A gain-of-function mutation in Tnni2 impeded bone development through increasing Hif3a expression in DA2B mice.

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.

Microarray analysis of two single-nucleotide polymorphisms of transforming growth factor alpha in patients with nonsyndromic cleft of north china.

OBJECTIVE: To investigate the association between the TGFA:c.3851T > C (rs11466285) and TGFA:c.3822G > A (rs3771523) single-nucleotide polymorphisms (SNPs) and nonsyndromic cleft lip and/or cleft palate (CL/P) with microarray in north China. DESIGN AND SETTING: Test association by both case-control and case-parent analysis. SUBJECTS AND METHODS: Two SNPs of 150 controls, 166 cases, and 271 of their parents were genotyped using microarray based on the allele-specific primer extension, and chi-square statistics and family-based association test analyses were performed. RESULTS: Both sequencing and microarray analysis produced identical results. We found significant evidence of overtransmission of the C allele of c.3851T > C and the A allele of c.3822G > A in case-parent trios for CL/P but not for cleft palate only (CP). Significant differences for both genotypic and allelic distributions between cases and controls were found at c.3822G > A and c.3851T > C for CL/P but not for CP. The TGFA [C; G] and [T; A] haplotypes showed significant overtransmission. CONCLUSIONS: These results support that two SNPs are associated with nonsyndromic CL/P but not for CP in northern Chinese populations. It was demonstrated that this microarray is suitable to test SNPs associated with nonsyndromic CL/P.

Elevated expression of EP4 in human decidua is associated with delayed embryo expulsion during medical abortion by promoting decidual cell proliferation.

Purpose: Mifepristone in conjunction with misoprostol, is widely used in China as an effective medical abortifacient. However, a small proportion of women experience the unpleasant side effects of prolonged vaginal bleeding caused by delayed embryo expulsion. The aims of this study were to determine whether the expression levels of prostanoid receptors in human decidua are associated with delayed embryo expulsion in mifepristone-misoprostol induced an early medical abortion.Methods: Discharged decidua tissues were collected from females undergoing an artificial abortion (AA) (n = 28), females with early embryo expulsion during a medical abortion (EEMA) (n = 20) and delayed embryo expulsion in medical abortion (DEMA) (n = 30). The expression levels of prostanoid receptors in human decidua were assessed with immunohistochemistry and real-time PCR methods. Further, the RNAi method was used to silence prostanoid receptors 4 (EP4) in the primary decidual cells and human endometrial adenocarcinoma cell line Ishikawa cells in vitro and cell cycle analysis of these cells was performed.Results: All five prostanoid receptors (EP1-4, FP) were observed in human early pregnancy decidua. The protein and mRNA expression level of EP4 in the DEMA group were all significantly higher than that in the EEMA group. EP4 silence induced G1/S arrest of primary decidual cells and Ishikawa cells in vitro.Conclusions: Elevated expression level of EP4 in human decidua was significantly associated with delayed embryo expulsion in early medical abortion by promoting decidual cell proliferation. Detailed studies on the nature of roles EP4 plays in human decidua will help us to develop more effective prevention and noninvasive intervention approaches for delayed embryo expulsion during a medical abortion.

Association of the G2014G genotype in estrogen receptor 1 gene with failure of the mifepristone-induced termination of early pregnancy.

Mifepristone is a synthetic steroid compound that has been applied to terminate early pregnancy for many years. However, about 15% of the women undergo failure in termination of early pregnancy, the causes of which remain largely unknown. We herein selected estrogen receptor 1 gene (ESR1) as a candidate gene to determine whether single nuclear polymorphisms (SNPs) in ESR1 were associated with the failure of mifepristone-induce abortion. The subjects recruited were 30 subjects that failed to abort and 60 subjects with a successful medical abortion. Three SNPs, T-397C (rs2234693), C325G (rs1801132), and G2014A (rs2228480), were analyzed by PCR, followed by restriction fragment length polymorphism (RFLP) analysis. The latter two polymorphic sites are located in the protein-coding region, but do not alter the amino acid. Among the three SNPs examined, we found the significant role of the G2014A polymorphism; namely, the distribution of the GG, AG and AA genotypes was different between the failure group and the success group. The frequency of the GG (G2014G) genotype was higher in the failure group (86.7%) than that in the success group (60.0%) (p = 0.030), while the frequency of the G2014A heterozygote was lower in the failure group (6.7%) than in the success group (28.3%) (p = 0.013). Moreover, the frequency of the G allele was higher in the failure group (90%) and lower in the success group (10.0%) (p = 0.013). These results indicate that the GG genotype of the G2014A polymorphism is associated with the risk of failure in the mifepristone-induced abortion.

