Physical mapping of distinct 7q22 deletions in uterine leiomyoma and analysis of a recently annotated 7q22 candidate gene

Uterine leiomyoma (UL) is a benign, smooth muscle tumor of the uterus affecting a significant proportion of women of reproductive age. Deletions involving chromosome 7q22 are common in UL and vary in length. Previously reported 7q22 deletion intervals were physically mapped using information from the recently completed human genome sequence. Four distinct deletion intervals, which included a microdeletion reported by our laboratory, were identified. This microdeletion contains two known genes, ORC5L and LHFPL3. The single deleted marker in the microdeletion was mapped within the LHFPL3 locus. The ORC5L gene has been studied in UL. Conversely, LHFPL3 has been annotated only recently, and has therefore not been studied in UL. The predicted LHFPL3 protein sequence contained a polyalanine domain, and a signature sequence for the PMP22 Claudin protein family. Members of this family are transmembrane proteins with roles in differentiation, proliferation, and extracellular matrix formation, and have been implicated in other tumors. Differences in LHFPL3 expression were observed in both human and Eker rat UL. Our results provide evidence for four distinct 7q22 deletion intervals, each with multiple candidate genes, including the recently identified LHFPL3 gene.     

Detection of a t(1;22)(q23;q12) translocation leading to an EWSR1-PBX1 fusion gene in a myoepithelioma

Chromosome banding as well as molecular cytogenetic methods are of great help in the diagnosis of mesenchymal tumors. Myoepithelial neoplasms of soft tissue including myoepitheliomas, mixed tumors, and parachordomas are diagnoses that have been increasingly recognized the last few years. It is still debated which neoplasms should be included in these morphologically heterogeneous entities, and the boundaries between them are not clear-cut. The pathogenetic mechanisms behind myoepithelial tumors are unknown. Only five parachordomas and one mixed tumor have previously been karyotyped, and nothing is known about their molecular genetic characteristics. We present a mesenchymal tumor classified as a myoepithelioma that had a balanced translocation t(1;22)(q23;q12) as the sole karyotypic change. A novel EWSR1-PBX1 fusion gene consisting of exons 1-8 of the 5'-end of EWSR1 and exons 5-9 of the 3'-end of PBX1 was shown to result from the translocation. Both genes are known to be targeted also by other neoplasia-specific translocations, PBX1 in acute lymphoblastic leukemia and EWSR1 in several solid tumors, most of which are malignant. Based on the structure of the novel fusion gene detected, its transforming mechanism is thought to be the same as for other fusion genes involving EWSR1 or PBX1.     

Inversion (X)(p22q13) in a uterine leiomyoma.

We report a case of uterine leiomyoma which showed a karyotype 46,X,inv(X)(p22q13) as the only clonal change in most of the cells. A few cells had an additional del(7), though del(7) has been found to be a primary change in leiomyomas. These findings indicate that the abnormality involving the X chromosome and particularly Xp22 can be considered as a primary chromosomal abnormality. We discuss the findings together with few reports of cases involving chromosome X in leiomyomas.     

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. © 2012 Wiley Periodicals, Inc.     

High resolution mapping of consistent leiomyoma breakpoints in chromosomes 12 and 14 to 12q15 and 14q24.1

A substantial percentage of uterine leiomyomas are cytogenetically characterized by consistent, clonal chromosome abnormalities, including t(12;14)(q14-15;q23-24) and other rearrangements of 12q14-15 that occur without any visible 14q changes. The partly similar banding characteristics of these two regions have hitherto precluded exact mapping of the 12q and 14q breakpoints to any particular band, let alone their assignment to subbands. In the series of four myomas presented here, in which one tumor had inv(12q), two t(12;14), and one a three-way t(7;12;14), we were able to achieve high resolution banding (550 band stage) of the rearranged chromosomes in several metaphases. This enabled us to assign a 12q breakpoint to 12q15 in all tumors and, in the three cases informative in this regard, the 14q breakpoint to 14q24.1. The more precise breakpoint mapping considerably narrows down the area that must be examined with molecular genetic methods in order to identify the gene loci that are rearranged in leiomyomas with 12q and 14q aberrations. It will also help determine to what extent leiomyoma rearrangements of 12q involve the same loci that are affected in 12q changes in other tumor types, e.g., in pleomorphic adenomas of the salivary gland, in lipomas, and in myxoid liposarcomas. At present it seems that the breakpoint in 12q may be cytogenetically identical in the three benign tumors, whereas it in myxoid liposarcomas appears to be more proximal.     

