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Analysis of telomere status in patients with Fanconi anaemia (FA) has previously been carried out by measurement of telomere restriction fragment (TRF) length by Southern blotting and densitometry. Results from these studies indicated that FA patients had significant reduction in telomere length compared with age-matched controls. This paper confirms and extends these findings using a direct FISH technique, which showed that 15 out of 16 FA patients had increased loss of telomere signals compared with controls. In 12 out of the 16 patients, decrease in telomere signal intensity could also be detected using a Q-FISH approach. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A dicentric chromosome 17 is described in a 5-year-old girl with minor malformations and severe mental retardation. The anomaly is interpreted as a de novo duplication of band 17p11 and centromere 17. [ABSTRACT FROM PUBLISHER]

A 13 year old male with mild mental retardation, obesity, and poor secondary sexual differentiation was found to have a 46,X,+,mar karyotype. In situ hybridisation with X and Y specific probes proved the marker to be composed of Y centromeric and short arm material. [ABSTRACT FROM PUBLISHER]

A variant chromosome 4 with a large G positive heterochromatic block is described and discussed in relation to chromosome 4 heteromorphisms observed with other banding techniques. The extra heterochromatin is C positive and fluoresces brilliantly with Q banding, but differs from Yqh with some methods of staining. [ABSTRACT FROM PUBLISHER]

Abstract Background Several studies have demonstrated that array comparative genomic hybridisation (CGH) for genome-wide imbalance provides a substantial increase in diagnostic yield for patients traditionally referred for karyotyping by G-banded chromosome analysis. The purpose of this study was to demonstrate the feasibility of and strategies for, the use of array CGH in place of karyotyping for genome imbalance, and to report on the results of the implementation of this approach. Results Following a validation period, an oligoarray platform was chosen. In order to minimise costs and increase efficiency, a patient/patient hybridisation strategy was used, and analysis criteria were set to optimise detection of pathogenic imbalance. A customised database application with direct links to a number of online resources was developed to allow efficient management and tracking of patient samples and facilitate interpretation of results. Following introduction into our routine diagnostic service for patients with suspected genome imbalance, array CGH as a follow-on test for patients with normal karyotypes (n = 1245) and as a first-line test (n = 1169) gave imbalance detection rates of 26% and 22% respectively (excluding common, benign variants). At least 89% of the abnormalities detected by first line testing would not have been detected by standard karyotype analysis. The average reporting time for first-line tests was 25 days from receipt of sample. Conclusions Array CGH can be used in a diagnostic service setting in place of G-banded chromosome analysis, providing a more comprehensive and objective test for patients with suspected genome imbalance. The increase in consumable costs can be minimised by employing appropriate hybridisation strategies; the use of robotics and a customised database application to process multiple samples reduces staffing costs and streamlines analysis, interpretation and reporting of results. Array CGH provides a substantially higher diagnostic yield than G-banded chromosome analysis, thereby alleviating the burden of further clinical investigations.

Abstract Background Microdeletion syndromes are generally identified because they usually give rise to specific phenotypic features; many of these deletions are mediated by duplicons or LCRs. The phenotypes associated with subtelomeric deletions are also becoming recognised. However, reciprocal duplication events at these loci are less easily recognised and identified, as they may give rise to milder phenotypic features, and the individuals carrying them may not therefore be referred for appropriate testing. 403 patients with developmental delay and/or dysmorphism, referred to our Genetics Centre for karyotyping and Fragile X expansion testing, were assessed for chromosome imbalance by Multiplex Ligation-dependent Probe Amplification (MLPA). Two MLPA kits were used, one containing probes for the subtelomere regions, and one containing probes for common microdeletion loci. 321 patients were tested with both kits, 75 with the subtelomere kit alone, and 7 with the microdeletion kit alone. Results 32 patients had abnormal results; the overall abnormality detection rate was 2.5% for karyotype analysis and 7.2% for MLPA testing; 5.5% of subtelomere tests and 2.1% of microdeletion tests gave abnormal results. Of the abnormal MLPA results, 5 were in cases with cytogenetically visible abnormalities; of the remaining, submicroscopic, changes, 3 results were established as de novo and 8 were inherited; parental samples were not available for the remaining cases. None of the patients was found to have a Fragile X expansion. Conclusion Karyotype analysis in combination with MLPA assays for subtelomeres and microdeletion loci may be recommended for this patient group.

