Possible role of GLI3 gene in the pathogenesis of idiopathic congenital talipes equinovarus / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV).</p><p><b>METHOD</b>Potential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR. Following generation of rat model for ICTEV, mRNA and protein levels of GLI3 were evaluated by real-time PCR and immunohistochemistry and Western blotting.</p><p><b>RESULTS</b>No mutation was found in exons 1 - 8 and 13 of GLI3 gene among the 84 ICTEV patients. No expression of GLI3 gene was detected in the flexor hallucis longus of ICTEV patients or normal controls. Expression of Gli3, in terms of both mRNA and protein, was stronger in the hindlimb of ICTEV rat embryos compared with normal controls.</p><p><b>CONCLUSION</b>Mutation in the coding region of GLI3 may not be responsible for the occurrence of ICTEV. However, there may still be connection between abnormal expression of the gene and pathogenesis of ICTEV.</p>

Mechanism of GLI3 gene transcription regulation in idiopathic congenital talipes equinovarus / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of transcription regulation of GLI3 gene in idiopathic congenital talipes equinovarus.</p><p><b>METHODS</b>pGL3-Gli3 luciferase report vectors were constructed, and the activity of Gli3 promoter was explored. A P-Match software was used to analyze the sequence upstream of the transcription start site of rat Gli3 gene, which was subsequently verified with chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). Expression of the Gli3 gene was analyzed in L6 cells transfected with Hoxd13 small interference RNA(siRNA) and Hoxd13 expression vectors.</p><p><b>RESULTS</b>The 5' region of rat Gli3 gene contains two potential binding sites for the Hoxd13 protein. CHIP and EMSA assays both confirmed that Hoxd13 can directly bind with site 2. As shown in L6 cells, expression of Gli3 may be enhanced with silencing of Hoxd13, whilst exogenous expression of Hoxd13 can down-regulate transcription of Gli3.</p><p><b>CONCLUSION</b>Hoxd13 can directly regulate the expression of Gli3 gene through a Hoxd13 binding site in the limb of rat embryo.</p>

Expression of COL9A1 gene and its polymorphism in children with idiopathic congenital talipes equinovarus / 中国当代儿科杂志

<p><b>OBJECTIVE</b>COL9A1 gene is located in the susceptibility region of idiopathic congenital talipes equinovarus (ICTEV) (6q12-13). This study aimed to investigate the expression of the COL9A1 gene and the distribution of single nucleotide polymorphism (SNP) of COL9A1 gene in patients with ICTEV and normal controls.</p><p><b>METHODS</b>Immunohistochemistry was used to detect the expression of COL9A1 in 25 children with ICTEV and 5 normal controls. The frequencies of genotypes and allele of two SNPs in COL9A1 gene rs35470562 and rs1135056 were investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing in 118 patients with ICTEV and 100 normal controls.</p><p><b>RESULTS</b>The COL9A1 protein expression was significantly higher in 22 (88%) out of 25 children with ICTEV than normal controls. There were significant differences in the frequencies of genotypes and allele of rs1135056 in COL9A1 gene between the ICTEV and the control groups: the G allele frequency was higher, the frequency of AA genotype was lower, and the frequencies of AG and GG genotypes were higher in ICTEV patients than those in healthy controls (P<0.05).</p><p><b>CONCLUSIONS</b>COL9A1 protein is highly expressed in patients with ICTEV and rs1135056, which is located in the coding region of COL9A1 gene, may be associated with the pathogenesis of ICTEV.</p>

Gene diagnosis for spinal muscular atrophy and its application study / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Twenty-six patients with SMA were directly tested with PCR-RFLP for exon 7 deletion in the SMN1 gene. Carrier detection was carried out with multi-PCR-DHPLC. Amniotic fluid was taken at the middle stage of gestation from pregnant women who had given birth to affected children.</p><p><b>RESULTS</b>Twenty-five out of 26 patients were diagnosed as having SMN1 gene deletion. Fifty-two of their parents were found to be carriers of exon 7 deletion. Eight of 20 fetuses were diagnosed as having SMN1 gene deletion by PCR-RFLP.</p><p><b>CONCLUSION</b>PCR-RFLP and multi-PCR-DHPLC techniques can provide rapid diagnosis for exon 7 deletion detection and carrier detection. PCR-RFLP may also be adapted for prenatal gene diagnosis of exon 7 deletion in SMN1 gene.</p>

Detection of new mutations in the dystrophin gene by denaturing high-performance liquid chromatography / 中华儿科杂志

