Novel mutations in the lipase H gene lead to secretion defects of LIPH in Chinese patients with autosomal recessive woolly hair/hypotrichosis (ARWH/HT).

Autosomal recessive woolly hair/hypotrichosis (ARWH/HT: OMIM #278150/604379) is a rare hereditary hair disease characterized by tightly curled hair at birth which can lead to sparse hair later in life. The mutations in both LIPH and LPAR6/P2RY5 are responsible for autosomal recessive woolly hair with or without hypotrichosis (ARWH/HT). To conduct clinical and genetic investigations in four patients from three unrelated Chinese Han families with ARWH/HT, we performed mutation screening of LIPH and LPAR6/P2RY5 gene and identified four mutations in LIPH: c.454G>A, c.614A>G, c.736T>A, c.742C>A. c.736T>A and c.742C>A mutations were reported in previous studies, and c.454G>A, c.614A>G were identified for the first time. We carried out functional studies of the two mutants with c.454G>A (p.Gly152Arg, G152R) or c.614A>G (p.His205Arg, H205R). Interestingly, both of them lead to secretion defects of LIPH, which are involved in the pathogenesis of ARWH/HT.

A novel non-stop mutation in MSX1 causing autosomal dominant non-syndromic oligodontia.

Oligodontia, which is the congenital absence of six or more permanent teeth, excluding the third molars, may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. Msh homeobox 1 (MSX1) was the first gene identified as causing non-syndromic oligodontia. In this study, we identified a novel heterozygous non-stop mutation (c.910_911dupTA, p.*304Tyrext*48) in MSX1 in a Chinese family with autosomal dominant non-syndromic oligodontia. This novel mutation substitutes the stop codon with a tyrosine residue, potentially adding 48 amino acids to the C-terminus of MSX1. Further in vitro study found that mutant MSX1 could be expressed but had lost its ability to enter the nucleus. This is the first report indicating that a non-stop mutation in MSX1 is responsible for oligodontia. This study broadens the mutation spectrum for MSX1 and provides a new way to clarify the mechanism of MSX1 in tooth agenesis.

Mutation detection of type II hair cortex keratin gene KRT86 in a Chinese Han family with congenital monilethrix.

BACKGROUND: Monilethrix is an autosomal dominant hair disorder characterized clinically by alopecia and follicular papules. In this study, we collected a Han monilethrix family to detect the mutations in patients and investigated the correlation between the genotype and phenotype of monilethrix. METHODS: In this study, we identified a Chinese family with monilethrix through light microscopic and scanning electron microscopic (SEM) examination. Genomic DNA from peripheral blood samples was prepared. DNA samples from controls and monilethrix patients were subject to polymerase chain reaction (PCR) amplification. Two pairs of primers were used to amplify the seventh exon of KRT86. Mutation screening of the PCR products was detected using direct sequencing. RESULTS: Light microscopic examination showed a regular alternate enlargement and narrow area. SEM examination showed that part of the cuticle of the nodules shed and disappeared gradually in the narrow area with granular protrusions on the surface similar to the erosion-like structure. Parallel longitudinal ridge and groovepattern appeared, and the ridges varied in width, like dead wood. A heterozygous transversion mutation c.1204G > A (p.E402K) in the seventh exon of KRT86 was identified in both patients. CONCLUSIONS: The mutation of extron 7 of KRT86 identified plays a major role in the pathogenesis of this pedigree with monilethrix, and is a mutation hot spot of KRT86. Further research is needed to explore the relationship between the phenotype and the mutation of the type II hair keratin gene KRT86 of monilethrix.

Novel deletion mutation of TRPS1 gene in a Chinese patient of trichorhinophalangeal syndrome type I.

