Research Area:	Genetics and Genomics
Technology Exchange:	Chromosome mapping
Keywords:	learning disability, genome imbalance, copy number variation, diagnosis and array CGH

Our previous work within the Oxford Genetics Knowledge Park showed that array based comparative genomic hybridization (aCGH) is a powerful technique for detecting clinically relevant genome imbalance, offering 40-1000 times the resolution of karyotyping and making at least 10-15% more diagnoses in patients with idiopathic learning disability (ILD). In collaboration, this work has also led to the identification of novel syndromes, most notably a recurrent 17q21.31 microdeletion syndrome and more recently, 15q24 and 15q13 microdeletion syndromes. In addition, our evaluation of aCGH platforms for clinical diagnostic use has resulted in recommendation and implementation within the ORH Trust's Clinical Cytogenetics Laboratory at the Churchill Hospital, Oxford.

Building upon the achievements and expertise gained within the Oxford GKP, our current goal is to maximise the potential of microarray technology within the NHS. This is being undertaken as part of the Oxford Partnership Comprehensive Biomedical Research Centre (BRC), a newly funded Government initiative established to drive the development, evaluation, optimization and transfer of clinically useful research tools into the clinical diagnostic environment so that ill-health may be prevented, diagnosed and treated much sooner than previously possible. Genetics is a key cross-cutting theme of the Oxford BRC, with application in variety of disease themes including Immunology, Diabetes, Cardiology, Cancer and Neurology. Currently, our laboratory is situated within the Wellcome Trust Centre for Human Genetics, housing state of the art technologies including microarray, genotyping and high-throughput sequencing facilities. Initial aims include evaluating multi-sample array formats and investigating the use of high-resolution oligonucleotide arrays for the diagnosis of clinically important conditions such as idiopathic learning disability and disorders of speech and language impairment (working with Dr Simon Fisher and Professor Anthony Monaco). We are also embarking on collaborative studies with Professor Shoumo Bhattacharya investigating genome imbalance in congenital heart disease and with Dr Anna Schuh investigating chronic lymphocytic leukaemia. Soon, the work will expand to include other approaches such as gene expression, genotyping, biomarker and sequencing studies with clinical application in other diseases. Ultimately, we hope to achieve significant advances in diagnosis, genetic counselling, education, therapeutics and preventative strategies thereby resulting in the improved welfare of patients and their families.

Name	Department	Institution	Country
Evan Eichler		Howrad Hughes Medical Institute	USA
Andrew Sharp		Department of Genome Sciences, Washington	USA
Corrado Romano		Oasi Institute for Research on Mental Retardation and Brain Aging	Sicily
Dr Simon E Fisher	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Jane Hurst		Oxford Radcliffe Hospitals NHS Trust	UK
Andrew Wilkie		Weatherall Institute of Medical Genetics, University of Oxford	UK

Sharp AJ, Mefford HC, Li K, Baker C, Skinner C, Stevenson RE, Schroer RJ, Novara F, De Gregori M, Ciccone R, Broomer A, Casuga I, Wang Y, Xiao C, Barbacioru C, Gimelli G, Bernardina BD, Torniero C, Giorda R, Regan R, Murday V, Mansour S, Fichera M, Castiglia L, Failla P, Ventura M, Jiang Z, Cooper GM, Knight SJ, Romano C, Zuffardi O, Chen C, Schwartz CE, Eichler EE et al. 2008. A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures. Nat Genet, 40 (3), pp. 322-328. Read abstract | Read more

We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4-BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is approximately 0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes. Hide abstract

Sharp AJ, Hansen S, Selzer RR, Cheng Z, Regan R, Hurst JA, Stewart H, Price SM, Blair E, Hennekam RC, Fitzpatrick CA, Segraves R, Richmond TA, Guiver C, Albertson DG, Pinkel D, Eis PS, Schwartz S, Knight SJ, Eichler EE et al. 2006. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet, 38 (9), pp. 1038-1042. Read abstract | Read more

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions. Hide abstract

Price TS, Regan R, Mott R, Hedman A, Honey B, Daniels RJ, Smith L, Greenfield A, Tiganescu A, Buckle V, Ventress N, Ayyub H, Salhan A, Pedraza-Diaz S, Broxholme J, Ragoussis J, Higgs DR, Flint J, Knight SJ et al. 2005. SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data. Nucleic Acids Res, 33 (11), pp. 3455-3464. Read abstract | Read more

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides. Hide abstract

Knight SJ, Lese CM, Precht KS, Kuc J, Ning Y, Lucas S, Regan R, Brenan M, Nicod A, Lawrie NM, Cardy DL, Nguyen H, Hudson TJ, Riethman HC, Ledbetter DH, Flint J et al. 2000. An optimized set of human telomere clones for studying telomere integrity and architecture. Am J Hum Genet, 67 (2), pp. 320-332. Read abstract | Read more

