Congenital Generalized Hypertrichosis Terminalis: Genetic Mapping And Pathogenic Mutation Identification

IntroductionCongenital generalized hypertrichosis terminalis(CGHT) is a rare condition characterized by androgen-independent,excessive pigmented terminal hair growth all over the body as compared to the normal persons of the same age,sex and race.The incidence of CGHT is unknown;however,reported incidence is about 1 in a billion. The involoved individuals were famous for their nicknames,"Werewolf syndrome", "hair-man" or "hairy boy",given by the mass media.Observations of CGHT were documented now and then since the Middle Ages,as a fascination for science and for science fiction.It was once regarded as an example of atavism,and at one time even thought to be the missing ape-human link required to prove Darwin's theory.Modern studies suggest that a genetic alteration plays a central,but still unresolved role,in CGHT or other "atavism" cases.It is generally believed that most affected individuals were suffering from an unknown genetic defect resulting in manifesting the ape-like clinical features.So the discovery of biological basis of CGHT will contribute to the scientific understanding of this intriguing riddle of nature with scuh a long histroy. However,the intensive researches of CGHT were seldom lauched because of the extreme rareness of suitable individuals or pedgirees as well as its genetically heterogeneous conditions by applying the classic researching means.And of cause,it is expectable that the convincing genetic findings of CGHT were extraordinarily rare in all avialble publications.Fortunately,the collection of academic valued pedigrees and the emergence of modern high-resolution microarray-based technologies combined with genome-wide linkage analysis skills provided the researchers a great opptunity to localize the genetic loci of CGHT and to investigate its pathogenic mutations in detail. Genetic diseases are caused by a variety of different possible alterations(mutations) in DNA sequences.The gains and losses of large chunks of DNA sequence large than 1kb,known as copy number variation(CNVs),were often overlooked in previous surveys of mutations that cause genetic diseases.All disease-causing CNVs,copy number mutation,may alter gene dosage,interrupt a gene or exert long-range positional effects on expression pattern of the genes outside the CNV region,by the gain,loss or disruption of a gene(s) for which dosage is critical,leading to the genomic disorders.Our work,for the first time,identifies CGHT as a genomic disorder by utilizing these advanced technologies.Materials and Methods1,Subject.Family investigations were performed in the three Han Chinese CGHT families (SY,GD and BJ).After obtaining written informed consent,DNA was extracted from peripheral blood leukocytes of all available individuals by standard methods.2,Exclusive mapping in pedigree SY with markers on or near the loci that have been reported beforeThere were two loci on chromosome 3 and 8 respectively related to CGHT in previous reports.Specific microsatellite markers on or near these loci were chosen by using the UCSC Genome Browser,for haplotype analysis.The PCR products of the microsatellite markers were separated by electrophoresis on denaturing polyacrylamide gel and allele fragments were detected with routine silver staining,then on the basis of individual's genotype and kinship,the haplotype was deduced according to the Mendel's law of inheritance.3,Genome-wide scan for localization of candidate genesDNA samples of the four-generation pedigree SY were genotyped on the Affymatrix GeneChip Human Mapping 50K Array Hind 240 containing 57,244 SNPs.Array experiments were carried out according to the protocol of the manufacturer.The Affymatrix GeneChip Operating Software(GCOS) and Affymatrix GeneChip Genotyping Analysis Software(GTYPE 4.0) were applied for genotying.4,Genome-wide linkage analysis studyParametric multipoint linkage analysis was performed using the dChip Linkage software under the assumptions of autosomal dominant inheritance with 99% penetrance,a disease allele frequency of 0.1%,and equal SNP allele frequency(50%). Other softwares were also used for verifying the reults5,Fine