Low Grip Strength is a Strong Risk Factor of Osteoporosis in Postmenopausal Women.

OBJECTIVE: To investigate the effect of grip strength on bone mineral density (BMD) in postmenopausal women. Low BMD is related to risk of fracture and falling is the strongest factor for fragility fractures. Handgrip strength is a reliable indicator of muscle strength and muscle strength is associated with falling. METHODS: For the present study 120 women were divided into two groups: those ≤65 years and those >65 years. Serum 25 hydroxyvitamin D (25OHD), BMD, and handgrip strength were measured to observe the effect of age on 25OHD, grip strength, and BMD, as well as the effect of 25OHD on grip strength and BMD. The correlation between grip strength and BMD was investigated. RESULTS: In the 120 patients, 25OHD was 24.31 ± 8.29 ng/mL. There were 37 cases with 25OHD <20 ng/mL and 83 cases with 25 OHD ≥20 ng/mL. The patients with 25OHD <20 ng/mL had significantly lower femoral neck BMD, most of them with a T score ≤-2.5 (P < 0.05). BMD measurement showed 66 patients with femoral neck T ≤-2.5, 30 cases with total hip T ≤-2.5 and 90 cases with lumbar BMD T ≤-2.5. The maximum grip strength in the group is 22.28 ± 6.17 kg. There were 38 cases with the maximum grip strength <20 kg and 82 cases with the maximum grip strength ≥20 kg. Patients >65 years had lower 25OHD, lower maximum grip strength, and lower BMD. The osteoporosis risk in postmenopausal women with a maximum grip strength <20 kg and who were >65 years was significantly elevated. CONCLUSION: Handgrip strength and 25OHD decrease with aging in postmenopausal women. The patients with lower 25OHD level had significantly lower BMD of femoral neck. The patients with lower handgrip strength had significantly lower BMD of lumbar spine, femoral neck, and total hip. Grip strength measurement is the simplest muscle strength measurement method. Our study confirmed that low grip strength was correlated with low BMD and was a strong risk factor for osteoporosis in postmenopausal women.

Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China.

OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. MATERIALS AND METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband's cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

[Mechanism of GLI3 gene transcription regulation in idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the mechanism of transcription regulation of GLI3 gene in idiopathic congenital talipes equinovarus. METHODS: 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. RESULTS: 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. CONCLUSION: Hoxd13 can directly regulate the expression of Gli3 gene through a Hoxd13 binding site in the limb of rat embryo.

[Possible role of GLI3 gene in the pathogenesis of idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV). METHOD: 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. RESULTS: 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. CONCLUSION: 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.

[Expression of COL9A1 gene and its polymorphism in children with idiopathic congenital talipes equinovarus].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[The regulation of UTROPHIN expression by EN1].

To investigate possible factors up-regulating the expression of UTROPHIN, potential regulatory elements in the promoter of the human UTROPHIN was predicted by P-match software and verified by EMSA and ChIP. The mechanism of EN1 regulation of the human UTROPHIN expression was evaluated by RNA interference and real-time PCR analyses. Two potential EN1 binding sites in UTROPHIN promoter region were predicted by P-Match software but only the second site was verified to interact directly with EN1 by EMSA and ChIP. The results from RNA interference and real-time PCR showed that the mRNA level of UTROPHIN increased in HeLa cells after EN1 was knockdowned by siRNA. It indicated that EN1 might be a negative regulatory factor for UTROPHIN. Our study suggested that UTROPHIN might be a new target for DMD therapy.

[Prenatal diagnosis of Duchenne and Becker muscular dystrophy by multiplex ligation-dependent probe amplification].