Presents an abstract of the article "Radiosensitivity in Breast Cancer Susceptibility," by Julian Barwell, A. Georgiou, I. Kesterton, L. Pangon, C. Langman, J. Berg, Z. Kote-Jarai, P. Green, N. Sodha, J. Morris, E. Solomon, Z. Docherty, R. Camplejohn, R. Eeles, and S. Hodgson.

Comparative Genomic Hybridization (CGH) is an established method for detection of genomic imbalance. CGH has been introduced into this laboratory as a tool in the characterisation of constitutional chromosome abnormalities. Following DNA extraction, labelling and hybridization of test and control DNA onto normal human metaphases, regions of genomic imbalance were detected using a fluorescence microscope, CCD image analysis system and dedicated software. We have used CGH in six cases where partial chromosome duplication was identified by G-banded chromosome analysis and fluorescence in situ hybridization (FISH), but where the breakpoints could not be definitively assigned due to ambiguous banding patterns. CGH analysis was informative for these six cases and showed: dup(1)(q31q32.1); dup(4)(q28.2q33); dup(11)(q23.2q25); dup(16)(q21q24); dup(X)(q26.1q28); dup(6)(q23.3q24.3). In two further cases, CGH was uninformative: one case of a small amount of additional material on the short arm of chromosome 8, and one case of del(11)(q14.3q21), dup(10)(q26.11q26.13). It is concluded that these imbalances were beyond the sensitivity of CGH. In another case, a tissue sample from a fetus with raised nuchal translucency failed to grow in culture. Quantitative Fluorescence PCR (QF-PCR) analysis was consistent with a monosomy X (Turner syndrome) karyotype, but interphase FISH with an X chromosome centromeric probe showed two signals. CGH analysis of fetal DNA indicated the presence of only a single X chromosome. A small pericentromeric X marker chromosome may have been the cause of the second FISH signal. We conclude that CGH is a useful diagnostic tool in the elucidation of constitutional chromosome abnormalities. [ABSTRACT FROM AUTHOR]

Chromosome instability syndromes are a diverse group of genetic disorders characterised by the abnormal behaviour of their chromosomes. This can be elevated spontaneous chromosome breakage; hypersensitivity to chromosome damaging agents; elevated sister chromatid exchanges (SCE); or clonal chromosome rearrangements. We report the experience of the South East Thames Regional Genetics Centre over the last 10 years in pre- and postnatal diagnosis of Fanconi anaemia (FA), Ataxia Telangiectasia (AT), Nijmegen breakage syndrome (NBS) and Bloom syndrome. FA testing detects hypersensitivity to diepoxybutane while AT and NBS testing detects hypersensitivity to ionising radiation and the presence of characteristic rearrangements. Bloom syndrome testing detects raised SCE levels. 830 postnatal tests for FA were performed with 117 (14%) positive of which 17 (14%) had a two cell population; 33 prenatal tests detected 3 {9%) positive cases. For AT, 130 postnatal tests were performed with 19 (14.6%) positive of which 3 showed no significant radiosensitivity only clonal rearrangements, and 13 prenatal tests, all negative. For NBS over 36 postnatal tests have been performed with 1 (2%) positive, and 1 prenatal test gave a positive result. A total of 21 postnatal tests have been performed for Bloom syndrome of which one (5%) was positive. Our experience shows that interpretation is not always straightforward; cases showing intermediate levels of breakage and those with two cell populations pose interesting diagnostic challenges. Chromosome instability testing should therefore be carried out only in centres with established experience in offering this specialist service. [ABSTRACT FROM AUTHOR]

