Genetic Mapping And Mutation Analysis In Human Skin Genetic Disorders

X-linked ichthyosis (XLI) (MIM,308100) is a relatively common genetic skin disease. It's estimated that deletions of the entire STS gene are found in more than 90% of the patients with XLI. This makes STS deletion in XLI a good model for the studies of genetic disorders caused by gene deletions. In this paper, we characterized 3 Chinese XLI cases with STS deletions.Congenital generalized hypertrichosis (CGH) is a rare disease with unknown genetic defects. The study of this disease may help in the understanding of the mechanism regulating hair follicle development and hair cycle. It may also provide precious clues for the treatment of alopecia. In this paper, we studied a large Chinese family with CGH accompanied with spina bifida and congenital scoliosis.Part I:Molecular characterization of DNA deletions associated with X-linked ichthyosisXLI is a relatively common genetic disorder with the incidence of approximately 1/6000 in live males. It is manifested as generalized scaling of the skin with large, polygonal, dark brown scales, especially on the extensor aspects of the limbs. Other clinical features include deep corneal opacities, cryptorchidism and mental retardation. Steroid sulfatase deficiency due to mutations of the steroid sulphatase (STS) gene leads to the development of the disease in most of the XLI cases. It's estimated that deletions of the entire STS gene accounts for more than 90%of the STS mutations, which is the highest among the genetic disorders. STS is located in Xp22.3-Xpter and there exists a highly homologous pseudogene on the long arm of chromosome Y. X-Y translocations have been proposed to be the major mechanism of STS deletions. However, further studies have showed that most of the deletions are interstitial and frequent deletions of the STS gene might result from homologous recombination between low copy repetitive elements in the region near the breakpoints. So far, only 3 XLI cases with STS deletions have been well characterized by the sequencing of the breakpoints. The DNA deletion in XLI often involves other genes near STS, leading to the phenotypes of other gene defect and the occurrence of the Xp22.3 contiguous gene deletion syndrome. Other genes involved in the Xp22.3 contiguous gene deletion syndrome except for STS include genes for short stature SS, X-linked chondrodysplasia punctata CDPX, X-linked mental retardation MRX, and X-linked recessive Kallmann syndrome KALI.We have molecularly characterized three Chinese families with XLI. Family 1 was referred from the Pediatric Genetic Clinic at the PUMC Hospital. Besides the characteristic skin manifestation of ichthyosis, the proband in the family was also affected with genital defects including cryptorchidism and small penis. The detection of STS deletion in this patient was performed by a new method using X-Y chromosome homologues primers to amplify all the 10 exons of the STS gene. The result revealed a deletion of the entire STS gene in the patient. A female member in the family was excluded as the carrier of the deletion through genotype analysis and was at no need for prenatal diagnosis. The primary mapping of the breakpoints reveal that the deletion spans for 2.4Mb including KAL, the disease gene for X-linked Kallmann syndrome. Combined with the occurrence of genital defects, the proband in the family could be diagnosed as the Xp22.3 contiguous gene deletion syndrome characterized by XLI and X-linked Kallmann syndrome. Fine mapping and cloning of the breakpoints by gap-PCR finally defined the exact location of the deletion breakpoints. No apparent homology was discovered in the analysis of the breakpoints and nearby sequences, suggesting that the mechanism of the recombination might be non-homologous DNA end-joining rather than a homologous recombination. Genomic DNA from the other two patients with XLI was kindly provided by our collaborators in Guangzhou and Xi'an, respectively. Both of the patients were affected with ichthyosis without the involvement of other systems. Deletions of the entire STS gene have been proved in the patients by our collaborators. Primary mapping of the deletion breakpoints revealed that the breakpoints in both of two patients were within the reported breakpoint hotspots which would lead to a deletion of about 1.7 Mb including the STS locus. Fine mapping located the breakpoints in a range with two highly homologous repeat sequences of medium length upstream and downstream of the STS gene, respectively. This result suggested that the genomic deletions in these two patients were produced be means of non-allelic homologues recombination. In conclusion, fine mapping of the DNA deletion breakpoints in 3 patients with