Mutation Analysis Of Inherited Hair Disorders

Part Ⅰ Identification of Microduplitations on Chromosome 6q22 in A Sporadic Case with Congenital Generalized HypertrichosisCongenital Generalized Hypertrichosis (CGH) is a phenotypic and genetic heterogeneous group characterized by excessive hair growth all over the body independent of androgen, and can be classified into several types with phenotypes overlapping with each other. Rearrangements on chromosome 4,8,17 and X, as well as point mutations in ABCA5, which is also located on chromosome 17, have been reported to cause CGH.In this study, a family with CGH was collected. The proband presented phenotypes similar to the Ambras form of CGH, with congenital visual disabilityand scoliosis, and the teeth began to shed at the age of 30 with none left by now. His parents and siblings presented no similar abnormal phenotypes. Blood or oralepithelia cells were obtained from the proband, his mother and all the four siblings after informed consent. The genomic DNA was subsequently extracted. Array comparative genomic hybridization (aCGH) analysis revealed two microduplications (named as Dupl and Dup2 respectively) on 6q22, which were validated to cosegregate with the disease phenotype in the family by quatitive real-time polymerase chain reaction (qPCR). A series of qPCR experiments were conducted to narrow down the boundaries, followed by gap-PCR attempts with different primer pairs until the breakpoints were amplified. Direct Sanger sequencing of the products revealed that the two microduplications were linked to each other. The structure was assumed for the rearranged chromosome through a further dual fluorescence in situ hybridization (FISH) assay. STR markers analysis within the microduplications suggested that both microduplications originate de novo paternally.Six genes are located or partially located within the two microduplications. GJA1 is the causative gene of oculo-dento-digital dysplasia (ODDD), thus the variation in its copy number might be the cause of the ophthalmic and dental phenotypes in the proband; the function of RSPO3 and RNF146 is reported to be related to Wnt signaling pathway, which regulates hair follicle cycling, and copy number variation of the two genes might lead to hypertrichosis, alone or in combination.In all, it is the first time that chromosomal rearrangements on chromosome 6 are detected in patients with CGH. We also inferred the structure of the rearranged chromosome, and speculated them to originate de novo paternally.Part II Identification of Microduplications on Chromosome Xq26.1 in Bazex-Dupre-Christol SyndromeBazex-Dupre-Christol Syndrome (BDCS) is an extremely rare genetic disorder characterized by a triad of congenital hypotrichosis, follicular atrophoderma and early-onset basal cell carcinomas (BCCs). It should be differentiated carefully since the clinical symptoms vary from or within families and overlap with some other diseases. BDCS is X-linked dominantly inherited, and the causative genes were mapped to an 11.4Mb interval on Xq25-q27.1. Although 12 genes within this interval were closely related to cell proliferation and differentiation, no mutations were so far identified, and the possibility of their causality was therefore excluded.Two European BDCS families were collected at the beginning of the project. A duplicated region shared by patients from both families was detected through whole genome copy number variation (CNV) analysis. qPCR assay validated the microduplications and showed that the microduplications cosegregated with disease phenotype in both families. Boundaries of the microduplications were subsequently narrowed down, with F2 to a base-level, using a combined method of qPCR, gap-PCR and Sanger sequencing. Chromosomal rearrangement mechanisms were also inferred in the two families.In order to get a further insight into the pathogenicity of the duplicated region, another six BDCS families from Europe were collected. Microduplications were detected in all affected individuals and cosegregated with the disease phenotype in all families through qPCR, as well as gap-PCR and Sanger sequencing at SNP sites as a make-up. Meanwhile, no similar microduplications were detected in a cohort of 215 unrelated unaffected individuals, total 354 X chromosomes. Boundaries of the microduplications in the six families were subsequently narrowed down, with F4, F7 and F9 to a base-level, using a combined method of qPCR, gap-PCR and Sanger sequencing. Chromosomal rearrangement mechanisms were also inferred in the six families. Besides, through haplotype analysis, microduplications in all eight families were proved to occur independently. Finally, transgenic mice with Krtl5-specific overexpression of Igsfl were generated, in attempt to conduct the phenotype evaluation and functional studies. However, no overexpression of Igsfl was detected within the expected region.In all, microduplications on Xq26.1 may probably be the causative mutation for BDCS, with abundant lines of gentic evidence mentioned above. More work remains to be done to clarify the pathogeny of the microduplications.Part III Identification of Pathogenic Mutations in Cases with Autosomal Dominant Hereditary HypotrichosisHereditary hypotrichosis (HH) is a group of rare clinical features, which occur alone or with other phenotypes. Patients usually show normal hair at birth, and begin to shed in several months. The disease shows high phenotypic heterogenecity, which can be generalized or scalp-limited, with or without abnormalities in hair shaft and hair follicles. Also, the disease shows high genetic heterogenecity, which can mainly be autosomal dominantly (AD) or autosomal recessively (AR) inherited. ADHH with known causative genes can be classified into three types. The first type is hereditary hypotrichosis simplex (HSS), and the causative genes are APCDD1, RPL21 and SNRPE for the generalized form, as well as CDSN for the scalp-limited form. The second type is Marie Unna hereditary hypotrichosis (MUHH), and the causative gene is U2HR for subtype 1 and EPS8L3 for subtype 2. The third type is woolly hair (WH), which is caused by mutations in KRT74 and KRT71. In this research project, three families (F1, F2, F3) with ADHH were collected, diagnosed and studied in the genetic level.In F1, APCDD1, RPL21, CDSN, SNRPE and two clusters of KRT genes were excluded as the causative genes for the disease through allele-sharing analysis. A c.82G>C (D28H) heterozygous mutation in U2HR was identified to be responsible for the disease, and can be helpful for genetic counceling and prenatal genetic diagnosis.In F2, the c.2T>C (MIT) heterozygous mutation was firstly confirmed in the affected father. The fetus was subsequently diagnosed to be unaffected with neither the idendified mutation nor the maternal contamination.In F3, mutation screening for APCDD1 and RPL21 was performed in the proband, with a c.95G>A (R32Q) heterozygous mutation detected in RPL21. The mutation cosegregated with the disease in all family members, and was considered to be the causative mutation. Haplotype analysis in F3, F4 and F5 revealed that the identical mutations detected in the three families occurred independently, which further confirmed RPL21 to cause ADHHS and suggested the mutation possibly be a gain-of-function mutation.