Telomere-specific clones are a valuable resource for the characterization of chromosomal rearrangements. We previously reported a first-generation set of human telomere probes consisting of 34 genomic clones, which were a known distance from the end of the chromosome ( approximately 300 kb), and 7 clones corresponding to the most distal markers on the integrated genetic/physical map (1p, 5p, 6p, 9p, 12p, 15q, and 20q). Subsequently, this resource has been optimized and completed: the size of the genomic clones has been expanded to a target size of 100-200 kb, which is optimal for use in genome-scanning methodologies, and additional probes for the remaining seven telomeres have been identified. For each clone we give an associated mapped sequence-tagged site and provide distances from the telomere estimated using a combination of fiberFISH, interphase FISH, sequence analysis, and radiation-hybrid mapping. This updated set of telomeric clones is an invaluable resource for clinical diagnosis and represents an important contribution to genetic and physical mapping efforts aimed at telomeric regions. Hide abstract

Knight SJ, Regan R, Nicod A, Horsley SW, Kearney L, Homfray T, Winter RM, Bolton P, Flint J. 1999. Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet, 354 (9191), pp. 1676-1681. Read abstract | Read more

No explanation for moderate to severe mental retardation is apparent in about 40% of cases. Although small chromosomal rearrangements may account for some undiagnosed cases, a lack of genome-wide screening methods has made it impossible to ascertain the frequency of such abnormalities. Hide abstract

Knight SJ, Horsley SW, Regan R, Lawrie NM, Maher EJ, Cardy DL, Flint J, Kearney L. 1997. Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres. Eur J Hum Genet, 5 (1), pp. 1-8. Read abstract

We report an innovative fluorescence in situ hybridization technique which exploits a unique resource of 41 telomere-specific probes and allows the simultaneous analysis of the subtelomeric region of every chromosome for deletion, triplication and balanced translocation events. This technique requires only a single microscope slide per patient and is expected to be a useful diagnostic tool with applications in the fields of idiopathic mental retardation, the detection of congenital abnormalities and in some forms of cancer. This will lead to more accurate genetic counselling of patients and their families and will provide the basis for future diagnostic, therapeutic and preventative measures. Hide abstract

Chakrabarti L, Knight SJ, Flannery AV, Davies KE. 1996. A candidate gene for mild mental handicap at the FRAXE fragile site. Hum Mol Genet, 5 (2), pp. 275-282. Read abstract

The cytogenetic expression of the folate sensitive fragile site, FRAXE, is due to the expansion of a GCC repeat in proximal Xq28 of the human X chromosome and is associated with a mild form of mental handicap. Normal individuals have 6-35 copies of the repeat whereas cytogenetically positive, developmentally delayed males have > 200 copies and show methylation of the associated CpG island. Through the use of conserved sequences adjacent to the FRAXE GCC repeat, we have isolated a 1495 bp cDNA which begins 331 bp distal to the FRAXE site and extends to a region > 170 kb distal in Xq28. The cDNA sequence possesses both a putative start of translation and a poly-A tail. The predicted protein has amino acid motifs which share significant homologies with the human AF-4 gene which encodes a putative transcription factor. On northern analysis, the cDNA detects a 9.5 kb transcript in human brain, placenta and lung. This transcript is present in multiple human brain tissues, but is more abundant in the hippocampus and the amygdala, thus providing possible functional insights. RT-PCR of normal adult brain RNA provides evidence for the existence of the 1495 bp transcript represented by the isolated cDNA. Hide abstract

Knight SJ, Flannery AV, Hirst MC, Campbell L, Christodoulou Z, Phelps SR, Pointon J, Middleton-Price HR, Barnicoat A, Pembrey ME. 1993. Trinucleotide repeat amplification and hypermethylation of a CpG island in FRAXE mental retardation. Cell, 74 (1), pp. 127-134. Read abstract

We have cloned the fragile site FRAXE and demonstrate that individuals with this fragile site possess amplifications of a GCC repeat adjacent to a CpG island in Xq28 of the human X chromosome. Normal individuals have 6-25 copies of the GCC repeat, whereas mentally retarded, FRAXE-positive individuals have > 200 copies and also have methylation at the CpG island. This situation is similar to that seen at the FRAXA locus and is another example in which a trinucleotide repeat expansion is associated with a human genetic disorder. In contrast with the fragile X syndrome, the GCC repeat can expand or contract and is equally unstable when passed through the male or female line. These results also have implications for the understanding of chromosome fragility. Hide abstract