Karyotyping and analysis of 5α -reductase-2 gene mutation in 25 patients with hypospadias / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the karyotypes and SRD5A2 gene mutations in 25 patients with sporadic or familial hypospadias.</p><p><b>METHODS</b>The patients included 10 adults and 15 children, whose chromosomes were analyzed by G-banded karyotyping, and the SRD5A2 genes were sequenced.</p><p><b>RESULTS</b>Two patients were found to have an abnormal karyotype, while eight have carried compound heterozygous mutations of the SRD5A2 gene, which included 5 genotypes formed by 6 types of mutations, i.e., p.G203S/p.R227Q, p.R227Q/p.R246Q, p.Q6X/p.Q71X, p.L20P/p.G203S, and p.Q71X/p.R227Q. Mutations of the SRD5A2 gene were present in 32% (8/25) of all patients, 35% (8/23) in those with a normal karyotype, and 44.4% (8/18) in those with proximal type hypospadia. Bioinformatic analysis, literature review and pedigree analysis confirmed that all such mutations are pathogenic.</p><p><b>CONCLUSION</b>Chromosomal anomalies and mutations of the SRD5A2 gene are the main cause of hypospadias. Sequencing of the SRD5A2 gene may explain the etiology of nearly half of the patients with proximal type of hypospadas but a normal karyotype, which can facilitate genetic consulting.</p>

Analysis of FOXL2 gene mutations in 5 families affected with blepharophimosis, ptosis and epicanthus inversus syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen for FOXL2 gene mutations in 6 patients with blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), and explore their genotype-phenotype correlation.</p><p><b>METHODS</b>Peripheral venous blood samples were collected from the patients for the extraction of genomic DNA. PCR and Sanger sequencing were employed to analyze the coding region and flanking sequences of the FOXL2 gene. Pathogenicity of the identified mutations was verified through literature review and bioinformatic analysis.</p><p><b>RESULTS</b>A heterozygous c.672_701dup30 mutation was found in the probands from the two familial cases, while three heterozygous mutations (two were novel), namely c.462_468del (p.Pro156Argfs*113), c.251T to A (p.Ile84Asn) and c.988_989insG (p.Ala330Glyfs*204) were detected in the three sporadic cases. Literature review and bioinformatic analysis indicated that all these mutations are pathogenic.</p><p><b>CONCLUSION</b>Identification of causative mutations in the BPES patients has provided a basis for genetic counseling and reproductive guidance. The novel mutations have enriched the mutation spectrum of the FOXL2 gene.</p>

Study of a Bethlem myopathy pedigree resulted from a novel mutation of COL6A3 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the molecular etiology for a muscular dystrophy pedigree with target region sequencing platform using hereditary myopathy capture array.</p><p><b>METHODS</b>Specific gene testing was performed based on the clinical diagnosis. Since no pathogenic mutation was found, target region sequencing with hereditary myopathy capture array combined with Sanger sequencing and bioinformatics analysis were employed in turn. PolyPhen and NCBI were used to evaluate the pathogenicity of identified mutation and conservation of the gene.</p><p><b>RESULTS</b>Target region sequencing indicated the proband has carried a heterozygous c.3353 A>C mutation of COL6A3 gene, which was confirmed by Sanger-sequencing in 4 affected individuals from the family. The same mutation was not detected in healthy members of the pedigree. Bioinformatics analysis suggested that the mutation has caused a highly pathogenic amino acid substitution from Histidine to Proline. The affected patients featured normal intelligence with mild myogenic damage by muscle biopsy, slightly increased serum creatine kinase and slow disease progression, which was consistent with Bethlem myopathy.</p><p><b>CONCLUSION</b>Target region sequencing is an effective and efficient method for genetic testing. The heterozygous c.3353A>C mutation in exon 8 of the COL6A3 gene probably underlies the Bethlem myopathy with autosomal dominant inheritance.</p>

Preliminary exploration of the influence factors on amplification of single cell duplex-nested PCR / 北京大学学报(医学版)

Objective:To explore the influence factors on amplification of single cell duplex-nested PCR.Methods:The mutational loci region CD41-42 and IVS-Ⅱ654 of ?-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 ℃ before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR.Results:TaKaRa EX Taq was the most efficient Taq DNA ploymerase among different Taq DNA ploymerases; primer pair R1+F1 at final concertration of 0.25 ?mol/L and R2+F2 at 0.3 ?mol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P0.05). Conclusion:There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 ℃ before the single cell PCR amplification could not improve the PCR amplification efficiency

The detection of the frequent mutations of iduronate-2-sulphatase gene in mucopolysaccharidosis type II patients in Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate and establish the gene diagnosis methods for the frequent mutations of iduronate-2-sulphatase(IDS) gene in mucopolysaccharidosis type II patients.</p><p><b>METHODS</b>polymerase chain- reaction-single strand conformation polymorphism PCR-SSCP) analysis was applied to detect the mutations of exons 3, 8 and 9 which were hot spots in the iduronate-2-sulfatase gene; DNA sequencing was applied to analyze the mutations which had been detected by PCR-SSCP; PCR-restriction fragment length polymorphism (PCR-RFLP) was applied to detect the results of DNA sequencing.</p><p><b>RESULTS</b>Obvious and abnormal bands in exon 9 of the IDS gene were found by applying PCR-SSCP; the mutation(C1672T) of exon 9 was found in the patient through DNA sequencing, which led to amino acid replacement(R468W); the PCR-restriction enzyme digestion showed that only one band(554 bp) appeared in the patient, but there were two bands (257 bp and 297 bp) in his parents, and it verified the results of sequencing analysis.</p><p><b>CONCLUSION</b>PCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion are effective methods for MPS II diagnosis. Combined applications of these methods can verify and complement each other and improve the accuracy of diagnosis.</p>