Tricho-rhino-phalangeal syndrome (TRPS) is a rare autosomal dominant disorder. Deletion or mutation of the TRPS1 gene leads to the tricho-rhino-phalangeal syndromes type I or type III. In this article, we describe a Chinese patient affected with type I TRPS and showing prominent pilar, rhinal and phalangeal abnormalities. Mutational screening and sequence analysis of TRPS1 gene revealed a previously unidentified four-base-pair deletion of nucleotides 1783-1786 (c.1783_1786delACTT). The mutation causes a frame shift after codon 593, introducing a premature stop codon after 637 residues in the gene sequence. This deletion is an unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein. Our novel discovery indicates that sparse hair and metacarpal defects of tricho-rhino-phalangeal syndromes in this patient are due to this TRPS1 mutation. And this data further supports the critical role of TRPS1 gene in hair and partial skeleton morphogenesis.

[Study on the mechanism of C17orf62 induced cell death].

OBJECTIVE: To isolate the long coding sequence of human novel gene C17orf62 which is named as C17orf62-L by us, and analyze its effects on cell viability, subcellular localization, and expression profile in multiple cell lines. METHODS: RT-PCR (reverse transcription polymerase chain reaction ) was used to clone C17orf62-L which encoded 187 amino acids from human multi-tissue cDNA library. We used bioinformatics analysis to identify structural characteristics of C17orf62-L, and RT-PCR to detect its expression. By Laser Scanning Confocal Microscopy we identified its subcellular localization of C17orf62-L. Furthermore, flow cytometry experiment was used to validate whether overexpression of C17orf62-L could influence cell phenotypes and Western blot was used to study related mechanisms. RESULTS: C17orf62-L was cloned and constructed into the pcDNA-and pEGFP-expression plasmids. C17orf62-L had signal peptide and transmembrane domain.C17orf62-L was widely expressed in multiple cell lines and was validated partial co-localization with Golgi apparatus. Functional studies showed C17orf62-L could induce cell death accompanied with rising of cleaved PARP(poly ADP-ribose polymerase). CONCLUSION: Human C17orf62 is a novel cell death inducing gene.

[Correlation study on polymorphisms of vitamin D receptor gene in patients with periodontitis].

OBJECTIVE: To investigate the relationship between vitamin D receptor (VDR) gene polymorphisms and periodontitis. METHODS: Ninety patients with aggressive periodontitis(AgP), 34 patients with chronic periodontitis and 91 healthy controls were recruited in this study. VDR gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with Taq I restriction endonuclease. RESULTS: The detection frequency of Tt genotype was higher in the aggressive periodontitis patients than in the healthy controls (16.7% vs 7.7%, P>0.05). The difference between the female aggressive periodontitis patients and healthy controls (20.8% vs 4.2%, P<0.05) was statistically significant, but no difference was found between the male patients and controls (10.8% vs 11.6%, P>0.05). There was a strong association between aggressive periodontitis and Tt genotype in females (AgP patients vs healthy controls, OR=6.02). The detection frequency of Taq I ER-alpha genotypes was not statistically different between the chronic periodontitis patients and healthy controls. CONCLUSION: In female Han Chinese population, the Tt VDR genotype may be associated with aggressive periodontitis.

Adenovirus-mediated PDCD5 gene transfer sensitizes K562 cells to apoptosis induced by idarubicin in vitro and in vivo.

PDCD5 (programmed cell death 5) accelerates apoptosis of certain tumor cells and is expressed at low levels in marrow-nucleated cells of AML and CML patients. In the present study, we evaluated the effects of PDCD5 overexpression on drug sensitivity of leukemia cells. K562 cells were treated with idarubicin (IDR) alone or in combination with adenoviral vectors expressing PDCD5 (Ad-PDCD5). As shown by annexin-V-FITC/PI dual labeling, apoptosis rates were markedly increased after combined treatment with Ad-PDCD5 compared to IDR treatment alone. We observed that PDCD5 overexpression significantly improves the antitumor effects of low dose IDR treatment in vivo. Tumor sizes were significantly decreased in combined Ad-PDCD5 and low dose IDR treatment groups compared with single IDR treatment groups. Similar results were obtained with combined systemic treatment of Ad-PDCD5 and low dose IDR, and combined treatment with Ad-PDCD5 local injection and low dose IDR i.p. injection. These results indicate that Ad-PDCD5 may be a promising agent for enhancing chemosensitivity.

