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1.  Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome 
PLoS ONE  2013;8(4):e60482.
Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.
doi:10.1371/journal.pone.0060482
PMCID: PMC3626651  PMID: 23596509
2.  Maternally Inherited Partial Monosomy 9p (pter → p24.1) and Partial Trisomy 20p (pter → p12.1) Characterized by Microarray Comparative Genomic Hybridization 
We report on a 17-year-old patient with midline defects, ocular hypertelorism, neuropsychomotor development delay, neonatal macrosomy, and dental anomalies. DNA copy number investigations using a Whole Genome TilePath array consisting, of 30K BAC/PAC clones showed a 6.36 Mb deletion in the 9p24.1–p24.3 region and a 14.83 Mb duplication in the 20p12.1–p13 region, which derived from a maternal balanced t(9;20)(p24.1;p12.1) as shown by FISH studies. Monosomy 9p is a well-delineated chromosomal syndrome with characteristic clinical features, while chromosome 20p duplication is a rare genetic condition. Only a handful of cases of monosomy 9/trisomy 20 have been previously described. In this report, we compare the phenotype of our patient with those already reported in the literature, and discuss the role of DMRT, DOCK8, FOXD4, VLDLR, RSPO4, AVP, RASSF2, PROKR2, BMP2, MKKS, and JAG1, all genes mapping to the deleted and duplicated regions.
doi:10.1002/ajmg.a.34168
PMCID: PMC3428835  PMID: 21948691
multiple congenital anomalies; midline; chromosomal aberration; array-CGH; monosomy 9p; trisomy 20p
3.  Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays 
Nature protocols  2009;4(12):1722-1736.
Aarray painting is a technique that uses microarray technology to rapidly map chromosome translocation breakpoints. previous methods to map translocation breakpoints have used fluorescence in situ hybridization (FIsH) and have consequently been labor-intensive, time-consuming and restricted to the low breakpoint resolution imposed by the use of metaphase chromosomes. array painting combines the isolation of derivative chromosomes (chromosomes with translocations) and high-resolution microarray analysis to refine the genomic location of translocation breakpoints in a single experiment. In this protocol, we describe array painting by isolation of derivative chromosomes using a MoFlo flow sorter, amplification of these derivatives using whole-genome amplification and hybridization onto commercially available oligonucleotide microarrays. although the sorting of derivative chromosomes is a specialized procedure requiring sophisticated equipment, the amplification, labeling and hybridization of Dna is straightforward, robust and can be completed within 1 week. the protocol described produces good quality data; however, array painting is equally achievable using any combination of the available alternative methodologies for chromosome isolation, amplification and hybridization.
doi:10.1038/nprot.2009.183
PMCID: PMC3330750  PMID: 19893508
4.  Auditory Function in the Tc1 Mouse Model of Down Syndrome Suggests a Limited Region of Human Chromosome 21 Involved in Otitis Media 
PLoS ONE  2012;7(2):e31433.
Down syndrome is one of the most common congenital disorders leading to a wide range of health problems in humans, including frequent otitis media. The Tc1 mouse carries a significant part of human chromosome 21 (Hsa21) in addition to the full set of mouse chromosomes and shares many phenotypes observed in humans affected by Down syndrome with trisomy of chromosome 21. However, it is unknown whether Tc1 mice exhibit a hearing phenotype and might thus represent a good model for understanding the hearing loss that is common in Down syndrome. In this study we carried out a structural and functional assessment of hearing in Tc1 mice. Auditory brainstem response (ABR) measurements in Tc1 mice showed normal thresholds compared to littermate controls and ABR waveform latencies and amplitudes were equivalent to controls. The gross anatomy of the middle and inner ears was also similar between Tc1 and control mice. The physiological properties of cochlear sensory receptors (inner and outer hair cells: IHCs and OHCs) were investigated using single-cell patch clamp recordings from the acutely dissected cochleae. Adult Tc1 IHCs exhibited normal resting membrane potentials and expressed all K+ currents characteristic of control hair cells. However, the size of the large conductance (BK) Ca2+ activated K+ current (IK,f), which enables rapid voltage responses essential for accurate sound encoding, was increased in Tc1 IHCs. All physiological properties investigated in OHCs were indistinguishable between the two genotypes. The normal functional hearing and the gross structural anatomy of the middle and inner ears in the Tc1 mouse contrast to that observed in the Ts65Dn model of Down syndrome which shows otitis media. Genes that are trisomic in Ts65Dn but disomic in Tc1 may predispose to otitis media when an additional copy is active.