mapping on chromosome 17q24.2-q24.3 in pedigree SY,GD and BJBased on the results of genome wide scan,linkage and haplotype analysis using 11 polymorphic microsatellite markers selected from 17q24.2-q24.3 were performed for verifying and fine mapping the critical region in all three pedigrees.6,DNA sequencing of the candidate gene coding sequenceTo determine the candidate genes by haplotyping,PCR was performed to amplify the exons of the gene,and then the amplicons were sequenced.7,Copy number analysis using array comparative genomic hybridizationTo detect genomic alteration,the copy number analysis of the three familial CGHT cases,one affected individual per family,were performed using the Affymatrix Genome-Wide Human SNP Array 6.0 following the manufacturer's protocols.The Affymatrix Genotyping Console 3.0 software was used for quality control,results exportation and CNV identification by the Shanghai Biochip Co.,Ltd.The data of 434 Han Chinese normal controls were generously provided by Shanghai Jiao Tong University. 8,Real-Time Quantitative PCR(qPCR) for CNV analysisEleven qPCR assays were designed using the Primer Express v2.0 software for validation of verified copy number variation.9,Statistical Analyses.Data were analyzed by using SPSS versions 14.0.SNP and CNV logarithm were imbedded in corresponding softwares.A P-value of＜0.05 was considered to be significant.Results1,Subject.Family investigation demonstrated that CGHT in the three Han Chinese families (SY,GD and BJ) is inherited in a classic autosomal dominant mode.2,Exclusive mapping in pedigree SY with markers on or near the loci that have been reported beforeThe results of linkage analysis of the markers on chromosome 3 and 8 showed no linkage relationship between these loci and the phenotype in pedigree SY.These were confirmed by genome wide scan.3,Genome-wide scan32,424 effective SNPs results at overall call rate 98%of all 19 available individuals of pedigree SY were used for downstream analysis.4,Genome-wide linkage analysisParametric multipoint linkage analysis produced a genomic extent of 4 Mb (64.37Mb- 67.73Mb) containing 9 RefSeq genes with a maximum logarithm of the odds(LOD) score of 2.37 with markers on chromosome 17q24.2-q24.3. 5,Fine mapping on chromosome 17q24.2-q24.3 in pedigree SY,GD and BJGenotyping and two-point linkage analysis demonstrated a maximum LOD score of 3.9,confirming the genetic linkage,consistent with the genome scan result.By genotyping the same microsatellite marker set,we detected haplotype sharing among affected members in families GD and BJ.Notably,all affected individuals in the three Chinese CGHT families showed a null allele for markers D17S2182 and D17S1786 on the disease haplotypes,suggesting the presence of a recurrent genomic microdeletion6,DNA sequence of the candidate gene coding sequenceNo mutation identified when sequencing the SOX9 related regions.7,Copy number variation analysis using array comparative genomic hybridizationThe details of genomic microdeletions of different sizes in the three familial cases were detected(SY:64.33Mb -65.33Mb,GD:64.5Mb-65.19Mb,BJ:64.48Mb-65.22Mb), comparing to the reference data immbeded in Affymatrix Genotyping Console 3.0 and the 434 Han Chinese normal controls data provided by Shanghai Jiao Tong University.8,Real-time quantitative PCR(qPCR) for CNV analysisA relative copy number of 0.5 for the corresponding qPCR targets in all analyzed individuals was confirmed.The four CNVs identified in there familial CGHT cases shared a common overlapping genomic region that contains four genes,ABCA5(MIM 612503),ABCA6(MIM 612504),ABCA10(MIM 612508) and MAP2K6(MIM 601254).And the microdeletions perfectly cosegregated with the disease phenotype in the three Han CGHT families.Conclusion(1) Localization of candidate genes in three Han Chinese CGHT pedigrees is on 17q24.2-q24.3.(2) Autosomal dominate CGHT is a genomic disorder,and the pathogenic copy number mutations on 17q24.2-q24.3 are responsible for CGHT.(3) The array linkage platform we setup is an effective tool for other gentic disease reaserches.(4) Array comparative genomic hybridization combined with real-time quantitative PCR is an effective method to detect copy number variation in genome.