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Contains fulltext : 108163.pdf (publisher's version ) (Open Access), In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.

1 mei 2012, Item does not contain fulltext, Genetic factors play a significant role in the etiology of intellectual disability (ID). The goal of this study was to identify microscopically visible chromosomal abnormalities in an Indonesian ID population and to determine their frequency, pattern, and clinical features. A total of 527 intellectually disabled individuals from special schools and institutions in 4 different areas on Java Island, Indonesia, were screened for cytogenetic abnormalities. Additional analyses were carried out for verification or further characterization by using fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, or analysis of the FMR1 promoter CGG(n) repeat. Of the 527 individuals with ID, chromosomal abnormalities were found in 87 (16.5%). Trisomy 21 was the major chromosomal abnormality, identified in 74 patients (14%). Other chromosome abnormalities included 8 X-chromosomal and 5 autosomal aberrations. Details on chromosome aberrations and confirmation analyses are discussed. This study shows that chromosomal abnormalities are an important cause of ID in Indonesia. Cytogenetic analysis is important for an adequate diagnosis in patients and subsequent genetic counseling for their families, especially in developing countries with limited facilities, such as Indonesia.

Contains fulltext : 88153.pdf (publisher's version ) (Open Access), The kidney has a high capacity to regenerate after ischemic injury via several mechanisms, one of which involves bone marrow-derived (stem) cells. The ATP binding cassette transporters, P-glycoprotein and breast cancer resistance protein, are determinants for the enriched stem and progenitor cell fraction in bone marrow. Because they are upregulated after acute kidney injury, we hypothesized that both efflux pumps may play a role in protecting against renal injury. Surprisingly, transporter-deficient mice were protected against ischemia-induced renal injury. To further study this, bone marrow from irradiated wild-type mice was reconstituted by bone marrow from wild-type, P-glycoprotein- or breast cancer resistance protein-deficient mice. Four weeks later, kidney injury was induced and its function evaluated. Significantly more bone marrow-derived cells were detected in kidneys grafted with transporter-deficient bone marrow. A gender mismatch study suggested that cell fusion of resident tubular cells with bone marrow cells was unlikely. Renal function analyses indicated an absence of renal damage following ischemia-reperfusion in animals transplanted with transporter-deficient bone marrow. When wild-type bone marrow was transplanted in breast cancer resistance protein-deficient mice this protection is lost. Furthermore, we demonstrate that transporter-deficient bone marrow contained significantly more monocytes, granulocytes, and early outgrowth endothelial progenitor cells.

1 september 2010, Contains fulltext : 88320.pdf (publisher's version ) (Closed access), Microcephaly, mental retardation and congenital retinal folds along with other systemic features have previously been reported as a separate clinical entity. The sporadic nature of the syndrome and lack of clear inheritance patterns pointed to a genetic heterogeneity. Here, we report a genetic analysis of a female patient with microcephaly, congenital bilateral falciform retinal folds, nystagmus, and mental retardation. Karyotyping revealed a de novo pericentric inversion in chromosome 6 with breakpoints in 6p12.1 and 6q21. Fluorescence in situ hybridization analysis narrowed down the region around the breakpoints, and the breakpoint at 6q21 was found to disrupt the CDK19 gene. CDK19 was found to be expressed in a diverse range of tissues including fetal eye and fetal brain. Quantitative PCR of the CDK19 transcript from Epstein-Barr virus-transformed lymphoblastoid cell lines of the patient revealed ~50% reduction in the transcript (p = 0.02), suggesting haploinsufficiency of the gene. cdk8, the closest orthologue of human CDK19 in Drosophila has been shown to play a major role in eye development. Conditional knock-down of Drosophila cdk8 in multiple dendrite (md) neurons resulted in 35% reduced dendritic branching and altered morphology of the dendritic arbour, which appeared to be due in part to a loss of small higher order branches. In addition, Cdk8 mutant md neurons showed diminished dendritic fields revealing an important role of the CDK19 orthologue in the developing nervous system of Drosophila. This is the first time the CDK19 gene, a component of the mediator co-activator complex, has been linked to a human disease.