doi:10.1371/journal.pone.0031433
PMCID: PMC3279367  PMID: 22348087
5.  Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants 
Nature Biotechnology  2011;29(6):512-520.
We have systematically compared copy number variant (CNV) detection on eleven microarrays to evaluate data quality and CNV calling, reproducibility, concordance across array platforms and laboratory sites, breakpoint accuracy and analysis tool variability. Different analytic tools applied to the same raw data typically yield CNV calls with <50% concordance. Moreover, reproducibility in replicate experiments is <70% for most platforms. Nevertheless, these findings should not preclude detection of large CNVs for clinical diagnostic purposes because large CNVs with poor reproducibility are found primarily in complex genomic regions and would typically be removed by standard clinical data curation. The striking differences between CNV calls from different platforms and analytic tools highlight the importance of careful assessment of experimental design in discovery and association studies and of strict data curation and filtering in diagnostics. The CNV resource presented here allows independent data evaluation and provides a means to benchmark new algorithms.
doi:10.1038/nbt.1852
PMCID: PMC3270583  PMID: 21552272
6.  Reduced TFAP2A function causes variable optic fissure closure and retinal defects and sensitizes eye development to mutations in other morphogenetic regulators 
Human genetics  2009;126(6):791-803.
Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37 highly selected individuals with severe ocular anomalies plus variable defects associated with BOFS for mutations or deletions in TFAP2A. We identified one individual with a de novo TFAP2A four amino acid deletion, a second individual with two non-synonymous variations in an alternative splice isoform TFAP2A2, and a sibling-pair with a paternally inherited whole gene deletion with variable phenotypic expression. Second, we determined that TFAP2A is expressed in the lens, neural retina, nasal process, and epithelial lining of the oral cavity and palatal shelves of human and mouse embryos—sites consistent with the phenotype observed in patients with BOFS. Third, we used zebrafish to examine how partial abrogation of the fish ortholog of TFAP2A affects the penetrance and expressivity of ocular phenotypes due to mutations in genes encoding bmp4 or tcf7l1a. In both cases, we observed synthetic, enhanced ocular phenotypes including coloboma and anophthalmia when tfap2a is knocked down in embryos with bmp4 or tcf7l1a mutations. These results reveal that mutations in TFAP2A are associated with a wide range of eye phenotypes and that hypomorphic tfap2a mutations can increase the risk of developmental defects arising from mutations at other loci.
doi:10.1007/s00439-009-0730-x
PMCID: PMC3083835  PMID: 19685247
7.  Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations 
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.