Identification of the keratin 9 (KRT9) N161S mutation in a Chinese kindred with epidermolytic palmoplantar keratoderma.

We present a family from Northeast China affected by epidermolytic palmoplantar keratoderma (EPPK) in which we confirmed the presence of the N161S mutation as the result of a 548A>G transition in exon1 of the keratin 9 gene. Genomic DNA from peripheral blood of all available members in this family was used for amplification of exon 1 of KRT9 by polymerase chain reaction. The mutation was detected by direct sequence analysis and identified by restriction endonuclease DdeI digestion. The finding of the same mutation in all available patients, together with the previous reports of the disease, strongly suggested that position 161 of the KRT9 gene also represents a mutation "hotspot" for EPPK. Our result is an important contribution to the investigation of the genotype/phenotype correlation and affords molecular genetic knowledge for future clinical diagnosis and gene therapy of EPPK.

Elevated mRNA expression of PGF2α receptor splice variant 2(FP-V2) in human decidua is associated with incomplete mifepristone-misoprostol-induced early medical abortion by regulation of interleukin-8.

OBJECTIVE: The combination of mifepristone and misoprostol is an established method for the induction of early abortion, but 15% of women still experience the unpleasant side effect of incomplete medical abortion. The purpose of this study was to determine whether prostaglandin (PG) F2α receptor (FP) and its two isoforms (FP-V1 and FP-V2) in human decidua are associated incomplete abortion. METHODS: Forty women who underwent medical abortion were recruited. Among them, there were 20 cases of incomplete abortion. The other 20 cases of complete abortion were used as controls. The expression levels of FP, FPV1 and FP-V2 in the decidua between of the two groups was detected by quantitative real-time polymerase chain reaction (PCR). Additionally, FP-V2 was knocked down using specific small interfering RNAs (siRNAs) in the primary cultures of decidual cells. The expression levels of cytokines in FP-V2 knockdown primary decidual cells and control decidual cells were detected by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: The FP and FP-V2 mRNA expression in the incomplete group was significantly higher than that in the complete group (p < 0.05). IL-8 was up-regulated by FP-V2 knockdown in primary-cultured decidual cells (p < 0.05). CONCLUSIONS: These results suggested that the elevated expression of FP-V2 in human decidua is significantly associated with incomplete mifepristone-misoprostol-induced early medical abortion and that IL-8 could be lined to this process.

[Report on 16 rare species of human chromosomal].

By examining the lymphocytic chromosomes of peripheral blood from patients with amenorrhea, spontaneous abortion and stillbirth history, .the 16 rare species of human chromosomal abnormal karyotypes were discovered. They were 46,XY,t(6;11)(q25;p15);46,XY,inv(3)(p25;q29);46,XY,t(7;18)(q10;p10);46,X,t(X;13)(q24;q14);46,XY,t(4;7)(q33;q22);46,XY,t(8;15)(q24;q15);46,XY,t(2;17)(q33;q25);46,XX,t(4;7)(q34;q11);46,XX,t(1;3)(p36;p23);46,XX,t(4;6)(q35;p11);46,X,inv(X)(q22;q28);46,XX,t(7;10)(p11;q26);46,XX,t(3;6)(p21;q23);46,XX,t(8;16)(p21;p13);46,XX,t(8;9)(q21;q34);46,XY,t(17;22)(q21;q11). Their clinical situation were described. Discussion on the relationship between the chromosomal aberrations and phenotype effect indicates the importance of chromosome karyotyping in patients with abnormal reproductive history.