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked lethal recessive disease caused by mutation in the DMD gene. There is no efficient treatment for this serious and disabling disease. We established a combination method to detect carriers and performed prenatal diagnosis. Using multiplex ligation-dependent probe amplification (MLPA) and linkage analysis of short tandem repeats (STR) methods, 26 prenatal diagnosis were performed for pregnancies at risk of having a DMD/BMD baby through amniocentesis. Seven out of 26 male fetuses were affected and the pregnancies were terminated. Four out of 26 female fetuses were found to be carriers. MLPA can be the method of choice for initial screening of DMD/BMD patients for deletions and duplications mutations. When combined with STR-based analysis, it can improve the rate of DMD/BMD prenatal diagnosis.

[Gene diagnosis for spinal muscular atrophy and its application study].

OBJECTIVE: To establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus].

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.

[Association study between mutations of transcription regulator sequences of COL1A1 gene and idiopathic con-genital talipes equinovarus].

RT-PCR was used to detect the expressions of COL1A1 mRNA in 20 patients with idiopathic congenital talipes equinovarus (ICTEV). The primers were designed by Primer 5 according to sequences of -1 031 bp~ +30 bp and the first intron of COL1A1. PCR-DGGE was used to screen the mutations in COL1A1 gene. Expression of COL1A1 on mRNA levels showed significantly higher in patients with ICTEV than in normal persons (t=12.680, P < 0.05). By DNA sequencing, a -161(T--> C) heterozygous mutation and a+ 274(C-->G) homozygous mutation were detected, and both were new identified mutations. These results indicated that the mutations in transcription regulator sequences of COL1A1 could cause ICTEV.

[Detection of new mutations in the dystrophin gene by denaturing high-performance liquid chromatography].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Application study on inversion diagnosis of F8 gene in hemophilia A].

OBJECTIVE: To establish an effective method of genetic diagnosis on hemophilia A (HA) by detecting the inversion mutation in intron 22 of F8 gene. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Analysis of association between COL9A1 gene and idiopathic congenital talipes equinovarus].

Genotypes of 2 SNPs(rs592121 and rs1135056) within COL9A1 gene in 84 ICTEV nuclear pedigrees were analyzed by restriction fragment length polymorphism and DNA sequencing. Analysis of association between SNP locus and ICTEV was performed using ETDT software. Haplotypes and their frequencies in 84 nuclear pedigrees were established and analyzed by TRANSMIT software. RT-PCR was used to detect the expressions of COL9A1 mRNA in 25 patients with ICTEV. The results showed that rs592121 and rs1135056 loci within COL9A1 gene existed transmission disequilibrium in 84 nuclear pedigrees (P<0.05). Expression of COL9A1 on mRNA levels showed significantly higher in patients with ICTEV than in normal person (t=4.7500, P<0.05) . These results indicate that COL9A1 gene may be important suscept-ble genes of ICTEV.

[Non-invasive prenatal genetic diagnosis using multiple displacement amplification].

OBJECTIVE: To investigate the feasibility of multiple displacement amplification (MDA) to apply in the non-invasive prenatal genetic diagnosis of Duchenne muscular dystrophy (DMD). METHODS: 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. RESULTS: 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. CONCLUSION: The MDA method which provides a highly uniform representation across the genome, representing the entire genome with minimal amplification bias, shows good application prospects.

[Detection of fetal nucleated red blood cells in the maternal circulation by Kleihauer test].

Maternal blood was obtained from 18 pregnant women at 7 to 25 weeks of gestation. After Percoll discontinuous density gradient centifugation, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. Positive fetal cells appeared with an intense red cytoplasmic staining while maternal cells with adult haemoglobin were colourless. Individual positive NRBC was collected by micromanipulator and whole genome amplification was then performed to determine sex and STR status. This allowed the simultaneous verification of the fetal origin of NRBC and prenatal diagnosis of genetic diseases. The non-invasive prenatal genetic diagnosis of 9 fetuses at high risk of Duchenne muscular dystrophy (DMD) was completed successfully. The Kleihauer test is a rapid, simple and direct chemical staining method to select fetal cells and can be applied in prenatal diagnosis.

[Relationship of phenotype with type of deletion of dystrophin gene].

