Mutation screening of the F VIII gene in 10 hemophilia A families / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the F VIII gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis.</p><p><b>METHODS</b>PCR, denaturinghigh performance liquid chromatogramphy (DHPLC) and DNA sequencing technologies were applied to screen the F VIII gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14(DXS 52), intron 13 (CA)n and EX18/Bcl I of the F VIII gene in the HA families. In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F VIII gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism.</p><p><b>RESULTS</b>Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism (SNP) were identified in 10 the HA families. Among them, c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT, c.4880_4881insA and c.5000G to A were novel mutations or polymorphism. No missense mutations c.878A G, c.1015A to G and c.6870G to T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined to in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient.</p><p><b>CONCLUSION</b>Six novel mutations, i.e., c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT and c.4880_4881insA, were identified in this study. PCR, DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.</p>

Mutation screening and prenatal diagnosis of tuberous sclerosis complex / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen mutations of tuberous sclerosis complex (TSC) patients to confirm a clinical diagnosis of TSC, and to perform prenatal diagnosis for families with mutations.</p><p><b>METHODS</b>In this study, PCR-denaturing high-performance liquid chromatography(DHPLC), supplemented with sequencing when necessary, was used to screen TSC1 and TSC2 mutations in 21 patients from 19 pedigrees visited author's hospital in the last five years. For novel mutations, one hundred unrelated healthy individuals were screened to exclude the possibility of polymorphism.</p><p><b>RESULTS</b>Seventeen different mutations were found in 21 patients of 19 pedigrees with 13 being novel mutations, including c. 2672delA, c. 2672insA of TSC1 gene and c.4918insCGCC, c.1143delG, Intron27+1 G>A, c.1957-1958delAG, Intron5+1 G>A, c.910insCT, c.2753 C>G, c.4078dupAGCAAGTCCAGCTCCTC, Intron 11 -1 G>A, Intron 14+1 G>A, c.684 C>A of TSC2 gene, indicating a high frequency of de novo mutations in TSC. Three of these mutations were in the TSC1 gene (N762S, c.2672insA and c. 2672delA), while all remaining 14 were in the TSC2 gene. Prenatal diagnosis for TSC was performed for 7 fetuses from these pedigrees. The six fetuses that tested negative for TSC mutations were carried to term and, to date, none of these children has shown symptoms of TSC.</p><p><b>CONCLUSION</b>Author's data showed that a mutation detection rate of tuberous sclerosis was 89.5%(17/19) among patients in author's hospital. The ratio of TSC2 and TSC1 mutations was about 1:1 in the familial cases, but TSC2 mutation was more common than TSC1 mutation in sporadic cases. Author's data demonstrated that birth of TSC children for those with familial history of TSC could be prevented through prenatal diagnosis.</p>

Mutation detection of PKD1 gene in patients with autosomal dominant polycystic kidney diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect gene mutation in the patients with autosomal dominant polycystic kidney disease (PKD).</p><p><b>METHODS</b>Polymerase chain reaction (PCR)-denaturing high-performance liquid chromatography (DHPLC) analyses were performed in 3o single copy region of PKD 1 gene (PKD1). DNA sequencing were carried out on PCR products with abnormal peak shape afterwards.</p><p><b>RESULTS</b>A new nonsense mutation (C11901A in exon 42 of PKD1 was identified to cause serine in position 3897 turning to a stop codon. A missense mutation, C10737T, was detected in exon 35 which caused threonine in position 3509 turn to methionine. Two kinds of samesense mutation, G11824A and C11860T in exon 42, were found in normal control.</p><p><b>CONCLUSION</b>PKD1 mutation were detected successfully by PCR-DHPLC. A new nonsense mutation, a missense mutation and two polymorphisms are identified in this study.</p>

Mutation screening and prenatal diagnosis of hidrotic ectodermal dysplasia in a Chinese family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the mutations in Cx30 gene in a Chinese family with hidrotic ectodermal dysplasia (HED) and to make prenatal diagnosis on the embryo which has been pregnant for 5 months.</p><p><b>METHODS</b>A family including 2 affected and 4 unaffected individuals was collected, and their informed consents were obtained. The affected woman had a five-month pregnancy. An 884 bp fragment containing the whole GJB6 coding sequence was amplified by PCR and the products were bi-direction sequenced directly. The mutation was further confirmed with restriction endoenzyme digesting. On the base of successful gene diagnosis, the following detection procedure on the pregnant baby was performed. First the whole coding region of Cx30 was amplified using primers Cx30-F and Cx30-R and the PCR products were digested by Hae II. Then the PCR products were cloned into pUCm-T vector. Blue-white blot screening method and PCR-restriction endoenzyme digesting technique were used to identify the correct clones. The mutant allele clone was sequenced to confirmed the mutation.</p><p><b>RESULTS</b>A heterozygous missense mutation 263C --> T in the Cx30 gene was detected in the affected little girl and her affected mother, which led to an amino acid substitution (A88V) in the second transmembrane domain of GJB6. The mutation was confirmed by Hae II digestion. A88V mutant allele cannot be cut while the wild normal allele can be cut into two fragments, 520 and 278 bp. The result of analyse on the five-month pregnancy show the embryo carried the A88V mutation too. So the embryo will be a patient.</p><p><b>CONCLUSION</b>An A88V missense mutation in the Cx30 gene can also cause HED in Chinese Han population. Based on the gene diagnosis, prenatal diagnosis can be played using bi-direction sequencing and confirmed with restriction endoenzyme digesting.</p>