Genetic study of a Chinese pedigree affected with pachyonychia congenita / 中华医学遗传学杂志

Objective@#To explore the genetic basis for a Chinese pedigree affected with pachyonychia congenita (PC).@*Methods@#With informed consent obtained, peripheral blood samples were taken from the pedigree. Genomic DNA was extracted with a phenol/chloroform method. Based on the clinical manifestation of the patients, candidate genes for PC were selected. Potential mutation was screened by PCR and Sanger sequencing. Suspected mutation was verified in other family members by PCR-high resolution melting (HRM) analysis. Haplotype analysis using microsatellite markers was also carried out to determine the founder of the mutation.@*Results@#A heterozygous c. 275A>G (Asn92Ser) mutation was discovered in exon 1 of the KRT17 gene in the proband. PCR-HRM analysis showed that all affected members were heterozygous carriers of the mutation. The same mutation was found in none of the unaffected members. Haplotype analysis and sequencing indicated the mother of the proband to be the founder.@*Conclusion@#The c. 275A>G (Asn92Ser) mutation of the KRT17 gene probably underlies the disease in this pedigree. Above finding has facilitated genetic counseling and prenatal diagnosis for this pedigree.

Genetic study of a Chinese pedigree affected with pachyonychia congenita / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a Chinese pedigree affected with pachyonychia congenita (PC).@*METHODS@#With informed consent obtained, peripheral blood samples were taken from the pedigree. Genomic DNA was extracted with a phenol/chloroform method. Based on the clinical manifestation of the patients, candidate genes for PC were selected. Potential mutation was screened by PCR and Sanger sequencing. Suspected mutation was verified in other family members by PCR-high resolution melting (HRM) analysis. Haplotype analysis using microsatellite markers was also carried out to determine the founder of the mutation.@*RESULTS@#A heterozygous c.275A>G (Asn92Ser) mutation was discovered in exon 1 of the KRT17 gene in the proband. PCR-HRM analysis showed that all affected members were heterozygous carriers of the mutation. The same mutation was found in none of the unaffected members. Haplotype analysis and sequencing indicated the mother of the proband to be the founder.@*CONCLUSION@#The c.275A>G (Asn92Ser) mutation of the KRT17 gene probably underlies the disease in this pedigree. Above finding has facilitated genetic counseling and prenatal diagnosis for this pedigree.

Analysis of gross deletions of COL1A1/2 genes in Chinese families affected with osteogenesis imperfecta / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify deletion of large fragment in COL1A1/2 genes among patients with osteogenesis imperfecta (OI).</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples by a standard SDS-proteinase K-phenol/chloroform method. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect gross deletions of the COL1A1/2 genes among 46 patients affected with OI, in whom no mutation was detected in the sequences of the COL1A1/2 genes.</p><p><b>RESULTS</b>Heterozygous deletions of the entire COL1A1 gene and exon 20 of the COL1A2 gene were detected in probands A and B, respectively, and no gross deletion was found in the remaining 44 samples. The MLPA result of proband A was confirmed by fluorescence quantitative PCR (Q-PCR) in his family. A further conjunction point analysis through gap-PCR and DNA sequencing revealed deletion of exons 17 to 23 in the COL1A2 gene, and a 637 bp-insertion from chromosome 5 in the proband B.</p><p><b>CONCLUSION</b>Two gross deletions have been found in the genes coding for collagen type I in the Chinese OI population, and the deletion of exons 17 to 23 in the COL1A2 gene is a novel mutation. This work not only has expanded the mutation spectrum of the COL1A1/2 gene, but also provided a support for prenatal genetic diagnosis for the families.</p>

Detection of pathogenic mutations in Marfan syndrome by targeted next-generation semiconductor sequencing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders.</p><p><b>METHODS</b>Peripheral blood samples were collected from three MFS patients and a normal control with informed consent. Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion Ampliseq(TM) Inherited Disease Panel. Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA fragments from different samples were ligated with barcoded sequencing adaptors. Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were carried out using an Ion One Touch system. The ion sphere particles were sequenced on a 318 chip using the PGM platform. Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads. After sequence alignment and extraction of SNPs and indels, all the variants were filtered against dbSNP137. DNA sequences were visualized with an Integrated Genomics Viewer. The most likely disease-causing variants were analyzed by Sanger sequencing.</p><p><b>RESULTS</b>The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 × median sequencing depth and a coverage of > 98% for the targeted regions in all the four samples. After data analysis and database filtering, one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All mutations were verified by conventional Sanger sequencing.</p><p><b>CONCLUSION</b>Pathogenic FBN1 mutations have been identified in all patients with MFS, indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.</p>