Novel candidate tumour suppressor gene loci on chromosomes 11q23-24 and 22q13 involved in human insulinoma tumourigenesis

Insulinomas represent the predominant syndromic subtype of endocrine pancreatic tumours. Previous molecular studies have shown that gain of chromosome 9q rather than MEN1 gene mutation is an important early event in tumour development and that chromosomal instability is associated with metastatic disease. In order to identify new gene loci and to define further the critical genetic events in insulinoma tumourigenesis, 27 insulinomas were investigated by array-based comparative genomic hybridization (array CGH) on 3.7 k genomic BAC arrays (resolution < or =1 Mb). Fluorescence in situ hybridization was used to validate alterations in a subset of tumours. Array CGH most frequently detected loss of chromosomes 11q and 22q and gains of chromosome 9q. The chromosomal regions of interest (CRI) included 11q24.1 (56%), 22q13.1 (67%), 22q13.31 (56%), and 9q32 (63%). Evaluation of the simultaneous occurrence of these aberrations in the individual tumours revealed that gain of 9q32 and loss of 22q13.1 are early genetic events in insulinomas, occurring independently of the other alterations. In tumours with increased genomic complexity, these alterations were often detected simultaneously, occurring in the same tumour cells. Losses of 11q24.1 and 22q13.31 were also associated with these more advanced tumour cases. The CRIs identified most likely harbour crucial candidate genes important in insulinoma tumourigenesis.     

Analysis of a 1-megabase deletion in 15q22-q23 in an autistic patient: identification of candidate genes for autism and of homologous DNA segments in 15q22-q23 and 15q11-q13

We have identified a one megabase deletion in the 15q22-15q23 region in a patient with autism, developmental delay, and mild dysmorphism. Genes that map within the deletion region and genes that are interrupted or rearranged at the deletion breakpoints are candidate genes for autism. Fluroescence in situ hybridization studies in this patient revealed that part or all of the PML gene is absent from one chromosome 15 and a BAC clone containing the D15S124 gene locus hybridizes to only one chromosome 15. BAC clones containing the PTPN9, and SLP-1[hUNC24] genes showed markedly reduced hybridization in the 15q22-q23 region on one chromosome 15 in the patient. These BACs also hybridize to the 15q11-q13 region in close proximity to SNRPN and HERC2, and in this region there is equal intensity of signal on the normal and on the deleted chromosome. There are previous reports of deletions and duplications of the 15q11-q13 region in patients with autism. Our patient represents the first report of a 15q22-q23 deletion. Hybridization of the PTPN9 and Slp-1 Bac clones to the 15q11-q13 and the 15q22-q23 regions of chromosome 15 may be due to the presence of PTPN9 or SLP-1 gene sequences or to the presence of other gene sequences or to non-coding homologous DNA sequences. The PTPN9 gene encodes a non-receptor protein tyrosine phosphatase. The Slp-1 [hUNC24] gene is expressed mainly in the brain. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:765-770, 2000.     

Further evidence for an imprinted gene for neonatal diabetes localised to chromosome 6q22-q23

Transient neonatal diabetes mellitus (TNDM) is a rare form of childhood diabetes which usually resolves in the first 6 months of life but which predisposes to type 2 diabetes of adult onset. We recently reported paternal uniparental isodisomy of chromosome 6 (UPD6) in two children with TNDM and proposed that there may be an imprinted gene important in the aetiology of diabetes on chromosome 6. We now describe two unrelated families which independently suggest that the gene is imprinted, is paternally expressed and maps to 6q22-q23. One family has a duplication while the other, with familial TNDM, shows linkage to a marker in this region.      

Gain of 1q25-32, 12q23-24.3, and 17q12-22 facilitates tumorigenesis and progression of human squamous cell lung cancer