Contains fulltext : 89268.pdf (publisher's version ) (Closed access)

Contains fulltext : 80027.pdf (publisher's version ) (Closed access), Uterine leiomyomas are the most common neoplasms in women of reproductive age. Approximately 40% of these neoplasms show recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 or 10q22. Using positional cloning strategies, we and others had previously identified HMGA1, HMGA2, RAD51L1, and MYST4 (previously referred to as MORF); as primary target (fusion) genes associated with tumor development in three of these distinct cytogenetic subgroups. Here, we report the positional cloning of a single, recurrent, leiomyoma-associated anomaly, t(1;2)(p36;p24). Molecular characterization of the reciprocal breakpoint intervals showed that that AJAP1 (alias SHREW1) and NPHP4 flank the breakpoint on chromosome 1 and that ITSN2 and NCOA1 flank the breakpoint on chromosome 2. Detailed analysis of the breakpoint regions revealed that in this particular case the translocation was associated with a 27-bp deletion on chromosome 1 and a 136-bp duplication on chromosome 2. No breakpoint-spanning (fusion) genes were identified. In silico prediction of transcription factor binding sites, however, indicated the presence of several such sites in the respective breakpoint regions, and major changes therein as a result of the t(1;2)(p36;p24) under investigation. We postulate that transcriptional deregulation of one or more of these breakpoint-flanking genes may contribute to the development of human uterine leiomyomas.

Contains fulltext : 52430.pdf (publisher's version ) (Closed access), OBJECTIVES: The multitarget fluorescence in situ hybridization probe set Vysis UroVysion, consisting of probes for chromosomes 3, 7, and 17 and for the 9p21 band, was studied to evaluate its value in the follow-up of patients with bladder cancer. The results were compared with conventional cytology and quantitative cytology (Quanticyt). The aim of this study was to evaluate whether UroVysion is a better adjunct to urethrocystoscopy than cytology and quantitative cytology. METHODS: UroVysion, cytology, and quantitative cytology were performed on 113 voided urinary samples of 105 patients under surveillance for non-muscle-invasive bladder cancer. Before urethrocystoscopy or transurethral resection of the bladder, a voided urinary sample was obtained. Results of all tests were compared to evaluate the value of UroVysion. RESULTS: Sixty-four patients had biopsy-proven urothelial cell carcinoma. Sensitivity and specificity were, respectively, 39.1% and 89.7% for UroVysion, 40.6% and 89.7% for cytology, and 42.1% and 67.9% for quantitative cytology. When the UroVysion test and cytology were combined, sensitivity increased to 53.1%, but specificity decreased to 79.5%. Detection of Ta tumours was equal for cytology and UroVysion (26.7%), detection of T1 and T2-T4 samples by UroVysion was 60% and 50%, respectively. Detection of grade 1, 2, and 3 tumours by UroVysion was 21.4%, 36.8%, and 66.7%, respectively. In four cases the UroVysion test was positive, but no abnormalities were seen at cystoscopy. CONCLUSIONS: Our data suggest that the use of UroVysion provides no improvement over cytology or quantitative cytology in the diagnosis of recurrent non-muscle-invasive bladder tumours.

Item does not contain fulltext, We recently described a transgenic mouse strain overexpressing hamster alphaA-crystallin, a small heat shock protein, under direction of the hamster vimentin promoter. As a result myelin was degraded and axonal dystrophy in both central nervous system (especially spinal cord) and peripheral nervous system occurred. Homozygous transgenic mice developed hind limb paralysis after 8 weeks of age and displayed progressive loss of myelin and axonal dystrophy in both the central and peripheral nervous system with ongoing age. Pathologically the phenotype resembled, to a certain extent, neuroaxonal dystrophy. The biochemical findings presented in this paper (activity of the enzymes superoxide dismutase, catalase and transglutamase, myelin protein zero expression levels and blood sugar levels) confirm this pathology and exclude other putative pathologies like Amyothrophic Lateral Sclerosis and Hereditary Motor and Sensory Neuropathy. Consequently, an excessive cytoplasmic accumulation of the transgenic protein or a disturbance of the normal metabolism are considered to cause the observed neuropathology. Therefore, extra-ocular alphaA-crystallin-expressing transgenic mice may serve as a useful animal model to study neuroaxonal dystrophy.

Item does not contain fulltext, The SSX gene family is composed of at least five functional and highly homologous members, SSX1 to SSX5, that are normally expressed in only the testis and thyroid. SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. In addition, the SSX1, SSX2, SSX4, and SSX5 genes were found to be aberrantly expressed in several other malignancies, including melanoma. The SSX proteins are localized in the nucleus and are diffusely distributed. In addition, they may be included in polycomb-group nuclear bodies. Other studies have indicated that the SSX proteins may act as transcriptional repressors. As a first step toward the elucidation of the cellular signaling networks in which the SSX proteins may act, we used the yeast two-hybrid system to identify SSX2-interacting proteins. By doing so, two novel human proteins were detected: RAB3IP, the human homolog of an interactor of the Ras-like GTPase Rab3A; and a novel protein, SSX2IP. RAB3IP did not interact with either SSX1, SSX3, or SSX4 in the yeast two-hybrid system, whereas SSX2IP interacted with SSX3 but not with either SSX1 or SSX4. Further analysis of deletion mutants showed that both RAB3IP and SSX2IP interact with the N-terminal moiety of the SSX2 protein. Immunofluorescence analyses of transfected cells revealed that the RAB3IP protein is normally localized in the cytoplasm. However, coexpression of both RAB3IP and SSX2 led to colocalization of both proteins in the nucleus. Likewise, the SSX2IP protein was found to be colocalizing with SSX2 in the nucleus. By performing glutathione-S-transferase pull-down assays, we found that both RAB3IP and SSX2IP interact directly with SSX2 in vitro. These newly observed protein/protein interactions may have important implications for the mechanisms underlying normal and malignant cellular growth.

