Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

Lee

American J of Med Genetics Pt A

et al. 2010

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Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous congenital disorder characterized by severe growth retardation. Hypomethylation of the differentially methylated region (DMR) of the H19 gene and uniparental disomy of maternal chromosome 7 is present in ∼45% of the patients with SRS so more than half of these patients have no known genetic etiology. We combined several molecular technologies including multiplex methylation polymerase chain reaction, methylation-sensitive multiple ligation probe-dependent amplification, and methylation-sensitive high-resolution melting to assess the epigenetic status of 34 patients with SRS. Additionally, we applied a whole genome strategy to detect copy number changes and loss of heterozygosity. Thirteen patients (38.2%) had hypomethylation of the DMR of the H19 gene and none had uniparental disomy of maternal chromosome 7. The whole genome arrays identified five patients (14.7%) with microdeletions on chromosomes 1q23q24.3, 7p15.3, 13q31.3, 14q32.31, and 15q26.2qter, respectively. The overall mutation detection rate was 52.9% by the epigenetic study and the whole genome strategy. Although epimutation may be the major cause of SRS and can be identified by multiplex methylation polymerase chain reaction, the whole genome approach also provides information on the etiology of SRS. If no epimutation is identified in the patients with typical SRS, microdeletions should be suspected.

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Section: Methylation-sensitive High-resolution Melting (Ms-hrm)mentioning

confidence: 99%

“…All DNA was extracted from peripheral blood using the Chemagic DNA Blood Kit (Chemagen, Baesweiler, Germany), and the MethylCode Bisulfite Conversion Kit (Invitrogen, Carlsbad, CA) was used to treat the DNA (1 mg) with bisulfite according to the manufacturer's instructions [Hung et al, 2009].…”

Section: Bisulfite Modification Of Dnamentioning

confidence: 99%

Epigenetic profiling of the H19 differentially methylated region and comprehensive whole genome array‐based analysis in Silver–Russell syndrome

Lee

American J of Med Genetics Pt A

et al. 2010

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“…Faivre et al (2009) recently reported that exons 24-32 represent a hotspot for neonatal MFS and severe forms of MFS. The purpose of this study was to evaluate FBN1 mutations in Taiwanese individuals by using denaturing high performance liquid chromatography (DHPLC) and multiplex ligation-dependent amplification (MLPA) (Liu et al, 1997;Matyas et al, 2002;Kosaki et al, 2005;den Dunnen & White, 2006;Howarth et al, 2007;Kozlowski et al, 2008;Hung et al, 2005Hung et al, , 2006Hung et al, , 2007Hung et al, , 2008aHung et al, , 2008bHung et al, , 2009. DHPLC can detect heteroduplexes of denatured and reannealed PCR amplicons, and has a highcapacity and low-costs.…”

Section: Introductionmentioning

confidence: 99%

Mutation spectrum of the fibrillin‐1 (FBN1) gene in Taiwanese patients with Marfan syndrome

Annals of Human Genetics

Lee

et al. 2009

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SUMMARYThe aim of this study was to establish a national database of mutations in the fibrillin-1 (FBN1) gene that cause Marfan syndrome (MFS) in the Taiwanese population. In this study, we screened 294 patients from 157 families for the presence of FBN1 mutations using polymerase chain reaction/ denaturing high performance liquid chromatography (PCR/DHPLC). We identified 56 mutations in 62 of the 157 (40%) families including 49 single-base substitutions (36 missense mutations, seven nonsense mutations, and six splicing sites), one small insertion, four small deletions, one small indel (insertion and deletion), and one exonic deletion (Exon 36). When family history was taken into consideration, the mutation detection rate rose to 91% (29 of 32). We further investigated the phenotypic data and found that one third (47 of 157) of the families fit the Ghent criteria for MFS. Based on that data, the mutation rate was 98% (46/47). That finding implies that family history and the Ghent criteria play a more important role than clinical manifestations in establishing a clinical diagnosis of Marfan syndrome. Among the 56 mutations found in this study, 40 (71%) have not been registered in the Human Gene Mutation Database (HGMD) or in the Universal Mutation Database (UMD). This is the first study of the mutation spectrum of MFS in a cohort of patients in Taiwan. The database is expected to considerably improve genetic counseling for and medical care of MFS families.

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“…To resolve this problem, we designed methylation-specific multiplex PCR to determine copy number changes and methylation status in a single tube. 21 Recently, the melting curve analysis system was considered a promising tool for detecting sequence variation by the use of a saturating double-stranded DNA dye. Methylation-specific PCR and quantitative melting curve analysis can discriminate between deletional and nondeletional PWS and AS, 22 and it is based on a single-step procedure.…”

Section: Discussionmentioning

confidence: 99%

Quantitative and Qualitative Analyses of the SNRPN Gene Using Real-Time PCR with Melting Curve Analysis

The Journal of Molecular Diagnostics

et al. 2011

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