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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.