Lihua Cao

Copy-number mutations on chromosome 17q24.2-q24.3 in congenital generalized hypertrichosis terminalis with or without gingival hyperplasia.

Congenital generalized hypertrichosis terminalis (CGHT) is a rare condition characterized by universal excessive growth of pigmented terminal hairs and often accompanied with gingival hyperplasia. In the present study, we describe three Han Chinese families with autosomal-dominant CGHT and a sporadic case with extreme CGHT and gingival hyperplasia. We first did a genome-wide linkage scan in a large four-generation family. Our parametric multipoint linkage analysis revealed a genetic locus for CGHT on chromosome 17q24.2-q24.3. Further two-point linkage and haplotyping with microsatellite markers from the same chromosome region confirmed the genetic mapping and showed in all the families a microdeletion within the critical region that was present in all affected individuals but not in unaffected family members. We then carried out copy-number analysis with the Affymetrix Genome-Wide Human SNP Array 6.0 and detected genomic microdeletions of different sizes and with different breakpoints in the three families. We validated these microdeletions by real-time quantitative PCR and confirmed their perfect cosegregation with the disease phenotype in the three families. In the sporadic case, however, we found a de novo microduplication. Two-color interphase FISH analysis demonstrated that the duplication was inverted. These copy-number variations (CNVs) shared a common genomic region in which CNV is not reported in the public database and was not detected in our 434 unrelated Han Chinese normal controls. Thus, pathogenic copy-number mutations on 17q24.2-q24.3 are responsible for CGHT with or without gingival hyperplasia. Our work identifies CGHT as a genomic disorder.

Novel point mutations in GDF5 associated with two distinct limb malformations in Chinese : brachydactyly type C and proximal symphalangism

AbstractGrowth/differentiation factor 5 (GDF5) is a secreted growth factor that plays a key regulatory role in embryonic skeletal and joint development. Mutations in the GDF5 gene can cause different types of skeletal dysplasia, including brachydactyly type C (BDC) and proximal symphalangism (SYM1). We report two novel mutations in the GDF5 gene in Chinese families with distinct limb malformations. In one family affected with BDC, we identified a novel nonsense mutation, c.1461T > G (p.Y487X), which is predicted to truncate the GDF5 precursor protein by deleting 15 amino acids at its C-terminus. In one family with SYM1, we found a novel missense mutation, c.1118T > G (p.L373R), which changes a highly conserved amino acid in the prodomain of GDF5. We transfected COS-7 cells with retroviral constructs to express human wild-type or mutant GDF5 cDNAs. The mature GDF5 protein was detected, as in the wild-type, in supernatant derived from the p.L373R mutant GDF5 transfected cells, but not in the supernatant from the p.Y487X mutant transfected cells, indicating that the two mutations led to different fates of the mutant GDF5 proteins, thereby producing distinct limb phenotypes.

Novel vitamin D 1α-hydroxylase gene mutations in a Chinese vitamin-D-dependent rickets type I patient.

Hereditary rickets is classified into four types: X-linked hypophosphatemic vitamin-D-resistant rickets, autosomal dominant hypophosphatemic vitamin-D-resistant rickets, vitamin-D-dependent rickets type I, and vitamin-D-dependent rickets type II, and the genes responsible are PHEX, FGF23, CYP27B1 and VDR, respectively (Clausmeyer et al. 2009; Gribaa et al. 2010; Malloy et al. 2010). Vitamin-Ddependent rickets type I (VDDR-I, MIM 264700), also known as vitamin D 1α-hydroxylase deficiency or pseudovitamin D deficiency rickets, is a rare autosomal recessive disorder characterized by the early onset of rickets with hypocalcemia. VDDR-I is caused by mutations of the 25hydroxyvitamin D 1α-hydroxylase gene (CYP27B1, MIM 609506) (Wang et al. 1998; Kitanaka et al. 1998). We identified compound heterozygous mutations in CYP27B1 gene in a Chinese VDDR-I patient. Both mutations are novel and include a small 11 nucleotide deletion (c.311321delGGCCCGAGCGC, p.R104LfsX225) in exon 2 that alters the downstream reading frame and creates a premature TGA stop signal at codon 328, and a missense mutation (c.473T>C, p.L158P) in exon 3 causing the amino acid change L158P. This study confirms the relationship between mutations of the CYP27B1 gene and the clinical findings of vitamin-D-dependent rickets type I.

A novel single-base deletion in ROR2 causes atypical brachydactyly type B1 with cutaneous syndactyly in a large Chinese family.