Two novel mutations in myosin binding protein C slow causing distal arthrogryposis type 2 in two large Han Chinese families may suggest important functional role of immunoglobulin domain C2.

Distal arthrogryposes (DAs) are a group of disorders that mainly involve the distal parts of the limbs and at least ten different DAs have been described to date. DAs are mostly described as autosomal dominant disorders with variable expressivity and incomplete penetrance, but recently autosomal recessive pattern was reported in distal arthrogryposis type 5D. Mutations in the contractile genes are found in about 50% of all DA patients. Of these genes, mutations in the gene encoding myosin binding protein C slow MYBPC1 were recently identified in two families with distal arthrogryposis type 1B. Here, we described two large Chinese families with autosomal dominant distal arthrogryposis type 2(DA2) with incomplete penetrance and variable expressivity. Some unique overextension contractures of the lower limbs and some distinctive facial features were present in our DA2 pedigrees. We performed follow-up DNA sequencing after linkage mapping and first identified two novel MYBPC1 mutations (c.1075G>A [p.E359K] and c.956C>T [p.P319L]) responsible for these Chinese DA2 families of which one introduced by germline mosacism. Each mutation was found to cosegregate with the DA2 phenotype in each family but not in population controls. Both substitutions occur within C2 immunoglobulin domain, which together with C1 and the M motif constitute the binding site for the S2 subfragment of myosin. Our results expand the phenotypic spectrum of MYBPC1-related arthrogryposis multiplex congenita (AMC). We also proposed the possible molecular mechanisms that may underlie the pathogenesis of DA2 myopathy associated with these two substitutions in MYBPC1.

A novel FBN2 mutation in a Chinese family with congenital contractural arachnodactyly.

Congenital contractural arachnodactyly (CCA, OMIM: 121050) is an autosomal dominant condition that shares skeletal features with Marfan syndrome (MFS, OMIM: 154700), including contractures, arachnodactyly, dolichostenomelia, scoliosis, crumpled ears and pectus deformities but excluding the ocular and cardiovascular complications that characterize MFS. These two similar syndromes result from mutations in two genes belonging to the fibrillin family, FBN1 and FBN2, respectively. We successfully identified a novel FBN2 mutation (C1406R) in a Chinese family with CCA for over five generations. This mutation was detected in the patients of this family but not in the seven unaffected family members or 100 normal individuals. SIFT and PolyPhen analyses suggested that the mutation was pathogenic. We identified a missense mutation in the calcium binding-epidermal growth factor (cbEGF)-like domain. Our study extends the mutation spectrum of CCA and confirms a relationship between mutations in the FBN2 gene and the clinical findings of CCA.

[Report of a rare distal arthrogrophy large family].

We describe a seven-generation large family with talipomanus and talipes, 175 individuals in this family were involved. 32 affected individuals including 18 males and 14 females whose clinical features were different and 1 suspicious male were found. The talipomanus were symmetrical, and varus and valgus were caused by vertical talus. We investigated their living environment, the dietary habit, obstetrical history, physical status, lifespan, and studied cytogenetics and so on. We propose these defects were rare distal arthrogrophy genetic disease.

A further heterogeneous locus contained in 8q21 for Freeman-Sheldon syndrome may be suggested by Kristin Hofmann.

Letter to the editor on a recent publication of Hofmann, K., et al. (2011). "7 Mb de novo deletion within 8q21 in a patient with distal arthrogryposis type 2B (DA2B)." in the European Journal of Medical Genetics. The author may suggest a further heterogeneous locus contained in 8q21 for Freeman-Sheldon syndrome.

A novel TNNI2 mutation causes Freeman-Sheldon syndrome in a Chinese family with an affected adult with only facial contractures.