Background: Prenatal diagnosis for chromosome abnormality is routinely undertaken by full karyotype analysis of chromosomes from cultured cells; pregnant women must wait on average 13-14 days for their results. Autosomal trisomies, which account for around 80% of significant abnormalities, can be detected by quantitative fluorescence (QF) PCR. We report on the development and implementation of this technique as the first such routine service within a diagnostic department of the UK National Health Service (NHS). Methods: We designed a "one-tube test" comprising four primer pairs for polymorphic tetranucleotide repeat sequences on chromosome 21, four primer pairs for sequences on chromosome 18, three primer pairs for sequences on chromosome 13, and one primer pair to identify the sex chromosomes. All prenatal samples received by our NHS diagnostic department between April, 2000, and April, 2001, were tested. After DNA extraction, PCR amplification was done and the products separated on a capillary-based genetic analyser; the results were interpreted with dedicated software. Follow-up karyotype analysis was done on all samples. Findings: 1148 amniotic fluid samples, 188 chorionic villus samples, and 37 fetal tissue samples were tested; the amplification failure rate was zero with our current protocol. QF-PCR results were obtained and reported on 1314 (98%) of the prenatal samples; the remaining 22 (2%) were uninformative because of maternal-cell contamination. One case of mosaicism in a chorionic villus sample, and two cases indicating somatic expansion of a tetranucleotide repeat were found. No false positive or false negative results were obtained. The mean reporting time for the last 4 months of data collection was 1.25 working days. Interpretation: QF-PCR aneuploidy testing is an efficient and accurate technique for the detection of autosomal trisomies in prenatal samples. Implementation of this service has led to the rapid diagnosis of abnormalities and early reassurance for women with normal results. [ABSTRACT FROM AUTHOR]

Objective: To analyse the results of the first 2 years of a QF-PCR stand-alone testing strategy for the prenatal diagnosis of aneuploidy in the London region and to determine the advantages and disadvantages of this policy., Methods: A review of the results of 9737 prenatal samples received for exclusion of chromosome abnormalities. All samples were subjected to QF-PCR testing for common aneuploidies but only samples fulfilling specific criteria subsequently had a full karyotype analysis., Results: Of the 9737 samples received, 10.3% had a chromosome abnormality detected by QF-PCR testing. Of the 7284 samples received with no indication for karyotype analysis, 25 (0.3%) received a normal QF-PCR result but subsequently had an abnormal karyotype detected either prenatally as a privately funded test or postnatally. Of these samples, without subsequent abnormal ultrasound findings, five had a chromosome abnormality associated with a poor prognosis, representing 0.069% of samples referred for Down syndrome testing., Conclusion: While back-up karyotyping is required for some samples, using QF-PCR as a stand-alone prenatal test for pregnancies without ultrasound abnormalities reduces costs, provides rapid delivery of results, and avoids ambiguous and uncertain karyotype results, reducing parental anxiety.

Objectives: To replace G-banded chromosome analysis for miscarriage products with a combined molecular approach: QF-PCR and MLPA, to increase efficiency, reduce costs, and improve the diagnostic success rate for these samples., Methods: A review of 10 years of karyotype results for miscarriages products indicated that 2.7% of nonmosaic chromosome imbalance would not be detected by the molecular approach. The molecular approach was validated on 117 samples in parallel with karyotype analysis; no discrepancies were detected. The molecular approach was implemented in September 2007, and in the first 18 months 500 samples were processed., Results: In 500 samples, 117 samples (23%) were abnormal. Of these abnormalities, 64% were trisomies, 12% triploid, 11% monosomy X and 13% other abnormalities. When compared to karyotype analysis, the success rate was higher (95% cf 70%) and the reporting time was lower (88% within 28 days cf 79%). In addition, efficiency was higher as labour-intensive cell culture and karyotyping were replaced by batch testing and automated analysis., Conclusions: This molecular approach is less labour-intensive, allows a higher sample throughput and has a higher success rate than karyotype analysis; it is therefore an efficient and cost-effective diagnostic testing strategy for miscarriage products., ((c) 2009 John Wiley & Sons, Ltd.)

Karyotyping is currently the "gold standard" test for the detection of human chromosome abnormalities. Over the past 40 years, changes in techniques have improved the band definition of chromosomes; however, very little has changed with respect to improvements through automation. In this study, we compare chromosome analysis by traditional microscopy with semi-automatic karyotyping using robotic equipment from MetaSystems (Altlussheim, Germany). Analysis using MetaSystems was significantly quicker than using the microscope with an average reduction in analysis time of 26.5 minutes; for the average analyst, this equates to a reduction of 27 percent. Analysis checking times using MetaSystems showed even greater improvement with an average reduction in checking time of 11.4 minutes; for the average checker, this equates to a reduction of 48 percent. The MetaSystems semi-automatic karyotyping equipment offers increased throughput of cases for karyotype analysis while maintaining accuracy.