[Preparation and identification of monoclonal antibodies against human programmed cell death 10 (PDCD10)].

OBJECTIVE: To obtain monoclonal antibodies against programmed cell death 10 (PDCD10) for further study of the structure and function of PDCD10 protein. METHODS: Balb/c mice were immunized with recombinant PDCD10, hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by ELISA, Western blotting and Immunofluorescence assay. RESULTS: Three hybridoma cell lines (5G1, 4F7 and 3H5) stable in secreting specific monoclonal antibodies were successfully obtained. Subclass of IgG belonged to IgG1 (4F7 and 5G1) and IgG2b (3H5), respectively. The ascite titers of these monoclonal antibodies reached 1:10(7). They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting. They failed to react with E.coli lysates and glutathione S-transferase (GST). In addition, these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay. Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus. CONCLUSION: Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared, which has laid the foundation for further study of PDCD10 protein.

[PTCH gene mutations in odontogenic keratocysts].

OBJECTIVE: To investigate the frequency, type and distribution of PTCH mutations in odontogenic keratocysts (OKC) and to analyze the molecular pathological relationship between sporadic OKC and OKC associated with nevoid basal cell carcinoma syndrome (NBCCS). METHODS: Genomic DNA was extracted from 8 cases of OKC lesions (4 sporadic OKCs and 4 NBCCS-related OKCs). PTCH gene mutations were detected by PCR-direct sequencing. RESULTS: Six novel PTCH mutations were identified in 6 out of 8 cases (2 sporadic and 4 NBCCS-related OKCs). Two of these were missense mutations leading to substitution of an amino acid residue respectively. The other 4 mutations were identified as insertion or deletion ranging from one single base to 7 bases, three of which caused frame-shift leading to premature truncation of PTCH protein and one resulted in an insertion of 2 amino acid residues. All these identified mutations were novel and have not been previously described. CONCLUSIONS: PTCH gene mutation is a common event in NBCCS-related OKCs and could also be detected in some sporadic OKCs. Abnormalities of PTCH gene may be involved in the pathogenesis of OKC.

[Gene mutation detection in a cleidocranial dysplasia family].

OBJECTIVE: To study gene mutation in Chinese patients with cleidocranial dysplasia. METHODS: A three generation family with the clinical diagnosis of cleidocranial dysplasia was investigated in present study. Genomic DNA was extracted from peripheral blood samples of each of the family members. Direct sequencing of the PCR products of the coding region of CBFA1 gene was used to identify the mutations. RESULTS: In each patient of the family, a heterozygous missense mutation, cDNA 674 G > A (R225Q), was detected in CBFA1 exon 3. The mutation changed the sequence in runt domain of the protein. CONCLUSIONS: Our findings indicate that mutation in CBFA1 is responsible for the tooth agenesis and other phenotypes of cleidocranial dysplasia in this Chinese family. The mutation detection could be applied in prenatal diagnosis for the family.

[Interleukin-1 polymorphisms in patients with aggressive periodontitis].

PURPOSE: To study whether interleukin-1 (IL-1) genotypes and/or alleles were associated with aggressive periodontitis (AgP). METHODS: Anti-coagulated peripheral blood samples were obtained from 122 AgP patients and 95 healthy controls. Genomic DNA was extracted from each sample. Single nucleotide polymorphisms (SNPs) at IL-1A +4845 and IL-1B +3954 were analyzed by standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The polymorphism of a variable number tandem repeat (VNTR) in intron 2 of IL-1RN was detected by PCR amplification and fragment size analysis. The genotypes and/or alleles distribution differences between two groups were analyzed by logistic regression analysis. RESULTS: The frequencies of A2+ genotype and allele 2 at IL-1A +4845 were significantly increased in male AgP patients compared with male controls (genotype: 18.0% vs 4.3%, adjusted OR=5.58, 95% CI=1.09-28.68, P=0.039; allele: 9.0% vs 2.2%, adjusted OR=4.97, 95% CI=1.01-24.50, P=0.049). CONCLUSIONS: The IL-1A +4845 polymorphism may be associated with AgP susceptibility in Chinese males.