<p><b>OBJECTIVE</b>Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by dystrophin gene mutations; 55%-65% of these pathogenic mutations are large deletion and duplication mutations that can be detected by multiplexed polymerase chain reaction. However, finding the remaining micro-mutations (substitutions, deletions or insertions of one or several nucleotides) cannot be achieved in this way. The aim of the present study was to detect mutations of the dystrophin gene in individuals with Duchenne muscular dystrophy (DMD) by denaturing high-performance liquid chromatography (DHPLC) and to establish a rapid and sensitive screening platform for micro-mutations leading to DMD.</p><p><b>METHODS</b>Twenty patients negative for large deletions in the dystrophin gene by multiplex PCR were selected for further screening by DHPLC and 20 normal male without DMD family history as the control cohort. Dystrophin exons and their flanking sequences were individually amplified by genomic PCR and the amplicons showing abnormal DHPLC profile were directly sequenced to identify the position and the type of the mutations.</p><p><b>RESULTS</b>After screening 68 exons covering the two deletion hotspots and 3'UTR region, four pathogenic mutations, including c.6808_6811del TTAA, c.4959_4960insA, c.8656C > T and c.8608C > T, were found in four DMD patients. Moreover, c.6808_6811del TTAA, c.4959_4960ins and c.8656C > T have not been reported previously. The first two frameshift mutations were predicted to produce premature stop codons, p.Leu2270MetfsX9 and p.Ser1654LysfsX5, respectively. The remaining two were nonsense mutations, leading to p.R2886X and p.R2870X, respectively.</p><p><b>CONCLUSION</b>Three novel and one recurrent dystrophin mutations have been identified in Chinese DMD patients. This study has demonstrated that DHPLC is an effective screening method for micro-mutation associated with DMD.</p>

Non-invasive prenatal genetic diagnosis using multiple displacement amplification / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of multiple displacement amplification (MDA) to apply in the non-invasive prenatal genetic diagnosis of Duchenne muscular dystrophy (DMD).</p><p><b>METHODS</b>Maternal blood was obtained from 20 pregnant women at 7 to 25 weeks of gestation. After the discontinuous density gradient centrifugation with Percoll, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. All positive NRBCs were collected by micromanipulator and then performed with MDA. Sex and short tandern repeat (STR) analysis were determind from a small aliquot of the reaction. The origin of NRBCs was verified and prenatal diagnosis of DMD was made at the same time.</p><p><b>RESULTS</b>The product length of MDA was >15 kb, while primer extension preamplification (PEP) is only about 1 kb. We completed non-invasive prenatal genetic diagnosis of 6 fetus at high risk of DMD using MDA. The results were all coincident with amniotic fluid control.</p><p><b>CONCLUSION</b>The MDA method which provides a highly uniform representation across the genome, representing the entire genome with minimal amplification bias, shows good application prospects.</p>

Application study on inversion diagnosis of F8 gene in hemophilia A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish an effective method of genetic diagnosis on hemophilia A (HA) by detecting the inversion mutation in intron 22 of F8 gene.</p><p><b>METHODS</b>Intron 22 inversion mutation in F8 gene was detected by using long distance-polymerase chain reaction (LD-PCR) and inversion-PCR (I-PCR) in 31 HA patients. The mothers of HA patients with intron 22 inversion mutation were selected to carrier diagnosis and amniotic fluid of the pregnant women with inversion mutation was collected at intermediate stage of gestation, and used to prenatal genetic diagnosis.</p><p><b>RESULTS</b>Seven patients showed F8 gene inversion mutation in thirty-one patients. Three in four mothers of HA patients with intron 22 inversion mutation were diagnosed as carriers. The prenatal diagnosis result indicated that the fetus conceived in the HA-carrier woman was normal individual.</p><p><b>CONCLUSION</b>The detection of intron 22 inversion mutation by LD-PCR and I-PCR is time-saving, and can be used in prenatal diagnosis on HA.</p>

Analysis and application of SCA1 and SCA3/MJD gene CAG repeats in Han population in Northeastern China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the normal range of (CAG)n in spinocerebellar ataxia type 1 (SCA1) gene and spinocerebellar ataxia type 3 (SCA3/MJD) gene in 110 normal subjects of Han population in Northeastern China, to assess the genotypes for clinically diagnosed spinocerebellar ataxia(SCA) individuals including 25 patients from 8 families and 6 sporadic patients, and to make presymptomatic and prenatal diagnosis.</p><p><b>METHODS</b>DNA fragments from the normal subjects and the patients were detected by fluorescence-PCR. Homozygosities were selected for DNA sequencing.</p><p><b>RESULTS</b>The normal ranges of (CAG)n of SCA1 and SCA3/MJD were 20-39 and 14-38 repeats respectively, SCA1 was found mostly to be 26 and 27 repeats, allele frequency 34.09% and 20.91%; heterozygosity was 84.55%, SCA3/MJD was found mostly to be 14 repeats, allele frequency 39.55%, heterozygosity was 78.18%.(CAG)(68) of SCA3/MJD gene of one affected individual had been found in a family but no CAG mutative expansion in related members was observed.</p><p><b>CONCLUSION</b>The normal ranges of CAG repeats vary with areas and races. SCAs genotyping is the first choice in presymptomatic and prenatal diagnosis.</p>