There is conflicting evidence as to whether individuals who are heterozygous for germ-line BRCA1 or BRCA2 mutations have an altered phenotypic cellular response to irradiation. To investigate this, chromosome breakage and apoptotic response were measured after irradiation in peripheral blood lymphocytes from 26 BRCA1 and 18 BRCA2 mutation carriers without diagnosed breast cancer, and 38 unaffected age, ethnically and sex-matched controls. To assess the role of BRCA1 and BRCA2 in homologous recombination, an S phase enrichment chromosome breakage assay was used. BrdUrd incorporation studies allowed verification of the correct experimental settings. We found that BRCA1 mutation carriers without cancer had increased chromosome breaks as well as breaks and gaps per cell post irradiation using the classical G2 assay (p = 0.01 and 0.004, respectively) and the S phase enrichment assay (p = 0.01 and 0.01, respectively) compared to age-matched unaffected controls. BRCA2 mutation carriers without cancer had increased breaks as well as breaks and gaps per cell post irradiation using the S phase enrichment assay (p = 0.045 and 0.012, respectively). No difference was detected using the G2 assay (p = 0.88 and 0.40 respectively). BRCA1 and BRCA2 mutation carriers had normal cell cycle kinetics and apoptotic response to irradiation compared to age-matched controls. Our results show a demonstrable impairment in irradiation induced DNA repair in women with heterozygous germline BRCA1 and BRCA2 mutations prior to being diagnosed with breast cancer.

Large-scale copy number variation that is cytogenetically visible in normal individuals has been described as euchromatic variation but needs to be distinguished from pathogenic euchromatic deletion or duplication. Here, we report eight patients (three families and two individuals) with interstitial deletions of 9q13-q21.12. Fluorescence in situ hybridisation with a large panel of BACs showed that all the deleted clones were from extensive tracts of segmentally duplicated euchromatin, copies of which map to both the long and short arms of chromosome 9. The variety of reasons for which these patients were ascertained, and the phenotypically normal parents, indicates that this is a novel euchromatic variant with no phenotypic effect. Further, four patients with classical euchromatic variants of 9q12/qh or 9p12 were also shown to have duplications or triplications of this segmentally duplicated material common to both 9p and 9q. The cytogenetic boundaries between the segmentally duplicated regions and flanking unique sequences were mapped to 9p13.1 in the short arm (BAC RP11-402N8 at 38.7 Mb) and to 9q21.12 in the long arm (BAC RP11-88I18 at 70.3 Mb). The BACs identified in this study should in future make it possible to differentiate between clinically significant deletions or duplications and euchromatic variants with no established phenotypic consequences.

Purpose: Individuals who have been treated for breast cancer have been reported to have increased lymphocyte chromosomal sensitivity to ionizing radiation and a significantly lower apoptotic response to irradiation compared to controls. We set out to test these findings using a substantial number of cases sampled before treatment (which could alter the parameters measured), compared to age-matched controls with normal mammograms., Material and Methods: We used the G2 chromosome breakage, and apoptotic response assays of peripheral blood lymphocytes to ionizing radiation to compare 211 unselected newly diagnosed and untreated breast cancer patients, with 170 age, sex and ethnically matched controls., Results: We found no significant differences between breast cancer patients and their matched controls in the G2 assay or apoptotic response. However, there was some evidence that both cases and controls with a strong family history of breast cancer had higher radiosensitivity than those without., Conclusions: This is the largest and best controlled study of its kind, but it has not replicated previous reports of differences between chromosome breakage or apoptotic response in breast cancer cases vs. controls. However there was a suggestion of increased radiosensitivity in patients with a strong family history, which may indicate a heritable cancer susceptibility trait, warranting further study.

Molecular cytogenetic aneuploidy testing for pregnant women at increased risk of chromosome abnormality leads to rapid reassurance for those with normal results and earlier decisions on pregnancy management in the case of abnormality. We tested 9080 prenatal samples using a one-tube QF-PCR test for trisomies 13, 18, and 21; the abnormality rate was 5.9%. There were no misdiagnoses for non-mosaic trisomy. A sex chromosome multiplex was developed that detects structural sex chromosome abnormalities as well as aneuploidies. The sex chromosome test was targeted at pregnancies (272) with specific abnormalities suggestive of Turner syndrome; 13.2% showed 45,X, confirmed by follow-up analysis.