doi:10.1016/j.ajhg.2009.05.005
PMCID: PMC2694971  PMID: 19500772
8.  Recurrent Rearrangements of Chromosome 1q21.1 and Variable Pediatric Phenotypes 
Mefford, Heather C. | Sharp, Andrew J. | Baker, Carl | Itsara, Andy | Jiang, Zhaoshi | Buysse, Karen | Huang, Shuwen | Maloney, Viv K. | Crolla, John A. | Baralle, Diana | Collins, Amanda | Mercer, Catherine | Norga, Koen | de Ravel, Thomy | Devriendt, Koen | Bongers, Ernie M.H.F. | de Leeuw, Nicole | Reardon, William | Gimelli, Stefania | Bena, Frederique | Hennekam, Raoul C. | Male, Alison | Gaunt, Lorraine | Clayton-Smith, Jill | Simonic, Ingrid | Park, Soo Mi | Mehta, Sarju G. | Nik-Zainal, Serena | Woods, C. Geoffrey | Firth, Helen V. | Parkin, Georgina | Fichera, Marco | Reitano, Santina | Giudice, Mariangela Lo | Li, Kelly E. | Casuga, Iris | Broomer, Adam | Conrad, Bernard | Schwerzmann, Markus | Räber, Lorenz | Gallati, Sabina | Striano, Pasquale | Coppola, Antonietta | Tolmie, John L. | Tobias, Edward S. | Lilley, Chris | Armengol, Lluis | Spysschaert, Yves | Verloo, Patrick | De Coene, Anja | Goossens, Linde | Mortier, Geert | Speleman, Frank | van Binsbergen, Ellen | Nelen, Marcel R. | Hochstenbach, Ron | Poot, Martin | Gallagher, Louise | Gill, Michael | McClellan, Jon | King, Mary-Claire | Regan, Regina | Skinner, Cindy | Stevenson, Roger E. | Antonarakis, Stylianos E. | Chen, Caifu | Estivill, Xavier | Menten, Björn | Gimelli, Giorgio | Gribble, Susan | Schwartz, Stuart | Sutcliffe, James S. | Walsh, Tom | Knight, Samantha J.L. | Sebat, Jonathan | Romano, Corrado | Schwartz, Charles E. | Veltman, Joris A. | de Vries, Bert B.A. | Vermeesch, Joris R. | Barber, John C.K. | Willatt, Lionel | Tassabehji, May | Eichler, Evan E.
The New England journal of medicine  2008;359(16):1685-1699.
BACKGROUND
Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients.
METHODS
We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons.
RESULTS
We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P = 1.1×10−7). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in the nine children with mental retardation or autism spectrum disorder and other variable features (P = 0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies.
CONCLUSIONS
We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.
doi:10.1056/NEJMoa0805384
PMCID: PMC2703742  PMID: 18784092
9.  Ultra-high resolution array painting facilitates breakpoint sequencing 
Journal of medical genetics  2006;44(1):51-58.
The application of comparative genomic hybridization to DNA microarrays (array-CGH) has greatly improved the speed and resolution at which chromosome rearrangements involving genomic imbalance can be studied. For diagnosis of patients whose disease is suspected to be due to a balanced reciprocal translocation, we have developed a modification of array-CGH, termed array painting, which utilizes flow sorting of the derivative chromosomes before hybridization to an array to map the rearrangement breakpoints (Fiegler et al. 2003). However, current mapping resolution by array painting is rarely adequate to definitively identify disrupted genes which may be responsible for the disease phenotype and further time consuming and often technically challenging studies are required to clone and sequence the breakpoints. In this study, we describe the use of ultra-high resolution arrays to provide such an increased improvement in the resolution of breakpoint mapping by array painting that translocation breakpoints can be directly amplified and sequenced. This method of ultra-high resolution array painting enables rapid and definitive identification of gene disruption in balanced reciprocal translocations and will greatly improve the diagnosis for this group of patients.
doi:10.1136/jmg.2006.044909
PMCID: PMC2597908  PMID: 16971479
10.  Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations 
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.
doi:10.1016/j.ajhg.2009.05.005
PMCID: PMC2694971  PMID: 19500772
11.  Characterization of a 3;6 Translocation Associated with Renal Cell Carcinoma 
Genes, chromosomes & cancer  2007;46(4):311-317.
