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Amsterdam-Zuidoost, Netherlands

Zuiverloon T.C.M.,Erasmus MC | Beukers W.,Erasmus MC | Van Der Keur K.A.,Erasmus MC | Munoz J.R.,Erasmus MC | And 4 more authors.
BJU International | Year: 2012

OBJECTIVE To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. PATIENTS AND METHODS Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patientswith BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). RESULTS The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC-a, TERT-a, TERT-b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). CONCLUSIONS We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance. © 2011 BJU INTERNATIONAL.

Raja Rayan D.L.,UK National Institute for Medical Research | Haworth A.,University College London | Sud R.,University College London | Matthews E.,UK National Institute for Medical Research | And 10 more authors.
Neurology | Year: 2012

Objective: To assess whether exon deletions or duplications in CLCN1 are associated with recessive myotonia congenita (MC). Methods: We performed detailed clinical and electrophysiologic characterization in 60 patients with phenotypes consistent with MC.DNAsequencing ofCLCN1followed by multiplex ligation-dependent probe amplification to screen for exon copy number variation was undertaken in all patients. Results: Exon deletions or duplications in CLCN1 were identified in 6% of patients with MC. Half had heterozygous exonic rearrangements. The other 2 patients (50%), with severe disabling infantile onset myotonia, were identified with both a homozygous mutation, Pro744Thr, which functional electrophysiology studies suggested was nonpathogenic, and a triplication/homozygous duplication involving exons 8-14, suggesting an explanation for the severe phenotype. Conclusions: These data indicate that copy number variation in CLCN1 may be an important cause of recessive MC. Our observations suggest that it is important to check for exon deletions and duplications as part of the genetic analysis of patients with recessive MC, especially in patients in whom sequencing identifies no mutations or only a single recessive mutation. These results also indicate that additional, as yet unidentified, genetic mechanisms account for cases not currently explained by either CLCN1 point mutations or exonic deletions or duplications. Copyright © 2012 by AAN Enterprises, Inc.

Nillesen W.M.,RUNMC | Yntema H.G.,RUNMC | Moscarda M.,Catholic University | Verbeek N.E.,UMC Utrecht | And 12 more authors.
Human Mutation | Year: 2011

The core phenotype of Kleefstra syndrome (KS) is characterized by intellectual disability, childhood hypotonia, and a characteristic facial appearance. This can be caused by either submicroscopic 9q34 deletions or loss of function mutations of the EHMT1 gene. Remarkably, in three patients with a clinical suspicion of KS, molecular cytogenetic analysis revealed an interstitial 9q34 microdeletion proximal to the coding region of the EHMT1 gene based on the NM- 024757.3 transcript. Because we found a mono-allelic EHMT1 transcript suggestive for haploinsufficiency of EHMT1 in two of these patients tested, we hypothesized that a deletion of regulatory elements or so far unknown coding sequences in the 5' region of the EHMT1 gene, might result in a phenotype compatible with KS. We further characterized the molecular content of deletions proximal to the transcript NM- 024757.3 and confirmed presence of a novel predicted open reading frame comprising 27 coding exons (NM- 024757.4). Further analysis showed that all three deletions included the presumed novel first exon of the EHMT1 gene. Subsequent testing of 75 individuals without previously detectable EHMT1 aberrations showed one additional case with a deletion comprising only this 5' part of the gene. These results have important implications for the genetic screening of KS and for studies of the functional significance of EHMT1. © 2011 Wiley-Liss, Inc.

Hagag E.,Alexandria University | Hagag E.,Institut Universitaire de France | Shwaireb M.,Alexandria University | Coffa J.,MRC Holland BV | And 2 more authors.
Eastern Mediterranean Health Journal | Year: 2013

Approximately 5%-10% of all breast cancers are inherited as the result of germline mutations in the BRCA1 gene. Large genomic rearrangements (LGRs) in BRCA1 have not been well-researched in the Egyptian population. Using multiplex ligation-dependent probe amplification, we showed BRCA1 rearrangements in 4/22 cases (18.2%) of familial breast cancer. No influence of having multiple breast cancer cases within the family was observed in patients diagnosed at < or ≥ 45 years and having BRCA1-positive LGRs. However, focusing on cases with first- and second-degree relatives affected, we observed a significant difference between the percentage of patients with BRCA1-positive versus BRCA1-negative LGRs. Our results provide the first evidence that LGRs in BRCA1 exist in the Egyptian population. Screening for these alterations may be desirable when breast cancer patients are diagnosed at an early age, especially if these cases have first- and second-degree of relatives with breast cancer.

Alsum Z.,King Khalid University | Safieh L.A.,King Faisal Specialist Hospital And Research Center | Nygren A.O.H.,MRC Holland BV | Al-Hamed M.A.,King Faisal Specialist Hospital And Research Center | And 3 more authors.
Genetic Testing and Molecular Biomarkers | Year: 2010

Pseudohypoparathyroidism type 1b (PHP1b) is a rare metabolic bone disorder characterized by isolated renal parathyroid hormone resistance. The disorder is almost always associated with an imprinting defect or deletions in the differentially methylated region of the GNAS locus located on chromosome 20q13. In addition to the epigenetic and genetic aberrations of the differentially methylated region, PHP1b can also result from a deletion of STX16, a long-range control element of methylation at the GNAS locus located centromeric of GNAS. This report describes the utilization of a recently described methylation-specific multiplex-ligation-dependent probe amplification assay for high-throughput molecular analysis of a patient with the clinical diagnosis of PHP1b. Although more patients will need to be tested to confirm this, methylation-specific multiplex-ligation-dependent probe amplification in our hands proved to be a rapid, sensitive, and fairly easy-to-interpret assay that can be used in lieu of Southern blot analysis to diagnose PHP1b. © 2009, Mary Ann Liebert, Inc.

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