the MKS1 gene is highly associated with the campomelic variant of the Meckel-Gruber syndrome.

Meckel-Gruber syndrome (MKS) is an autosomal recessive disorder causing severe defects in the developing central nervous system and other organs. Recently, mutations in the MKS1 gene have been identified as disease causing in individuals of Finnish MKS families. The primary aim of the present study was to assess the frequency of the 'Finnish founder mutation' (29 bp IVS15-7_35) in the MKS1 gene in 20 aborted fetuses with a diagnosis of MKS. The secondary aim was to screen for novel mutations in the coding sequence of the MKS1 gene of MKS fetuses and to obtain genotype-phenotype correlations where possible. Furthermore, we evaluated the carrier rate of a deletion of 29 bp in intron 15 of the MKS1 gene in a German population. To identify and characterize mutations in the MKS1 gene, sequence analyses and quantitative real time polymerase chain reaction studies were performed. We could identify the same type of mutation, a deletion of 29 bp in intron 15 of the MKS1 gene, in 8 out of the 20 cases studied. Six out of the eight cases with such a mutation displayed the campomelic variant of MKS. The carrier frequency among 519 healthy German individuals was 1:260. This deletion in the MKS1 gene is highly associated with a distinct subtype of the MKS, namely the campomelic variant. In individuals of European origin suffering from the campomelic MKS variant, the described deletion is highly likely to be causative. Regarding the results of our study, the incidence of MKS in Germany can be estimated as 1:135,000. In families with a known mutation in the MKS1 gene, it is now possible to offer an early prenatal testing, for example with chorionic villus sampling and mutation analysis.

Prenatal diagnosis of a large de novo terminal deletion of chromosome 11q.

OBJECTIVE: To describe the prenatal phenotype of the 11q deletion syndrome (Jacobsen syndrome) and present the molecular characterization of the deletion in the case presented. CASE: Ultrasound at 18 and 20 weeks of gestation, on a 34-year-old woman who presented for amniocentesis, revealed slow movements, oligohydramnios and dilatation of the cerebral ventricles in the fetus. Maternal and paternal ages were 34 and 38 years, respectively. RESULTS: Prenatal karyotyping of cultured amniotic fluid cells revealed an 11q terminal deletion, 46,XX,del(11)(q23) (Jacobsen syndrome). Real-time quantitative PCR analysis was used to identify and map the breakpoint physically to a 45-kb region located 14.5 Mb from the 11q telomere. Polymorphic DNA marker analysis showed that DNA sequences on the paternally derived chromosome are deleted. At autopsy, facial dysmorphism without major malformations was recorded. Examination of the internal organs disclosed the following abnormalities: a Meckels' diverticulum of 4-mm length, adhesion between the gall bladder and the transverse colon, and bilaterally bilobed lungs without further situs anomalies. CONCLUSION: Our case demonstrates significant phenotypic variability of Jacobsen syndrome at midtrimester pregnancy; the syndrome may be manifested at this stage only by mild to moderate ventriculomegaly of the brain.