The most frequent cause of familial clear cell renal cell carcinoma (RCC) is von Hippel–Lindau disease and the VHL tumor suppressor gene (TSG) is inactivated in most sporadic clear cell RCC. Although there is relatively little information on the mechanisms of tumorigenesis of clear cell RCC without VHL inactivation, a subset of familial cases harbors a balanced constitutional chromosome 3 translocation. To date nine different chromosome 3 translocations have been associated with familial or multicentric clear cell RCC; and in three cases chromosome 6 was also involved. To identify candidate genes for renal tumorigenesis we characterized a constitutional translocation, t(3;6)(q22;q16.1) associated with multicentric RCC without evidence of VHL target gene dysregulation. Analysis of breakpoint sequences revealed a 1.3-kb deletion on chromosome 6 within the intron of a 2 exon predicted gene (NT_007299.434). However, RT-PCR analysis failed to detect the expression of this gene in lymphoblast, fibroblast, or kidney tumor cell lines. No known genes were disrupted by the translocation breakpoints but several candidate TSGs (e.g., EPHB1, EPHA7, PPP2R3A RNF184, and STAG1) map within close proximity to the breakpoints.
doi:10.1002/gcc.20403
PMCID: PMC2695133  PMID: 17205537
12.  The DNA sequence of the human X chromosome 
Ross, Mark T. | Grafham, Darren V. | Coffey, Alison J. | Scherer, Steven | McLay, Kirsten | Muzny, Donna | Platzer, Matthias | Howell, Gareth R. | Burrows, Christine | Bird, Christine P. | Frankish, Adam | Lovell, Frances L. | Howe, Kevin L. | Ashurst, Jennifer L. | Fulton, Robert S. | Sudbrak, Ralf | Wen, Gaiping | Jones, Matthew C. | Hurles, Matthew E. | Andrews, T. Daniel | Scott, Carol E. | Searle, Stephen | Ramser, Juliane | Whittaker, Adam | Deadman, Rebecca | Carter, Nigel P. | Hunt, Sarah E. | Chen, Rui | Cree, Andrew | Gunaratne, Preethi | Havlak, Paul | Hodgson, Anne | Metzker, Michael L. | Richards, Stephen | Scott, Graham | Steffen, David | Sodergren, Erica | Wheeler, David A. | Worley, Kim C. | Ainscough, Rachael | Ambrose, Kerrie D. | Ansari-Lari, M. Ali | Aradhya, Swaroop | Ashwell, Robert I. S. | Babbage, Anne K. | Bagguley, Claire L. | Ballabio, Andrea | Banerjee, Ruby | Barker, Gary E. | Barlow, Karen F. | Barrett, Ian P. | Bates, Karen N. | Beare, David M. | Beasley, Helen | Beasley, Oliver | Beck, Alfred | Bethel, Graeme | Blechschmidt, Karin | Brady, Nicola | Bray-Allen, Sarah | Bridgeman, Anne M. | Brown, Andrew J. | Brown, Mary J. | Bonnin, David | Bruford, Elspeth A. | Buhay, Christian | Burch, Paula | Burford, Deborah | Burgess, Joanne | Burrill, Wayne | Burton, John | Bye, Jackie M. | Carder, Carol | Carrel, Laura | Chako, Joseph | Chapman, Joanne C. | Chavez, Dean | Chen, Ellson | Chen, Guan | Chen, Yuan | Chen, Zhijian | Chinault, Craig | Ciccodicola, Alfredo | Clark, Sue Y. | Clarke, Graham | Clee, Chris M. | Clegg, Sheila | Clerc-Blankenburg, Kerstin | Clifford, Karen | Cobley, Vicky | Cole, Charlotte G. | Conquer, Jen S. | Corby, Nicole | Connor, Richard E. | David, Robert | Davies, Joy | Davis, Clay | Davis, John | Delgado, Oliver | DeShazo, Denise | Dhami, Pawandeep | Ding, Yan | Dinh, Huyen | Dodsworth, Steve | Draper, Heather | Dugan-Rocha, Shannon | Dunham, Andrew | Dunn, Matthew | Durbin, K. James | Dutta, Ireena | Eades, Tamsin | Ellwood, Matthew | Emery-Cohen, Alexandra | Errington, Helen | Evans, Kathryn L. | Faulkner, Louisa | Francis, Fiona | Frankland, John | Fraser, Audrey E. | Galgoczy, Petra | Gilbert, James | Gill, Rachel | Glöckner, Gernot | Gregory, Simon G. | Gribble, Susan | Griffiths, Coline | Grocock, Russell | Gu, Yanghong | Gwilliam, Rhian | Hamilton, Cerissa | Hart, Elizabeth A. | Hawes, Alicia | Heath, Paul D. | Heitmann, Katja | Hennig, Steffen | Hernandez, Judith | Hinzmann, Bernd | Ho, Sarah | Hoffs, Michael | Howden, Phillip J. | Huckle, Elizabeth J. | Hume, Jennifer | Hunt, Paul J. | Hunt, Adrienne R. | Isherwood, Judith | Jacob, Leni | Johnson, David | Jones, Sally | de Jong, Pieter J. | Joseph, Shirin S. | Keenan, Stephen | Kelly, Susan | Kershaw, Joanne K. | Khan, Ziad | Kioschis, Petra | Klages, Sven | Knights, Andrew J. | Kosiura, Anna | Kovar-Smith, Christie | Laird, Gavin K. | Langford, Cordelia | Lawlor, Stephanie | Leversha, Margaret | Lewis, Lora | Liu, Wen | Lloyd, Christine | Lloyd, David M. | Loulseged, Hermela | Loveland, Jane E. | Lovell, Jamieson D. | Lozado, Ryan | Lu, Jing | Lyne, Rachael | Ma, Jie | Maheshwari, Manjula | Matthews, Lucy H. | McDowall, Jennifer | McLaren, Stuart | McMurray, Amanda | Meidl, Patrick | Meitinger, Thomas | Milne, Sarah | Miner, George | Mistry, Shailesh L. | Morgan, Margaret | Morris, Sidney | Müller, Ines | Mullikin, James C. | Nguyen, Ngoc | Nordsiek, Gabriele | Nyakatura, Gerald | O’Dell, Christopher N. | Okwuonu, Geoffery | Palmer, Sophie | Pandian, Richard | Parker, David | Parrish, Julia | Pasternak, Shiran | Patel, Dina | Pearce, Alex V. | Pearson, Danita M. | Pelan, Sarah E. | Perez, Lesette | Porter, Keith M. | Ramsey, Yvonne | Reichwald, Kathrin | Rhodes, Susan | Ridler, Kerry A. | Schlessinger, David | Schueler, Mary G. | Sehra, Harminder K. | Shaw-Smith, Charles | Shen, Hua | Sheridan, Elizabeth M. | Shownkeen, Ratna | Skuce, Carl D. | Smith, Michelle L. | Sotheran, Elizabeth C. | Steingruber, Helen E. | Steward, Charles A. | Storey, Roy | Swann, R. Mark | Swarbreck, David | Tabor, Paul E. | Taudien, Stefan | Taylor, Tineace | Teague, Brian | Thomas, Karen | Thorpe, Andrea | Timms, Kirsten | Tracey, Alan | Trevanion, Steve | Tromans, Anthony C. | d’Urso, Michele | Verduzco, Daniel | Villasana, Donna | Waldron, Lenee | Wall, Melanie | Wang, Qiaoyan | Warren, James | Warry, Georgina L. | Wei, Xuehong | West, Anthony | Whitehead, Siobhan L. | Whiteley, Mathew N. | Wilkinson, Jane E. | Willey, David L. | Williams, Gabrielle | Williams, Leanne | Williamson, Angela | Williamson, Helen | Wilming, Laurens | Woodmansey, Rebecca L. | Wray, Paul W. | Yen, Jennifer | Zhang, Jingkun | Zhou, Jianling | Zoghbi, Huda | Zorilla, Sara | Buck, David | Reinhardt, Richard | Poustka, Annemarie | Rosenthal, André | Lehrach, Hans | Meindl, Alfons | Minx, Patrick J. | Hillier, LaDeana W. | Willard, Huntington F. | Wilson, Richard K. | Waterston, Robert H. | Rice, Catherine M. | Vaudin, Mark | Coulson, Alan | Nelson, David L. | Weinstock, George | Sulston, John E. | Durbin, Richard | Hubbard, Tim | Gibbs, Richard A. | Beck, Stephan | Rogers, Jane | Bentley, David R.
