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Genetic Services-Learning Disability Project Summary This report summarises the technology development aspects of the Learning Disability Project, from the period 6th October, 2003 to 31st March, 2005, within the Regional Molecular Genetics Laboratory, Cambridge. Subtelomeric rearrangements have been shown as a significant causative abnormality in cases of learning disability (LD). Current diagnostic screening strategies based on fluorescent in situ hybridization (FISH) are laborious and costly. We have evaluated multiplex ligation dependent probe amplification (MLPA) as a replacement method, in direct comparison to FISH. MLPA represents a lower cost screening strategy for patients with learning disability, which is amenable to high-throughput screening approaches. We have evaluated MLPA in a group of 150 consecutive LD patients referred on a basis of learning disability and/or developmental delay. All patients were previously screened by FISH. Eighty normal patient control samples were also screened by MLPA alone, to evaluate variation within the MLPA technique. Nine subtelomeric abnormalities were detected in the LD patient sample group. Seven of these abnormalities were detected by FISH, and confirmed by MLPA. Two novel, de novo micro-deletions were detected by MLPA and subsequently confirmed by FISH. To our knowledge, neither deletion has been previously reported. Observation of variation within the control patient samples, has allowed us to evaluate performance of the MLPA method, and optimise application of the technique to the detection of subtelomeric rearrangements in patients with learning disability. As a result of this project, MLPA was implemented as the diagnostic screening method for subtelomeric abnormalities within the Cambridge Genetics Service, in August, 2004. Background Telomeres and LD Recent assessments of learning disability have suggested that the condition is common amongst the general population, with a prevalence of 2-3% (summarised in [1]). Rearrangements involving the subtelomeric regions of human chromosomes, have been identified as a significant cause of learning disability [2]. As these regions are of high gene density [3], abnormalities of copy number in genes at these loci, have a more pronounced phenotypic effect than at other loci throughout the genome. Project Aims Our primary aim has been to introduce telomere screening to a larger cohort of patients than currently offered screening, by the evaluation of MLPA as an alternative test to the present strategy of fluorescent in situ hybridization (FISH). As MLPA offers a number of advantages to FISH, principally in terms of cost and assay time, this may allow a broadening of referral criteria for patients with LD, and expand knowledge of subtelomeric abnormalities associated with this condition. In essence, the evaluation was aimed at determining whether MLPA could detect the same abnormalities as FISH, and if additional abnormalities could be identified and confirmed. Methods MLPA and FISH MLPA is a hybridization and PCR amplification-based method that allows one to determine the relative abundance of over 40 DNA sequences in a test sample [4]. By comparison of abundance to an unaffected individual, abnormalities in the number of copies of each DNA sequence can be identified and quantified. Careful choice of the DNA sequence to be screened, allows the chromosome ends (telomeres) to be tested for abnormal copy number changes associated with learning disability. The particular MLPA assay used throughout this assessment, consists of 2 sets of probes, each set containing 36 probes to quantify the copy number of telomere ends (Figure 1). FISH is a hybridization technique allowing the direct visual detection of the presence and location of a given DNA sequence, on the intact chromosome of a test patient. Chromosomes are prepared in such a way as to allow the presence of the normal complement of two gene copies of a given sequence, to be observed. Absence of a copy is readily detected whilst increases in copy number are more problematic to confirm, especially if the distance between the copies is small. This is a key difference between the methods, with MLPA having the potential to more accurately identify and quantify copy number increases. Patient Samples Approximately 150 referrals for telomere screening by FISH were re-screened by MLPA. The FISH patients were screened as part of the Cytogenetics diagnostic service, and all details about the referral (phenotype, family history etc) were therefore known to the Cytogenetics department. For the MLPA screen, patients were chosen as those with normal karyotype, referred for telomere screening on the basis of a broad phenotype of developmental delay and/or learning disability, and the availability of corresponding DNA and chromosomal material for analysis. Patient identifiers were removed and replaced by a numbering scheme allowing any abnormal samples to be traced back to the original patient. In this way, all MLPA analysis was performed without prior knowledge to any abnormalities identified by FISH. Once abnormal MLPA results had been identified and decoded, FISH was performed to either confirm or refute novel findings. After MLPA analysis was completed, all data were decoded to identify any samples with FISH abnormalities that were not detected by MLPA, and to allow further investigation as to possible causes for such discrepancies. Normal control samples used for the MLPA evaluation, were patients referred to the diagnostic laboratory for reasons other than developmental delay and dysmorphism. All controls were chosen on the basis of a low likelihood of telomeric abnormalities in such referrals. These control samples were de-identified and anonymised in such a manner that deciphering the original patient from the sample was impossible. MLPA Data Analysis The MLPA data were quantitative, and required an assessment of dosage quotient (DQ) ratios to determine whether an abnormality had arisen from deletion of telomeric DNA, or as an artefact within the assay system. The calculation of DQ ratios entailed a mathematical comparison between quantities of DNA generated from a test patient sample, to that generated in a normal control patient sample. Further analysis of DQ ratios allowed determination of statistical variance within the assay, and enabled confidence limits to be assigned to the generated data. To streamline the generation and manipulation of DQ ratio data, unique software was devised and written for this purpose. Results Summary A total of 9 abnormalities were detected by MLPA. Seven abnormalities were previously reported deletions and derivative chromosomes. Two abnormalities were novel, and not detected by FISH with the ToTel Vysion FISH probe panel. Subsequent FISH analysis with additional DNA sequences from the regions of abnormality, confirmed the MLPA results and allowed the extent of the deletions to be determined. Both deletions were de novo and have not been previously reported. The exact phenotypic consequences of each deletion could not be accurately determined due to the large number of genes deleted in each case coupled with the unknown consequence of altered copy number for those genes deleted. Interstitial deletions and duplications at 3q and 4p respectively, were not detected with the MLPA probe sets under evaluation. This was shown to be the result of MLPA probes being located within telomeric regions that did not have altered copy number in these patients. Additional MLPA with probes located within the deleted and duplicated regions, confirmed appropriate performance of the assay. The successful evaluation of MLPA to determine altered copy number of subtelomeric regions in patients with learning disability, has lead to MLPA being offered by this laboratory as the primary screening strategy for such patients. Our current screening strategy consists of MLPA on DNA samples, when referred from a clinical geneticist with a specific request for telomere analysis. Absence of telomeric abnormality is directly reported from the Molecular Genetics laboratory, with detection limitations of the assay being acknowledged on generated reports. Detected abnormalities however, are confirmed by FISH and subsequently reported from the Cytogenetics laboratory. References 1. Flint J, Knight S. The use of telomere probes to investigate submicroscopic rearrangements associated with mental retardation. Curr Opin Genet Dev 2003; 13(3):310-6. 2. Flint J, Wilkie AO, Buckle VJ, Winter RM, Holland AJ, McDermid HE. The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation. Nat Genet 1995; 9(2):132-40. 3. Saccone S, De Sario A, Della Valle G, Bernardi G. The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc Natl Acad Sci U S A 1992; 89(11):4913-7. 4. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002; 30(12):e57. Figure 1 Raw MLPA data showing the 36 telomere probe products (blue), whose peak areas are quantified and compared to those of normal control patients, to determine telomere copy number.