To explore the genetic changes involved in the stepwise development of lung cancer, we have determined the genetic events associated with the histological progression from normal bronchial epithelium to squamous cell carcinoma. Comparative genomic hybridization was used to identify chromosomal imbalances in 54 microdissected samples, including squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumour derived from 23 patients with squamous cell carcinoma of the lung. Histopathological progression was accompanied by an increased number of chromosomal abnormalities. Gains of 1q25-32, 12q23-24.3, and 17q12-22, in particular, were detected at high frequencies in both carcinoma in situ and invasive tumours and were found more often in the cases with lymph node metastases than in those without. Our previous expression profiling of squamous cell carcinomas had identified overexpression of laminin5 gamma2, a gene located at 1q25-31. Therefore, this was investigated at the protein level by immunohistochemical analysis in 336 samples of squamous cell carcinoma of the lung. Consistent with the genomic data for this region, the expression level of laminin5 gamma2 was higher in the primary tumours with lymph node metastases than in tumours without metastases (p = 0.012). These data suggest that gains of genes from 1q25-32, 12q23-24.3, and 17q12-22 facilitate tumorigenesis and progression of squamous cell carcinoma of the lung, and may serve as potential predictors for this disease.     

USH1H, a novel locus for type I Usher syndrome, maps to chromosome 15q22-23

Usher syndrome (USH) is a hereditary disorder associated with sensorineural hearing impairment, progressive loss of vision attributable to retinitis pigmentosa (RP) and variable vestibular function. Three clinical types have been described with type I (USH1) being the most severe. To date, six USH1 loci have been reported. We ascertained two large Pakistani consanguineous families segregating profound hearing loss, vestibular dysfunction, and RP, the defining features of USH1. In these families, we excluded linkage of USH to the 11 known USH loci and subsequently performed a genome-wide linkage screen. We found a novel USH1 locus designated USH1H that mapped to chromosome 15q22-23 in a 4.92-cM interval. This locus overlaps the non-syndromic deafness locus DFNB48 raising the possibility that the two disorders may be caused by allelic mutations.     

Deletion at 14q22-23 indicates a contiguous gene syndrome comprising anophthalmia, pituitary hypoplasia, and ear anomalies

Anophthalmia and pituitary gland hypoplasia are both debilitating conditions where the underlying genetic defect is unknown in the majority of cases. We identified a patient with bilateral anophthalmia and absence of the optic nerves, chiasm and tracts, as well as pituitary gland hypoplasia and ear anomalies with a de novo apparently balanced chromosomal translocation, 46,XY,t(3;14)(q28;q23.2). Translocation breakpoint analysis using FISH and high-resolution microarray comparative genomic hybridization (CGH) has identified a 9.66 Mb deleted region on the long arm of chromosome 14 which includes the genes BMP4, OTX2, RTN1, SIX6, SIX1, and SIX4. Three other patients with interstitial deletions involving 14q22-23 have been described, all with bilateral anophthalmia, pituitary abnormalities, ear anomalies, and a facial phenotype similar to our patient. OTX2 is involved in ocular developmental defects, and the severity of the ocular phenotype in our patient and the other 14q22-23 deletion patients, suggests this genomic region harbors other gene/s involved in ocular development. BMP4 haploinsufficiency is predicted to contribute to the ocular phenotype on the basis of its expression pattern and observed murine mutant phenotypes. In addition, deletion of BMP4 and SIX6 is likely to contribute to the abnormal pituitary development, and SIX1 deletion may contribute to the ear and other craniofacial features. This indicates that contiguous gene deletion may contribute to the phenotypic features in the 14q22-23 deletion patients.     

Molecular cytogenetic analysis of consistent abnormalities at 8q12-q22 in breast cancer

Studies using comparative genomic hybridization (CGH) indicate that portions of chromosome arm 8q from 8q12 to 8qter are present at an increased relative copy number in a broad range of solid tumors. In this study we define an ∼ 1 Mb wide region that appears to be frequently abnormal in copy number or structure in breast cancer cell lines and primary tumors. This was accomplished by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs) mapped to 8q12-q22. Eleven breast cancer cell lines and ten primary tumors were analyzed. A minimal region of rearrangement was localized to the CEPH-YAC 928F9 in three breast cancer cell lines with unbalanced translocation breakpoints mapping in this region. Unbalanced translocations also were detected in two primary tumors mapping between CEPH-YAC clones 890C4 and 936B3, flanking 928F9. An increased copy number in the minimal region was detected in nine cell lines and in multiple primary tumors. This suggests the possibility that a single gene mapping to 928F9 is involved in breast cancer development or progression and may be deregulated by copy number increases in some tumors and by translocation in others. Four expressed sequence tags were mapped to YAC 928F9 and analyzed for rearrangements by Southern analysis and for abnormal expression by Northern analysis. Genes Chromosomes Cancer 22:105–113, 1998. © 1998 Wiley-Liss, Inc.