Mutations in ROR2, encoding the receptor tyrosine kinase-like orphan receptor 2, cause two distinct skeletal diseases: autosomal dominant brachydactyly type B1 (BDB1) and autosomal recessive Robinow syndrome. In a large Chinese family with a limb phenotype, consisting of atypical BDB1 and cutaneous syndactyly of varying degrees, we performed a two-point linkage analysis using microsatellite markers on 2q33–q37 and 9q22.31, and found a significant linkage to the ROR2 locus. We identified a novel single-base deletion in ROR2, c.2243delC (p.W749fsX24), and confirmed its segregation with the limb phenotype in the family. This deletion is predicted to produce a truncated ROR2 protein with an additional C-terminal polypeptide of 24 amino-acid residues. To the best of our knowledge, the deletion represents the second ROR2 mutation associated with a BDB1-syndactyly phenotype.

Novel and recurrent COL2A1 mutations in Chinese patients with spondyloepiphyseal dysplasia.

Spondyloepiphyseal dysplasia (SED) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses and flattened vertebral bodies. SED is mainly caused by mutations in the gene encoding the type II procollagen α-1 chain (COL2A1). We looked for mutations in COL2A1 in three unrelated Chinese families with SED. Putative mutations were confirmed by RFLP analysis. We identified three missense mutations (p.G504S, p.G801S and p.G1176V) located in the triple-helical domain; p.G801S and p.G1176V are novel mutations. The p.G504S mutation has been associated with diverse phenotypes in previous studies. Our study extends the mutation spectrum of SED and confirms a relationship between mutations in the COL2A1 gene and clinical findings of SED.

Identification of novel and recurrent mutations in the calcium binding type III repeats of cartilage oligomeric matrix protein in patients with pseudoachondroplasia.

Pseudoachondroplasia is an autosomal dominant osteochondrodysplasia characterized by disproportionate short stature, joint laxity, and early onset osteoarthrosis. Pseudoachondroplasia is caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). We looked for mutations in the COMP gene in three sporadic Chinese pseudoachondroplasia patients and identified two novel mutations, c.1189G>T (p.D397Y) and c.1220G>A (p.C407Y), and one recurrent mutation, c.1318G>C (p.G440R), in the calcium binding type III repeats of COMP. This study confirms the relationship between mutations of the COMP gene and clinical findings of pseudoachondroplasia; it also provides evidence for the importance of the calcium binding domains to the functioning of COMP.

A novel frameshift mutation in keratin 16 underlies pachyonychia congenita with focal palmoplantar keratoderma

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A novel mutation in the EXT1 gene identified in a Han Chinese kindred with hereditary multiple exostosis.

Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by growth of benign multiple exostoses. In our present study, we describe a four-generation Han Chinese kindred with eight members affected by HME. Haplotyping analysis and mutation detection was performed. The results linked the disease-causing gene to the EXT1 locus on chromosome 8. A novel mutation in EXT1, c.1897delC, which cosegregated with the disease phenotype, was detected. To further confirm this mutation, a mismatch primer was designed to introduce a ScaI restriction site into the normal allele by polymerase chain reaction, and the following restriction fragment length polymorphism analysis demonstrated that the mutation was not detected in any unaffected individuals of the family or 100 unrelated Han Chinese control individuals. This mutation leads to a frameshift from codon 633, resulting in a premature termination at codon 642 and loss of the highly conserved C terminal region of the protein. Therefore, this heterozygous mutation must be classified as pathogenic and can be regarded as the cause of HME in this Chinese family.

Mutations in mevalonate pathway genes in patients with familial or sporadic porokeratosis

Porokeratosis comprises heterogeneous keratinization disorders that are characterized by one or more atrophic patches surrounded by a ridge‐like cornoid lamella. In this study, we evaluated seven families affected by porokeratosis and five sporadic patients of the disease in a Chinese population. We performed Sanger sequencing of exons and flanking intron–exon boundaries of mevalonate pathway genes (MVD, MVK, PMVK and FDPS) and of SLC17A9. In five familial and three sporadic patients, we detected six variations, including four novel mutations (MVD c.1A>G; p.Met1?, c.916G>A; p.Ala306Thr, c.1013+1G>A, and PMVK c.65A>G; p.Lys22Arg) and two recurrent mutations (MVD c.746T>C; p.Phe249Ser, and MVK c.1028T>C; p.Leu343Pro). We then applied I‐TASSER and iGEMDOCK to assess these variants for probable functional impacts. The findings of this study extend the mutation spectrum of porokeratosis and provide further evidence for the genetic basis of this disease.

A missense mutation of HOXA13 underlies hand-foot-genital syndrome in a Chinese family

Hand-foot-genital syndrome (HFGS) is a rare autosomal dominant inherited syndrome characterized by limb malformations and urogenital defects. HFGS is caused by mutations in the HOXA13 gene. The aim of this study was to identify causative mutations in individuals and to explore the molecular pathogenesis in a Chinese family with HFGS. We performed Sanger sequencing and identified a recurrent missense mutation in the homeodomain (c.1123G>T, p.V375F) of HOXA13, molecular modelling predicted the mutation would affect DNA binding, and a luciferase reporter assay indicated that it impaired the ability of HOXA13 to activate transcription of the human EPHA7 promoter. This is the first report of the molecular basis for HFGS caused by missense mutations of HOXA13.