Item does not contain fulltext, Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, and miscarriages. Automated detection of subtle deletions or duplications involving telomeres is essential for high-throughput diagnosis, but impossible when conventional cytogenetic methods are used. Array-based comparative genomic hybridization (CGH) allows high-resolution screening of copy number abnormalities by hybridizing differentially labeled test and reference genomes to arrays of robotically spotted clones. To assess the applicability of this technique in the diagnosis of (sub)telomeric imbalances, we here describe a blinded study, in which DNA from 20 patients with known cytogenetic abnormalities involving one or more telomeres was hybridized to an array containing a validated set of human-chromosome-specific (sub)telomere probes. Single-copy-number gains and losses were accurately detected on these arrays, and an excellent concordance between the original cytogenetic diagnosis and the array-based CGH diagnosis was obtained by use of a single hybridization. In addition to the previously identified cytogenetic changes, array-based CGH revealed additional telomere rearrangements in 3 of the 20 patients studied. The robustness and simplicity of this array-based telomere copy-number screening make it highly suited for introduction into the clinic as a rapid and sensitive automated diagnostic procedure.

Item does not contain fulltext, Cystatin M/E (CST6 ), a new member of the cystatin gene family, has a restricted expression pattern in humans, which is largely limited to cutaneous epithelia. Although cystatin M/E possesses two distinct biochemical properties, being a cysteine proteinase inhibitor and a substrate for transglutaminase, its physiological function is unknown. Here we report the isolation and characterization of the mouse Cst6 orthologue and the assignment of the chromosomal localization to the proximal end of mouse chromosome 19. This region corresponds to the locus of the spontaneous harlequin ichthyosis (ichq) mouse mutation, for which no causative gene has been identified so far. We found a nonsense mutation in the Cst6 gene of BALB/cJ-ichq/+ mice, which precludes the synthesis of functional protein. Immunohistochemistry confirmed the absence of cystatin M/E at the protein level in ichq/ichq mice. Mice that are homozygous for two null alleles display a hyperplastic, hyperkeratotic epidermis and abnormal hair follicles, and die between 5 and 12 days of age. In wild-type mice, cystatin M/E was found in the stratum granulosum and in the infundibulum of the hair follicle indicating that the anatomical site in the skin where cystatin M/E is normally expressed correlates with the abnormalities at the tissue level in ichq/ichq mice. Our data provide evidence that cystatin M/E is required for viability and for correct formation of cornified layers in the epidermis and hair follicles. The ichq mouse mutation may serve as a model for human type 2 harlequin ichthyosis.

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Item does not contain fulltext, In a subset of papillary renal cell carcinomas a t(X;1)(p11;q21) chromosome translocation has repeatedly been reported. Positional cloning has demonstrated that, as a result of this translocation, the transcription factor TFE3 gene on the X-chromosome becomes fused to a novel gene, PRCC, on chromosome 1. Since as yet little is known about the function of PRCC, we sought to identify the mouse counterpart of the PRCC gene. Isolation and sequence analysis of a mouse Prcc cDNA revealed a high level of conservation between man and mouse, both at the nucleotide and protein level. As the human PRCC gene, the mouse Prcc gene is ubiquitously expressed. It shows low expression in all mouse fetal tissues examined. In addition, we identified a genomic cosmid clone containing the complete Prcc gene. The mouse Prcc gene consists of seven exons, all of which contain coding sequences. The small second exon, which was found to be located adjacent to the t(X;1) breakpoint in the human gene on chromosome 1, is also conserved between man and mouse. In mouse, Prcc is located on chromosome 3. These cDNA and genomic clones will be instrumental in the creation of mouse models for a further elucidation of the function of PRCC.

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Item does not contain fulltext, p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.

Item does not contain fulltext, We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus e

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Contains fulltext : 224430.pdf (publisher's version ) (Closed access)

Contains fulltext : 216411.pdf (Publisher’s version ) (Open Access)

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Contains fulltext : 142231.pdf (publisher's version ) (Open Access)

Contains fulltext : 25861___.PDF (publisher's version ) (Open Access)