Identification of Genes Involved in Causing Diseases of Ectodermal Appendages

Abstract

The ectoderm is the outermost layer of the developing embryo that gives rise to epidermis and its appendages. Ectodermal appendages share a common developmental program that relies on extensive coalition amongst epithelia and nearby mesenchyme. The genetic pathways involved in the regulation of appendage development are widely conserved between species and different appendages. In the present investigation nine consanguineous, multigeneration families (A-I) segregating autosomal recessive hereditary disorders of ectodermal appendages were ascertained from different zones of Pakistan. Homozygosity mapping via polymorphic microsatellite markers was used to elucidate the gene defect in eight families while for one family microarray technology was used. In family A, affected individuals showed characteristic clinical features of hereditary hypotrichosis including sparse hair on the scalp and rest of the body. Interestingly, eyebrows and eyelashes were not affected in any of the affected individuals. Human genome scan using 400 highly polymorphic microsatellite markers mapped the disease locus to a large region on chromosome 10. This novel locus maps to 29.18 cM (28.50 Mb) region, flanked by markers D10S538 and D10S2327 on chromosome 10q11.23–q22.3. A maximum multipoint LOD score of 3.26 was obtained at recombination fraction zero with several markers in this region. Exons and splice-junction sites of five putative candidate genes (DKK1, MYST4, ZMYND17, P4HA1, ZNF365), located in the linkage interval, were sequenced but were negative for functional sequence variants and therefore their involvement in causing hypotrichosis phenotype at the present novel locus is not supported. In family B, affected individuals showed clinical features of autosomal recessive hypotrichosis (LAH2) including thin fragile hair on scalp, sparse to absent eyebrows and eyelashes, and sparse hair on rest of the body. Morphological inspection of hairs by light microscopy did not reveal any nodes or constriction. Histopathological examination of the biopsy showed complete absence of normal hair follicle structures. Genotyping data showed linkage of the family to LIPH gene on chromosome 3q27.3. Sequence analysis of LIPH gene revealed a novel homozygous missense mutation (c.2T>C, p.M1T) in all the affected individuals.Three families (C, D, E) showed archetypal clinical features of autosomal recessive hypotrichosis (LAH3). The patients showed sparse to absent twisted scalp hair, sparse eyebrows and eyelashes, sparse axillary and body hair. Genotyping data showed linkage of the families to LPAR6 gene on chromosome 13q14.2. Sequence analysis of the LPAR6 gene revealed one novel homozygous missense mutation (c.8G>C, p.S3T) in family C, and two previously reported mutations (c.436G>A, p.G146R; c.69insCATG, p.24insHfsX29) in the other two families (D and E). In family F, affected individuals showed clinical phenotypes of autosomal recessive hypotrichosis (LAH1). Out of 12 individuals over four generations, only two females were found to be affected. They showed complete loss of scalp hairs and eyebrows, however very thin eyelashes were present. Genotyping data showed linkage of the family to desmoglein and desmocollin gene cluster on chromosome 18q12.1. Sequence analysis of six candidate genes (DSG1-4, DSC1, DSC3) failed to detect any functional sequence aberration, that could be responsible for the disease phenotype in this family. Family G showed clinical features of atrichia with papular lesions. Patients showed complete hair loss of scalp, absent eyebrows and eyelashes, loss of axillary and pubic hair. Genotyping results showed linkage of the family to human hairless gene (HR) located on chromosome 8p21.3. Sequence analysis of the coding exons and splice junction sites of the HR and its upstream region (U2HR) failed to detect any functional sequence variant. In family H, affected individuals showed isolated nail dysplasia phenotype; thickening and hyperpigmentation of all finger- and toenails that become claw like around teenage. Genome scan using Illumina‟s Human 660W-quad chip was performed that showed a single homozygous region on chromosome 8q22.3. In parallel sequence analysis of five selected candidate genes (ATP6V1C1, BAALC, CTHRC1, KLF10, FZD6) in the linked region revealed the disease causing homozygous nonsense mutation (c.1750G>T, p.E584X) in the frizzled 6 (FZD6) gene, a member of Wnt signaling pathway. Expression analyses in nail sections showed a strong expression of FZD6 in the ventral nail matrix and to lesser extent, the nail bed. Manuscript describing “FZD6 encoding the Wnt receptor frizzled-6 is mutated in autosomal recessive nail dysplasia” was submitted for publication to American Journal of Human Genetics. However, before the manuscript could be sent for review, another group published FZD6 mutation paper in the same journal. Now the present manuscript has been submitted for publication to British Journal of Dermatology.In family I, affected individuals showed clinical features of ectodermal dysplasia (ED) of hair and nail type; characterized by total or partial hair loss and dystrophy of finger- and toenails since birth. Genotyping data showed linkage of the family to type II keratin gene cluster on chromosome 12p11.22–12q14.1. This region corresponds to 30.92 Mb (22.34 cM) according to the sequenced based physical map (Build 36.2) of the human genome and flanked by markers D12S1631 and D12S298. A maximum two-point LOD score of 3.59 was obtained with three markers while maximum multipoint LOD score exceeding 4.0 was obtained with nine markers along the disease interval at recombination fraction zero. KRT85 has previously been reported as a causative gene for ED of hair and nail type at chromosome 12q13.13. KRT85 along with twelve other genes (KRT81, KRT82, KRT83, KRT84, KRT86, KRT6C, KRT71, KRT72, KRT74, KRT75, KRT78, WNT10B) were sequenced. However sequence analysis failed to detect any functional sequence variants.