[The relationship between a nucleotide substitution of S100A8 gene and aggressive periodontitis].

OBJECTIVES: To screen polymorphisms in the upstream region of S100A8 gene and to detect whether the polymorphisms were associated with aggressive periodontitis. METHODS: Thirty aggressive periodontitis patients and twenty-eight healthy controls were recruited for the study with informed consent. All subjects were of Chinese descent and systemically healthy. The regions about 800 bp upstream from the ATG start codon in exon 2 of the S100A8 gene of 10 patients and 8 controls were amplified by polymerase chain reaction (PCR) and analyzed by direct sequencing. A single nucleotide polymorphism (SNP) at 94 bp upstream from the ATG start codon was selected, and then the shorter regions (about 250 bp upstream from the ATG start codon) of the rest subjects were also amplified by PCR and analyzed by direct sequencing. The frequency of the SNP and the distribution of the genotype were detected and compared between the two groups. RESULTS: A nucleotide substitution (A-->G) at 94 bp upstream from the ATG start codon was demonstrated in Chinese, which was in a cis-acting element, named gamma interferon response element (gamma-IRE) in intron 1 of S100A8 gene. All of the subjects that carried the polymorphism were heterozygous. There was no statistically significant difference in the frequency of allele 2 (corresponding to the nucleotide G) between patients and controls (11.7% vs. 17.9%, chi2 = 0.887, P > 0.05). The prevalence of the heterozygous genotype was 23.2% and 35.7% (chi2 = 1.07, P > 0.05) in patients and controls, respectively. CONCLUSIONS: This is the first report that a nucleotide substitution of S100A8 gene was demonstrated in Chinese. The frequencies of allele 2 and heterozygous genotype were lower in patients, but there is no statistically significant difference between the aggressive periodontitis patients and healthy controls in this preliminary study.

Association analysis between interleukin-1 family polymorphisms and generalized aggressive periodontitis in a Chinese population.

BACKGROUND: It has been suggested that aggressive periodontitis (AgP) has a genetic basis, but this theory has not been confirmed. The intent of this investigation was to study whether specific interleukin (IL)-1 genotypes and/or alleles could be used to predict susceptibility to generalized AgP (GAgP) in Chinese. METHODS: The GAgP group consisted of 122 patients, and the control group included 95 healthy subjects. Single nucleotide polymorphisms at IL-1A (+4845) and IL-1B (-511, +3954) were analyzed by standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The polymorphism of a variable number tandem repeat (VNTR) in intron 2 of IL-1RN was detected by PCR amplification and fragment size analysis. RESULTS: There was no significant association of IL-1 polymorphisms with GAgP in the unstratified subjects. However, when cases were stratified by gender, the frequencies of A2+ genotype and allele 2 at IL-1A +4845 were significantly increased in male patients compared to male controls (genotype: odds ratio [OR] 5.58, 95% confidence interval [CI]: 1.09 to 28.68, P = 0.039; allele: OR 4.97, 95% CI: 1.01 to 24.50, P = 0.049; adjusted for age and smoking status). The frequency of IL-1B -511 A1/A2 heterozygote was significantly increased in male GAgP group compared to male controls (adjusted OR 3.16, 95% CI: 1.01 to 9.89, P = 0.048). In females, no significant differences were found between patients and controls in corresponding analyses at all polymorphic loci. A possible combined effect of IL-1B -511 polymorphism and smoking on the elevated risk to GAgP was observed. The OR of GAgP for combined A2+ genotype and smoking was 12.45 (95% CI: 1.43 to 108.06, P = 0.022), and for combined allele 2 and smoking was 18.25 (95% CI: 2.32 to 143.86, P = 0.006). CONCLUSIONS: The polymorphisms of IL-1A +4845 and IL-1B -511 may play an important role in determining GAgP susceptibility in Chinese males. Furthermore, a possible combined effect of the polymorphism of IL-1B -511 and smoking on GAgP susceptibility was suggested.