Distal arthrogryposes (DAs), a clinically and genetically heterogeneous group of disorders characterized by congenital contractures with predominant involvement of the hands and feet, can be classified into at least 12 different forms. These autosomal dominant disorders are of variable expressivity and reduced penetrance. Mutations in sarcomeric protein genes, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosin 2 (TPM2), embryonic myosin heavy chain 3 (MYH3), and myosin binding protein C1 (MYBPC1), have been identified in distal arthrogryposis type 1 (DA1, MIM 108120), type 2B (DA2B, MIM 601680) and type 2A (DA2A)/Freeman-Sheldon syndrome (FSS, MIM 193700). However, mutations causing FSS have only been reported in MYH3. Herein we describe a Chinese DA family whose members meet classical strict criteria for FSS, as well as one member of the family who has isolated facial features consistent with FSS. No disease-causing mutation was found in MYH3. Segregation of microsatellite markers flanking the TNNI2 and TNNT3 genes at 11p15.5 was compatible with linkage. Subsequent sequencing of TNNI2 revealed a novel mutation, c.A493T (p.I165F), located in the C-terminal region, which is critical for proper protein function. This mutation was found to cosegregate with the FSS phenotype in this family, and assessment using SIFT and PolyPhen-2 predicted a damaging effect. To the best of our knowledge, we report the first TNNI2 mutation in classical FSS and describe an atypical adult FSS case with only facial contractures resulting from somatic mosaicism. We infer that DA1, DA2B and FSS represent a phenotypic continuum of the same disorder and provide further genetic evidence for this hypothesis.

TGFA and IRF6 contribute to the risk of nonsyndromic cleft lip with or without cleft palate in northeast China.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) are common birth defects with a complex etiology. Multiple interacting loci and possible environmental factors influence the risk of NSCL/P. 12 single nucleotide polymorphisms (SNPs) in 7 candidate genes were tested using an allele-specific primer extension for case-control and case-parent analyses in northeast China in 236 unrelated patients, 185 mothers and 154 fathers, including 128 complete trios, and 400 control individuals. TGFA and IRF6 genes showed a significant association with NSCL/P. In IRF6, statistical evidence of an association between rs2235371 (p = 0.003), rs2013162 (p<0.0001) and NSCL/P was observed in case-control analyses. Family based association tests (FBATs) showed over-transmission of the C allele at the rs2235371 polymorphism (p = 0.007). In TGFA, associations between rs3771494, rs3771523 (G3822A), rs11466285 (T3851C) and NSCL/P were observed in case-control and FBAT analyses. Associations between other genes (BCL3, TGFB3, MTHFR, PVRL1 and SUMO1) and NSCL/P were not detected.

Association of glutathione S-transferase M1 and T1 null polymorphisms with the development of cervical lesions: a meta-analysis.

OBJECTIVES: An accumulation of evidence suggests that gene-based self-susceptibility may contribute to the development of cancer. Some studies have found that particular polymorphisms of the glutathione S-transferase M1 and T1 genes are associated with increased risk of cervical lesions, but other studies have had contrary results. The present meta-analysis evaluated the association of glutathione S-transferase M1 and T1 null polymorphisms with the development of cervical lesions. In addition, stratified analyses were performed in an attempt to identify any race-specific effects. STUDY DESIGN: Twenty-one related studies were included in the meta-analysis, comprising glutathione S-transferase M1 data from 1423 patients with cervical lesions and 2415 healthy matched controls, and glutathione S-transferase T1 data from 2081 patients with cervical lesions and 2287 healthy matched controls. The fixed-effect model (Mantel-Haenszel method) and the random-effect (DerSimonian and Laird) model were used to examine the difference in frequency of glutathione S-transferase M1 and T1 null polymorphisms between pre- and invasive cervical lesions. Subgroup analyses were also conducted to evaluate any race-specific effect on the frequencies of glutathione S-transferase polymorphisms in cervical lesions. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. Heterogeneity was assessed using the heterogeneity metric (I²) and Chi-squared test. RESULTS: The glutathione S-transferase M1 null polymorphism was associated with increased risk of low-grade intra-epithelial lesions (OR 1.37, 95% CI 1.05-1.77), but no increased risk of high-grade intra-epithelial lesions (OR 1.29, 95% CI 0.87-1.8) or invasive cervical cancer (OR 1.20, 95% CI 0.99-1.46). The association seemed to be confined to Southeast Asians (OR 1.97, 95% CI 1.44-2.71). No significant associations were found for the glutathione S-transferase T1 null polymorphism for any of the populations. CONCLUSIONS: The glutathione S-transferase M1 null polymorphism significantly increases susceptibility to early-stage cervical lesions in Southeast Asians. However, the glutathione S-transferase T1 null polymorphism does not appear to be a risk factor for cervical lesions in any population.