Objectives: QF-PCR can be used to rapidly diagnose primary trisomy in prenatal samples. Our objectives were to estimate the prevalence of primary trisomy mosaicism for chromosomes 13, 18 or 21 in a cohort of prenatal samples, and to compare and contrast the detection of this mosaicism using both QF-PCR and karyotype analysis., Methods: Data was collated from all prenatal samples displaying mosaicism for a primary trisomy between June 2000 and March 2004. Levels of mosaicism were estimated and samples were categorised according to the cell population in which the mosaicism was detected., Results: In a total of 8983 samples, 18 samples (0.20%) displaying mosaicism were detected, including trisomy 13 (three samples), trisomy 18 (seven samples), trisomy 21 (seven samples) and mosaic triploidy (one sample). This included 7 amniotic fluid and 11 chorionic villus samples. Mosaicism was detected by QF-PCR in 12 samples and by karyotype analysis in 8 samples., Conclusions: QF-PCR can detect mosaicism when the abnormal cell line contributes at least 15% of the whole sample. Use of both karyotype and QF-PCR analysis leads to the detection of more cases of mosaicism than either test alone., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Objectives: To establish the genotype of cultured cells from a cohort of amniotic fluid and chorionic villus samples, and compare this genotype with that obtained from uncultured material from the same sample, in order to assess the frequency and significance of maternal cell contamination of prenatal samples., Methods: Quantitative fluorescence-polymerase chain reaction (QF-PCR) was carried out by amplification of microsatellite markers using fluorescence-labelled primers, followed by quantitative analysis of the allele peaks on a genetic analyser. A multiplex of 12 primer pairs for four loci on each of chromosomes 13, 18 and 21 was used., Results: A total of 307 prenatal samples were tested. Of the 254 amniotic fluid samples, 39.8% had some degree of bloodstaining, ranging from 5% bloodstaining in the cell pellet to heavily bloodstained fluid. Uncultured samples were tested by QF-PCR analysis and the cultured cells were tested by both QF-PCR and karyotype analysis. Of the samples, 90.2% had the same single genotype on direct and cultured material. Two samples (0.65%) were mosaic for an aneuploidy cell line. A second genotype, interpreted as maternal cell contamination, was identified in direct and/or cultured preparations in 9.1% of samples, 17.8% of which were not bloodstained. Seven amniotic fluid samples (2.8%) showed maternal cell contamination in cultured material., Conclusions: For heavily bloodstained amniotic fluid samples, a maternal blood specimen may help interpret the results of rapid trisomy testing, followed by confirmation of the fetal origin of cultured cells. QF-PCR analysis has established a higher incidence of maternal cell contamination of cultured amniocytes than previous reports; the presence of MCC (maternal cell contamination) in cultured cells from samples with no bloodstaining underlines the need for karyotype analysis of more than one XX culture., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Rapid diagnosis of common chromosome aneuploidies in raised risk pregnancies, usually prior to full karyotype analysis, is now carried out in a number of European genetic centres; several techniques for detecting genomic copy number changes have been described. Prenatal diagnosis of genetic disease requires accurate and robust assays; the invasive procedures are associated with a risk of pregnancy loss and an abnormal result may lead to termination of the pregnancy. The testing of prenatal material (amniotic fluid, chorionic villi or, more rarely, fetal blood) is associated with specific problems, including the quality and quantity of the tissue and difficulties of interpretation due to phenomena such as maternal cell contamination and mosaicism. In addition, there are 24-h, high-throughput demands on centres offering such a service. The extent to which existing and proposed strategies, including different PCR-based assays, a multiplex ligation-dependent probe amplification approach, and microarrays, fulfil the requirements of rapid prenatal testing is discussed. In the past 3 years, we have tested 7720 prenatal samples for trisomies 13, 18 and 21 using a quantitative fluorescence-PCR (QF-PCR) approach. The abnormality rate was 5.7%. There were no misdiagnoses for nonmosaic trisomy, the amplification failure rate was 0.09% of samples, and 97% of samples received a report on the working day following sample receipt. Maternal cell contamination and mosaicism were also detected. Our data recommend a QF-PCR approach as the current method of choice for rapid aneuploidy testing.