Nature  2005;434(7031):325-337.
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.
doi:10.1038/nature03440
PMCID: PMC2665286  PMID: 15772651
13.  Finishing the finished human chromosome 22 sequence 
Genome Biology  2008;9(5):R78.
A combination of approaches was used to close 8 of the 11 gaps in the original sequence of human chromosome 22, and to generate a total 1.018 Mb of new sequence.
Background
Although the human genome sequence was declared complete in 2004, the sequence was interrupted by 341 gaps of which 308 lay in an estimated approximately 28 Mb of euchromatin. While these gaps constitute only approximately 1% of the sequence, knowledge of the full complement of human genes and regulatory elements is incomplete without their sequences.
Results
We have used a combination of conventional chromosome walking (aided by the availability of end sequences) in fosmid and bacterial artificial chromosome (BAC) libraries, whole chromosome shotgun sequencing, comparative genome analysis and long PCR to finish 8 of the 11 gaps in the initial chromosome 22 sequence. In addition, we have patched four regions of the initial sequence where the original clones were found to be deleted, or contained a deletion allele of a known gene, with a further 126 kb of new sequence. Over 1.018 Mb of new sequence has been generated to extend into and close the gaps, and we have annotated 16 new or extended gene structures and one pseudogene.
Conclusion
Thus, we have made significant progress to completing the sequence of the euchromatic regions of human chromosome 22 using a combination of detailed approaches. Our experience suggests that substantial work remains to close the outstanding gaps in the human genome sequence.
doi:10.1186/gb-2008-9-5-r78
PMCID: PMC2441464  PMID: 18477386
14.  UBE2QL1 is Disrupted by a Constitutional Translocation Associated with Renal Tumor Predisposition and is a Novel Candidate Renal Tumor Suppressor Gene 
Human Mutation  2013;34(12):1650-1661.
Investigation of rare familial forms of renal cell carcinoma (RCC) has led to the identification of genes such as VHL and MET that are also implicated in the pathogenesis of sporadic RCC. In order to identify a novel candidate renal tumor suppressor gene, we characterized the breakpoints of a constitutional balanced translocation, t(5;19)(p15.3;q12), associated with familial RCC and found that a previously uncharacterized gene UBE2QL1 was disrupted by the chromosome 5 breakpoint. UBE2QL1 mRNA expression was downregulated in 78.6% of sporadic RCC and, although no intragenic mutations were detected, gene deletions and promoter region hypermethylation were detected in 17.3% and 20.3%, respectively, of sporadic RCC. Reexpression of UBE2QL1 in a deficient RCC cell line suppressed anchorage-independent growth. UBE2QL1 shows homology to the E2 class of ubiquitin conjugating enzymes and we found that (1) UBE2QL1 possesses an active-site cysteine (C88) that is monoubiquitinated in vivo, and (2) UBE2QL1 interacts with FBXW7 (an F box protein providing substrate recognition to the SCF E3 ubiquitin ligase) and facilitates the degradation of the known FBXW7 targets, CCNE1 and mTOR. These findings suggest UBE2QL1 as a novel candidate renal tumor suppressor gene.
doi:10.1002/humu.22433
PMCID: PMC4028990  PMID: 24000165
renal cell carcinoma; UBE2QL1; ubiquitin conjugating enzyme; FBXW7

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