Identification And Functional Analysis Of Pathogenic Genes In Families With Syndactyly And Pachyonychia Congenita

A single gene disorder is the result of a single mutated gene, also named monogenic disease, which is a natural model to study the relationship between disease and gene. Online Mendelian Inheritance in Man(OMIM) is an online catalog of human genes and genetic disorders, which is an important database for researcher.To date, 4368 entries have phenotype description and molecular basis is known, 1669 have phenotype description and disease gene locus but the molecular basis is unknown, and there are an amount of diseases with suspected Mendelian basis. So, it is necessary to excavate the genetic disease resources and carry out the relevant studies. Present study focused on monogenic diseases of syndactyly, and pachyonychia congenita. Clinical, genetic and functional analyses were carried out as following three parts:Part 1: Identification and functional analysis of pathogenic gene in families with syndactyly type I-cBackground:Syndactyly(SD) is a digital malformation in which adjacent fingers and/or toes are webbed because they fail to separate during limb development. It is one of the most common hereditary limb malformations in births. The current classification scheme defined nine types syndactyly at least on the basis of phenotype and genotype. SD1 is an autosomal dominant limb malformation characterized in its classical form by complete or partial webbing between the third and fourth fingers and/or the second and third toes. Its four subtypes(a, b, c, and d) are defined based on variable phenotypes, however, the responsible gene is yet to be identified. SD1-a has been mapped to chromosome 3p21.31 and SD1-b to 2q34–q36. SD1-c and SD1-d are very rare and, to our knowledge, no gene loci have been identified. In this study, two Chinese families with SD-1c were collected and related analyses for finding disease gene were carried out.Materials and Methods: After family investigation and pedigree analysis, clinical data were collected and blood samples of probands or family members and healthy controls were obtained. Genomic DNA was extracted and the whole coding regions and exon-intron boundaries of syndactyly-related gene were screened by polymerase chain reaction(PCR) and direct sequencing. Typing short tandem repeats(STR), linkage, haplotype analyses, bioinformatics and cytological experiments were perforem for pathogenic mutaions of SD1-c families. The gene-phenotype analysis for disease gene also was performed.Main Results: In two Chinese families with SD1-c, linkage and haplotype analyses mapped the disease locus to 2q31-2q32. Copy number variation(CNV) analysis using array-CGH excluded the possibility of microdeletion or microduplication. Sequence analyses of candidate syndactyly genes in this region identified c.917G>A(p.R306Q) in the homeodomain of HOXD13 in family A. Analysis on family B identified the mutation c.916C>G(p.R306G) and therefore confirmed the genetic homogeneity. Luciferase assays indicated that these two mutations affected the transcriptional activation ability of HOXD13. The spectrum of HOXD13 mutations suggested a close genotype-phenotype correlation between the different types of HOXD13-Syndactyly. Overlaps of the various phenotypes were found both among and within families carraying the HOXD13 mutation.Conclusions: Mutations(p.R306 Q and p.R306G) in the homeodomain of HOXD13 cause SD1-c. There are affinities between SD1-c and synpolydactyly. Different limb malformations due to distinct classes of HOXD13 mutations should be considered as a continuum of phenotypes and further classification of syndactyly should be done based on phenotype and genotype.Part 2: Identification and functional analysis of pathogenic gene in a family with syndactyly type IVBackground:SD4 is inherited in an autosomal dominant fashion and characterized by complete cutaneous syndactyly of all fingers accompanied with polydactyly. Triphalangeal thumb-polysyndactyly syndrome(TPTPS) consists of triphalangeal thumb, polydactyly and syndactyly is a variable phenotype of SD4. Genomic duplications of the long-range limb-specific cis-regulator(ZRS) of 7q36 region cause SD4 and TPTPS. In this part, one family with SD4 with TPTPS phenotype was indentified by q PCR and array-based comparative genomic hybridization(array CGH) for ZRS region.Materials and Methods After family investigation and pedigree analysis, clinical data were collected and blood samples of probands or family members and healthy controls were obtained. The q PCR and array-CGH for the ZRS region were carried out in SD4 family. In addition, the gene-phenotype analyses for disease genes also were performed.Main Results: In the collected family, one individual had overlapping clinical symptoms of TPTPS and SD4, while the other had a typical SD4 with postaxial polydactyly of the toe. Results of q PCR suggested that the duplication of ZRS involved all affected individuals and array-CGH detected its size as 115.3 kb.Conclusions: The work of SD4 family confirms the genetic homogeneity of SD4 and TPTPS. The result expands the spectrum of ZRS duplications. TPTPS and SD4 should be considered a continuum of phenotypes.Part 3: Mutation screening and functional analysis of pathogenic genes in families with pachyonychia congenitaBackground:Pachyonychia congenita(PC), a rare autosomal dominant disorder characterized by hypertrophic nail dystrophy, is classified into two main clinical subtypes, PC-1 and PC-2. PC-1 is associated with mutations in the KRT6 A or KRT16 genes, whereas PC-2 is linked to KRT6 B or KRT17 mutations. In addion, types of PC-16, PC-6a, PC-17, PC-6b and PC-U(U: unknown) based disease genes also were applied. In this part, two families with PC were collected and related research was carried out, in order to defining the gene diagnosis, expending the phenotype and genotype spectrums and providing evidence for mechanisms of disease research.Materials and Methods:Blood samples were collected from two Chinese families with PC(F01 and F02). The entire coding region of the KRT6 A, KRT16, KRT6 B and KRT17 genes were amplified by polymerase chain reaction and analyzed by direct sequencing. Functions of mutants of disease gene were assessed by Poly Phen-2, SIFTS, Swiss-Model and cytological experimental methods. In addition, the gene-phenotype analyses for disease genes also were performed.Main Results:(1) In family F01, a novel missense mutation p.Leu128Pro(c.383T>C) was identified in a highly conserved helix motif in domain 1A of K16. Poly Phen-2 and SIFTS analysis rated the substitution as probably damaging; Swiss-Model analysis indicated that the structure of the mutant protein contained an unnormal α-helix. Overexpression of mutant protein in cultured cells led to abnormal cell morphology. The wider spectrum of KRT16 mutations suggests that changes in codons 125, 127 and 132 are most commonly responsible for PC-1, and that proline substitution mutations at codons 127 or 128 may produce more severe disease.(2) In family F02, we did not find any mutation in these four PC genes and further work need to performd, and family F02 may belong to PC-U type.Conclusions: The wider spectrum of KRT16 mutations suggests that changes in codons 125, 127, and 132 are most commonly responsible for PC-1 and that proline substitution mutations at codons 127 or 128 may produce more severe disease. This study extends the PC mutation spectrum and adds new information on the clinical and genetic diversity of PC.In summary, present works indentify the disease gene of HOXD13 to syndactyly type I-c for the first time. Mutations in the homeodomain of HOXD13 cause SD1-c in Chinese families. In addition, novel muations of ZRS region and KRT16 gene for syndactyly type IV and pachyonychia congenita are found and reported. This study extends the phenotype and mutation spectrums, and also adds new information for clinicl genetic services in the diaseases of syndacyly and pachyonychia congenita.