A novel mutation in TNNT3 associated with Sheldon-Hall syndrome in a Chinese family with vertical talus.

Distal arthrogryposis (DA) is a group of rare, clinically and genetically heterogeneous disorders primarily characterized by congenital contractures of the limb joints. Recently, mutations in genes encoding the fast-twitch skeletal muscle contractile myofibers complex, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosine 2 (TPM2), and embryonic myosin heavy chain 3 (MYH3), and the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1) were confirmed to cause DA1, DA2A, and DA2B. DA2B, or Sheldon-Hall syndrome (SHS; MIM 601680), is intermediate to DA1 and DA2A, or Freeman-Sheldon syndrome (FSS; MIM193700), and shows prominent facial traits. This report describes a Chinese family with SHS over three generations in which all affected individuals showed vertical talus and one demonstrated preauricular tags on the face. Linkage analysis and PCR sequencing revealed a novel substitute mutation at a hot-spot site in TNNT3 (c.187C > T; p.R63C). This mutation was confirmed to cosegregate with the DA phenotype in affected individuals. SIFT and PolyPhen analyses suggest that the mutation is pathogenic. We report this mutation in TNNT3 and speculate that bilateral vertical talus, or severe clubfoot, might be a special characteristic for cases with the TNNT3 R63C mutation.

Molecular prenatal diagnosis for hereditary distal arthrogryposis type 2B.

Autosomal dominant distal arthrogryposes (DAs) are a group of muscle diseases characterized by congenital contractures of the limbs. Currently, prenatal diagnosis of DAs depends upon ultrasound examination during late gestation. Recently, five genes encoding fast switch proteins located at 9p13.2, 11p15.5 and 17q13.1 were identified. These included TPM2, TNNI2/TNNT3, and MYH3/MYH8. Last year, we discovered a novel heterozygous mutation c.523_525delAAG (p.K175del) in the TNNI2 gene, which encodes the isoform of troponinI, in a seven-generation Chinese family affected with distal arthrogryposis type 2B (DA2B). Here, we report the molecular prenatal diagnosis of 3 high-risk fetuses of two women in the family by two-point linkage inferential analysis and deletion detection of the TNNI2 gene with chorionic villus sampling (CVS) or amniocentesis. To our knowledge, this is the first description of molecular prenatal diagnosis for DAs.

A novel deletion in TNNI2 causes distal arthrogryposis in a large Chinese family with marked variability of expression.

Distal arthrogryposis (DA) is composed of a group of clinically and genetically heterogeneous disorders, characterized by multiple congenital contractures of the limbs. Point mutations in three genes encoding contractile fast-twitch myofibers, TPM2, TNNI2 and TNNT3, were recently identified in DA type 1 (DA1; MIM 108120) and DA type 2B (DA2B; MIM 601680). We have described a large Chinese DA family in which different individuals had phenotypes similar to DA1 or DA2B. To map the disease locus in this family, two-point linkage analysis was first performed using microsatellite markers selected from the genomic regions close to the TPM2, TNNI2/TNNT3 and TNNC2 genes. A positive LOD score of 3.61 at theta = 0 was obtained with the marker close to the TNNI2/TNNT3 genes, corresponding to the genetic mapping site of DA2B. Direct sequencing of the PCR-amplified DNA fragment spanning exon 8 of the TNNI2 gene showed a heterozygous deletion, c.523_525delAAG (p.K175del), in the proband. This novel mutation was confirmed to cosegregate with the DA phenotype in affected individuals but not detected in all unaffected individuals of the family and not in 50 healthy controls. In summary, we have found a novel TNNI2 mutation in a Chinese family with DA2B. Our work represents the first report on the link between TNNI2 and the DA phenotype in Chinese.