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Loughery J.E.,University of Ulster | Loughery J.E.,University of Dundee | Dunne P.D.,University of Ulster | Dunne P.D.,Queens University of Belfast | And 4 more authors.
Human Molecular Genetics | Year: 2011

DNA methyltransferase 1 (DNMT1) maintains methylation at CpG dinucleotides, important for transcriptional silencing at many loci. It is also implicated in stabilizing repeat sequences: DNMT1 deficiency causes microsatellite instability in mouse embryonic stem cells, but it is unclear how this occurs, how repeats lacking CpG become unstable and whether the effect is confined to stem cells. To address these questions, we transfected hTERT-immortalized normal human fibroblasts (hTERT-1604) with a short hairpin RNA construct targeting DNMT1 and isolated stable integrants with different levels of protein. DNMT1 expression levels agreed well with methylation levels at imprinted genes. Knockdown cells showed two key characteristics of mismatch repair (MMR) deficiency, namely resistance to the drug 6-thioguanine and up to 10-fold elevated mutation rates at a CA 17 microsatellite reporter, but had limited viability. The likely cause of MMR defects is a matching drop in steady-state protein levels for key repair components in DNMT1 knockdown cells, affecting both the MutLa and MutSa complexes. This indirect effect on MMR proteins was also seen using a different targeting method in HT29 colon cancer cells and did not involve transcriptional silencing of the respective genes. Decreased levels of MMR components follow activation of the DNA damage response and blocking this response, and in particular poly(ADP-ribose) polymerase (PARP) overactivation, rescues cell viability in DNMT1-depleted cells. These results offer an explanation for how and why unmethylated microsatellite repeats can be destabilized in cells with decreased DNMT1 levels and uncover a novel and important role for PARP in this process. © The Author 2011. Published by Oxford University Press. All rights reserved. Source

Berwouts S.,Catholic University of Leuven | Fanning K.,Catholic University of Leuven | Morris M.A.,University of Geneva | Barton D.E.,National Center for Medical Genetics | Dequeker E.,Catholic University of Leuven
European Journal of Human Genetics | Year: 2012

In the 2000s, a number of initiatives were taken internationally to improve quality in genetic testing services. To contribute to and update the limited literature available related to this topic, we surveyed 910 human molecular genetic testing laboratories, of which 291 (32%) from 29 European countries responded. The majority of laboratories were in the public sector (81%), affiliated with a university hospital (60%). Only a minority of laboratories was accredited (23%), and 26% was certified. A total of 22% of laboratories did not participate in external quality assessment (EQA) and 28% did not use reference materials (RMs). The main motivations given for accreditation were to improve laboratory profile (85%) and national recognition (84%). Nearly all respondents (95%) would prefer working in an accredited laboratory. In accredited laboratories, participation in EQA (P<0.0001), use of RMs (P<0.0014) and availability of continuous education (CE) on medical/scientific subjects (P=0.023), specific tasks (P<0.0018), and quality assurance (P<0.0001) were significantly higher than in non-accredited laboratories. Non-accredited laboratories expect higher restriction of development of new techniques (P=0.023) and improvement of work satisfaction (P<0.0002) than accredited laboratories. By using a quality implementation score (QIS), we showed that accredited laboratories (average score 92) comply better than certified laboratories (average score 69, P<0.001), and certified laboratories better than other laboratories (average score 44, P<0.001), with regard to the implementation of quality indicators. We conclude that quality practices vary widely in European genetic testing laboratories. This leads to a potentially dangerous situation in which the quality of genetic testing is not consistently assured. © 2012 Macmillan Publishers Limited. All rights reserved. Source

Dhar R.,Our Ladys Hospital for Sick Children | Reardon W.,National Center for Medical Genetics | McMahon C.J.,Our Ladys Hospital for Sick Children
Cardiology in the Young | Year: 2015

We report a baby girl with an antenatal diagnosis of biventricular non-compaction and complete heart block detected at 22 weeks' gestation. Postnatal echocardiography confirmed severe biventricular non-compaction hypertrophic cardiomyopathy, multiple muscular ventricular septal defects, and mild-moderate pulmonary valve stenosis. Skeletal muscle biopsy confirmed complex 1 mitochondrial respiratory chain deficiency. An epicardial VVI pacemaker was implanted on day 3 of life and revised at 7 years of age. She remains stable at 8 years of age following pacing and medical treatment with carvedilol, aspirin, co-enzyme Q10, and carnitine. This represents the first report of biventricular non-compaction hypertrophic phenotype in association with congenital complete heart block and complex 1 mitochondrial respiratory chain deficiency in a child. © Cambridge University Press 2014. Source

Biancalana V.,University of Monastir | Glaeser D.,Center for Human Genetics | McQuaid S.,National Center for Medical Genetics | Steinbach P.,University of Ulm
European Journal of Human Genetics | Year: 2015

Different mutations occurring in the unstable CGG repeat in 5' untranslated region of FMR1 gene are responsible for three fragile X-associated disorders. An expansion of over ∼200 CGG repeats when associated with abnormal methylation and inactivation of the promoter is the mutation termed 'full mutation' and is responsible for fragile X syndrome (FXS), a neurodevelopmental disorder described as the most common cause of inherited intellectual impairment. The term 'abnormal methylation' is used here to distinguish the DNA methylation induced by the expanded repeat from the 'normal methylation' occurring on the inactive X chromosomes in females with normal, premutation, and full mutation alleles. All male and roughly half of the female full mutation carriers have FXS. Another anomaly termed 'premutation' is characterized by the presence of 55 to ∼200 CGGs without abnormal methylation, and is the cause of two other diseases with incomplete penetrance. One is fragile X-associated primary ovarian insufficiency (FXPOI), which is characterized by a large spectrum of ovarian dysfunction phenotypes and possible early menopause as the end stage. The other is fragile X-associated tremor/ataxia syndrome (FXTAS), which is a late onset neurodegenerative disorder affecting males and females. Because of the particular pattern and transmission of the CGG repeat, appropriate molecular testing and reporting is very important for the optimal genetic counselling in the three fragile X-associated disorders. Here, we describe best practice guidelines for genetic analysis and reporting in FXS, FXPOI, and FXTAS, including carrier and prenatal testing. © 2015 Macmillan Publishers Limited All rights reserved. Source

Simpson M.A.,Kings College London | Deshpande C.,Clinical Genetics | Dafou D.,Kings College London | Vissers L.E.L.M.,Radboud University Nijmegen | And 13 more authors.
American Journal of Human Genetics | Year: 2012

Genitopatellar syndrome (GPS) is a rare disorder in which patellar aplasia or hypoplasia is associated with external genital anomalies and severe intellectual disability. Using an exome-sequencing approach, we identified de novo mutations of KAT6B in five individuals with GPS; a single nonsense variant and three frameshift indels, including a 4 bp deletion observed in two cases. All identified mutations are located within the terminal exon of the gene and are predicted to generate a truncated protein product lacking evolutionarily conserved domains. KAT6B encodes a member of the MYST family of histone acetyltranferases. We demonstrate a reduced level of both histone H3 and H4 acetylation in patient-derived cells suggesting that dysregulation of histone acetylation is a direct functional consequence of GPS alleles. These findings define the genetic basis of GPS and illustrate the complex role of the regulation of histone acetylation during development. © 2012 The American Society of Human Genetics. Source

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