OBJECTIVE: To detect the distribution characteristics of dystrophin gene deletions in the northeastern of China and the relationship of severity with type of deletion. METHODS: To screen deletion distribution of 124 DMD/BMD patients via multiplex PCR, male high-risk fetuses were detected deletion by the same method. RESULTS: The deletion frequency was 49%. Deletions located in the regions of exons 45 - 53 and exons 8 - 19 were 41 (67%) and 13 (21%) cases respectively, and in 5 (8%) cases deletions were scattered over both regions, still 2 cases (3%) were checked up deletions lying in exons 34 and 43; there were 9 cases of in-frame deletions and 49 frameshift mutations in all deletions; of 30 high-risk fetuses 10 male ones were screened deletions, who had the same deletion-segments as their probands. CONCLUSIONS: The distribution of dystrophin gene deletions in the northeastern of China cluster mainly in two hot-spots, neighboring regions of exon 8 might be a real deletion "hot spot" in this region; the phenotype is associated with the type of gene deletion, the phenotype is BMD when in-frame deletions occur; severe DMD when frameshift mutations occur. Multiplex PCR method provides the short-cuts for detecting patients and making prenatal gene diagnosis.

[DMDUse of fetal specific antibody-HbF to detect fetal erythroblasts for non-invasive prenatal diagnosis of DMD].

Maternal blood was obtained at 8-26 weeks of gestation. After discontinuous density gradient centrifugation with Percoll, HbF antibody was used to identify fetal NRBC. The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb). Fetal cells appear yellow cytoplasmic staining,while adult cells colorless. NRBCs were collected by micromanipulation and whole genome amplification was performed. DMD was prenatally diagnosed by using the combination of sex determination,multiplex PCR and linkage analysis of several STR sites of dystrophin. NRBCs stained with HbF were found in all of 20 maternal blood samples with gestations, and 7 fetuses with risk of DMD were diagnosed. We concluded that HbF antibody could identify fetal NRBC efficaciously, and this is a kind of more prospective application method.

[Studying dystrophin gene deletion in the northeast of China and applicating].

To detect the distribution characteristics of dystrophin gene deletions of the DMD/BMD patients in the northeast of China and apply for prenatal gene diagnosis, we have analyzed the distribution of the breakpoints of the deleted-patients and the optimized primer-assembly after screening deletions of 120 DMD/BMD patients via multiplex PCR with 12-pair primers and male high-risk fetuses have been detected deletion by multiplex PCR. Results indicated the deletion frequency was 49.2%, about 66.4% deletion breakpoints positioned in introns 44-52, intron 50 was the highest breakpoint (14.8%). The optimized four-primer-assembly was the primers of exon 48, 51, 45 and 8, which could detect 41.7% deletions of all cases; 9 deletions male ones of 29 high-risk fetuses were detected, who had the same deletion-segments as their probands. For the first time screening deletions of DMD/BMD patients in the northeast of China, we have found distribution of the deletions mainly lied in two hot-spots, neighboring regions of exon 8 might be a real deletion 'hot spot' in this area compared with other areas of our country; introns 44-52 of dystrophin gene were highly unstable and prone to break: intron 44 was more stable than the whole molecular region of 'central deletion hot spot' and the unstability of intron 50 changed along with the regional and ethnic difference; the optimized primer-assembly provided the short-cut for detecting patients and making prenatal gene diagnosis, especially it's feasible and advantageous for the isolated pedigrees.

[Analysis and application of SCA1 and SCA3/MJD gene CAG repeats in Han population in Northeastern China].

OBJECTIVE: 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. METHODS: DNA fragments from the normal subjects and the patients were detected by fluorescence-PCR. Homozygosities were selected for DNA sequencing. RESULTS: 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. CONCLUSION: The normal ranges of CAG repeats vary with areas and races. SCAs genotyping is the first choice in presymptomatic and prenatal diagnosis.