Telomeric repeat sequences (TTAGGG) are known to cap the termini of every human chromosome. Proximal to these repeat sequences are chromosome-specific repeat sequences, which in turn are distal to gene-rich regions. Submicroscopic, subtle, or cryptic abnormalities in these regions can now be investigated using commercial probe sets for all of the chromosome-specific subtelomeric regions of the human genome. Using this technology, previously unidentified genomic imbalance has been found in a proportion of patients with idiopathic developmental delay and learning difficulties. We have used these probe sets to investigate cases with apparently terminal anomalies detected on G-banded chromosome analysis. As a result of such investigations, we have found that 3 (19%) of 16 apparently terminal deletion cases were the result of more complex rearrangements involving other chromosome subtelomeres. The remaining 13 cases contained no chromosome-specific subtelomere repeats on the deleted arm, but in all 16 cases, the TTAGGG telomere repeat cap was present. A further case was investigated where extra material was found in the terminal region of the chromosome 12 short arm, found to represent a complex inversion/duplication/deletion rearrangement. Investigation of all cases with terminal anomalies, including apparently terminal deletions, is likely to uncover further cases involving complex rearrangements and should lead to a greater understanding of the mechanisms by which these abnormalities arise., (Copyright 2003 Wiley-Liss, Inc.)

Childhood autism is characterised by impairments in communication and reciprocal social interaction together with restricted/stereotyped interests, which are evident before 3 years of age. Specific developmental disorders of speech and language (SDDSL) are characterised by impairment in the development of expressive and/or receptive language skills which is not associated with intellectual, sensory, physical, or neurological impairment. Family and twin studies indicate a substantial genetic component in the aetiology of both disorders. They also reveal increased rates of SDDSL in relatives of autistic individuals, suggesting that this phenotype can represent one manifestation of the genetic liability for autism. Modelling of the recurrence risk for autism and milder phenotypes, such as SDDSL, suggest that three or four epistatic loci may be aetiologically involved. A recently published linkage study of an exceptional family with an apparently dominantly inherited SDDSL implicated chromosome band 7q31 as the site of the putative susceptibility locus (SPCH1). This region of chromosome 7 also shows strong linkage in multiplex families with autism. We present two individuals (one has autism, the other SDDSL) with different, apparently balanced chromosome rearrangements involving a breakpoint at 7q31.3. Fluorescence in situ hybridisation was used to localise the breakpoints to an approximately 1 cM interval between CFTR and D7S643. Our findings may be of interest and relevance to the genetic aetiology of autism, and helpful in the search for susceptibility loci for SDDSL and autism. Am. J. Med. Genet. (Neuropsychiatr. Genet. ) 96:228-234, 2000., (Copyright 2000 Wiley-Liss, Inc.)

A group of 103 subjects with a strict diagnosis of autism were tested for deletion of band q11.2 on the long arm of chromosome 22. No deletions were found, indicating that when a patient has been diagnosed with autism using strict and consistent criteria, in the absence of other indications, it is unlikely that this individual will have a 22q11 deletion. Testing for 22q11 deletions is therefore unlikely to be necessary in these patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:15-17, 2000., (Copyright 2000 Wiley-Liss, Inc.)

Menkes disease is a rare X-linked recessive disorder of copper metabolism, characterised by progressive neurological degeneration, abnormal hair and connective tissue manifestations. We report on a girl with classic Menkes disease, carrying a de novo balanced translocation 46,X,t(X;13)(q13.3; q14.3). The translocation breakpoints at Xq13.3 and 13q14.3 coincide with the Menkes disease and Wilson disease loci, respectively., (Copyright 1999 Wiley-Liss, Inc.)

The phenotype of four cases of Smith-Lemli-Opitz syndrome (SLO) with proven defects in cholesterol biosynthesis are compared, and shown to be markedly disparate even between sibs, and demonstrate the dilemma for the clinician. The advent of a biochemical test for SLO has been enormously valuable in determining which patients are truly affected by the condition, but because of the wide phenotypic variation, a diagnosis on clinical features alone remains problematic.