XLI was performed with the successful cloning of the breakpoint in one patient through gap-PCR. A new method to detect STS deletions by PCR amplification was designed. One of the patients was proved to be affected with both XLI and X-linked recessive Kallmann syndrome and was diagnosed as Xp22.3 contiguous gene deletion syndrome with the evidence of KAL1 deletion. This might represent the first Chinese case reported with Xp22.3 contiguous gene deletion syndrome diagnosed through genetic testing. Our results have demonstrated that both of non-homologous end-joining and non-allelic homologues recombination could be the mechanisms underlying the genomic deletions associated with XLI. PartⅡ:Positional cloning of the disease gene for congenital generalized hypertrichosisHypertrichosis is defined as an excessive growth of hair (terminal, vellus or lanugo) in areas of the body that are not predominantly androgen dependent, and is independent of age, race or sex. Congenital generalized hypertrichosis (CGH) is a rare condition with the incidence of as low as 1 in 1000 million. X-linked CGH is a type of CGH characterized by generalized hypertrichosis with or without dental anomalies and hearing loss. Only two large Mexican pedigrees have been reported all around the world and the disease gene was mapped to Xq26-27 in both pedigrees. However, the disease gene has not yet been cloned since the first genetic mapping in 1994. The identification of the disease gene will not only bring new insight into hair follicle biology but also shed new light on the treatment of hypotrichosis. Spina bifida belongs to the phenotypic spectrum of neural tube defects (NTDs) which are mainly caused by the failure of neural tube closure during the neural plate development at the third week after conception. The incidence of spinal bifida is 1/1,500-2,000 which makes it the second most common type of birth defects after congenital heart diseases. It is estimated that genetic factors account for～60% of the risk of human NTDso Although genetic diseases with NTDs have been recognized with some chromosome abnormalities, the disease gene of NTDs has not been discovered so far. Congenital scoliosis (CS) is the lateral curvature of the spine caused by abnormal embryonic vertebrae development. Both environmental factors and genetic factors are thought to be associated with the development of CS. Many mouse models of CS have been generated, but the genes responsible for human CS have not been found. In two studies of CS, a sibling risk for neural tube defects of approximately 5%was reported, suggesting an association between vertebral malformations and NTDs in etiology.We have performed positional cloning in a large Han Chinese family with CGH, spina bifida and CS. In the family, females were much less severely affected than males by the absence of spina bifida and scoliosis. This is the first report of a genetic syndrome that is a combination of the three manifestations. We performed genotyping and two-point linkage analysis using microsatellite markers to define the disease locus. The susceptible region was mapped to a 5.6cM interval at Xq26.3-27.2. Mutation screening was preformed in all of the annotated genes (25 known genes with 222 exons), the pseudogenes (25genes wih 41 exons), some of the highly conserved non-coding sequences (54), all of the annotated non-coding RNA sequences (3), and some predictive transcription factors binding sites (6). The results didn't reveal any disease-causing mutation. To exclude the possibility of small deletions, primers were designed for 162 short sequences hundreds basepairs apart near the candidate FGF13 gene and PCR amplifications were performed both in the patients and normal controls. No deletions were found. We also extracted mRNA from skin fibroblast cells and skin tissues both from patients and normal controls, and performed RT-PCR to examine several candidate genes. Comparison between the amplified fragments in both subjects revealed no significant differences. In addition, prenatal diagnosis was made in a female carrier. Haplotype analysis indicated that the chromosome segment linked with the disease gene was present in the male fetus.In summary, we reported a new genetic syndrome of hypertrichosis accompanied with spina bifida and congenital scoliosis. The genetic locus was mapped to a 5.6cM region at Xq26.3-27.2 by two point linkage analysis, confirming the presence of a candidate hypertrichosis gene in this chromosomal region which could be also related to human NTDs. Extensive mutation screening by genomic DNA sequencing and RT-PCR detection have not found potential pathogenic alterations. Further screening for other types of DNA changes such as DNA copy number variations should be performed.