Identification of CUX1 as the recurrent chromosomal band 7q22 target gene in human uterine leiomyoma.

Uterine leiomyomas are benign solid tumors of mesenchymal origin which occur with an estimated incidence of up to 77% of all women of reproductive age. The majority of these tumors remains symptomless, but in about a quarter of cases they cause leiomyoma-associated symptoms including chronic pelvic pain, menorrhagia-induced anemia, and impaired fertility. As a consequence, they are the most common indication for pre-menopausal hysterectomy in the USA and Japan and annually translate into a multibillion dollar healthcare problem. Approximately 40% of these neoplasms present with recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 and/or 10q22. Using positional cloning strategies, we and others previously identified HMGA1, HMGA2, RAD51L1, MORF, and, more recently, NCOA1 as primary target (fusion) genes associated with tumor initiation in four of these distinct cytogenetic subgroups. Despite the fact that the del(7)(q22) subgroup is the largest among leiomyomas, and was first described more than twenty years ago, the 7q22 leiomyoma target gene still awaits unequivocal identification. We here describe a positional cloning effort from two independent uterine leiomyomas, containing respectively a pericentric and a paracentric chromosomal inversion, both affecting band 7q22. We found that both chromosomal inversions target the cut-like homeobox 1 (CUX1) gene on chromosomal band 7q22.1 in a way which is functionally equivalent to the more frequently observed del(7q) cases, and which is compatible with a mono-allelic knock-out scenario, similar as was previously described for the cytogenetic subgroup showing chromosome 14q involvement.

CDK19 is disrupted in a female patient with bilateral congenital retinal folds, microcephaly and mild mental retardation.

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.

Human male meiotic sex chromosome inactivation.

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.

A cytogenetic study in a large population of intellectually disabled Indonesians.

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.

Deficiency of either P-glycoprotein or breast cancer resistance protein protect against acute kidney injury.

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.

Characterization of a recurrent t(1;2)(p36;p24) in human uterine leiomyoma.

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.

UroVysion compared with cytology and quantitative cytology in the surveillance of non-muscle-invasive bladder cancer.

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.

A null mutation in the cystatin M/E gene of ichq mice causes juvenile lethality and defects in epidermal cornification.

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.

The cancer-related protein SSX2 interacts with the human homologue of a Ras-like GTPase interactor, RAB3IP, and a novel nuclear protein, SSX2IP.

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.

High-throughput analysis of subtelomeric chromosome rearrangements by use of array-based comparative genomic hybridization.

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.