Mutation Identification In A Han Chinese Kindred With Hereditary Multiple Exostosis And Three Acne Inversa Families

Paper 1 Mutation Identification in A Han Chinese Kindred with Hereditary Multiple ExostosisIntroductionHereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by growth of multiple exostoses-benign cartilage-capped bone tumors that grow outward from the metaphyses of long bones. Exostoses may also occur at other sites, such as the ribs, scapula and pelvis. Prevalence rate is 1/100,000 in Europe, and 1/50,000 in Washington. HME is inherited in an autosomal dominant manner, with a penetrance of 95%. About 90% of individuals with HME have an affected parent; about 10% of patients develop HME as the result of a de novo gene mutation. Three genes are known to be associated with HME:EXT1, EXT2 and EXT3, located on 8q24.11-q24.13, 11p11-p12 and 19p, respectively. However, a second mutational hit may arise in a related gene such as the EXT-like (EXTL) genes or other genes involved in the signaling cascade of chondrocyte proliferation. The EXTL family of genes is related to EXTs by sequence homology. The EXTL family of genes currently consists of three members:EXTL1, EXTL2 and EXTL3, respectively located on 1p36.1, 1p11-p12 and 8p12-p22. To date, no HME disorder has been attributed to mutations in EXT3, EXTL1, EXTL2 and EXTL3.In this study, we recruited one Han Chinese family with typical HME for research. First of all, haplotyping was performed to locate the chromosome position of pathopoiesis gene. Then, we undertook DNA sequencing to identify the mutation. At last, restriction fragment length polymorphism was undertaken to confirm this mutation.Materials and Methods1. SubjectsA four-generation Han Chinese kindred in which HME had affected 8 members (five males and three females) was investigated. Blood samples were collected from the 12 family members by standard procedures with written informed consent.2. Haplotype analysisGenomic DNA was extracted from venous blood samples using proteinase K digestion and standard phenol-chloroform extraction. Using the UCSC Genome Browser on Human 2006 May assembly (http://www.genome.ucsc.edu), two perfect microsatellite repeat sequences each close to the EXT1 gene and the EXT2 gene were selected as genetic markers for haplotyping. The PCR products of the microsatellite markers were separated by electrophoresis on 8% denaturing polyacrylamide gel and allele fragments were detected with routine silver staining, then on the basis of individual's genotype and kinship, the haplotypes were deduced according to the Mendel's law of inheritance.3. DNA sequencing of the candidate geneAfter determining the candidate gene by haplotyping, PCR was performed to amplify the exons of the gene, and then the amplicons were sequenced.4. Restriction fragment length polymorphism (RFLP) analysisTo confirm this mutation, we employed a mismatch primer which creates a Scal restriction site in the deletion mutant allele, but not in the normal allele. The PCR products using the mismatch primer were digested with restriction enzyme Scal, separated by 8% neutral polyacrylamide gel electrophoresis and showed by routine silver staining. All the 12 available family members and 100 unrelated Han Chinese normal controls were included in the above-mentioned PCR-RFLP analysis.Results1. Haplotype analysisHaplotype analysis indicated the causative gene of the Han Chinese HME family was possibly the EXT1 gene located on chromosome 8.2. Mutation screeningWe identified a heterozygous deletion, c.1897delC, in exon 10 of the EXT1 gene in the proband. This deletion will result in a frameshift from codon 633 and a premature termination at codon 642, therefore designated as p.Leu633TyrfsX10 at protein level. To confirm this deletion mutation, in all the other available family members, the exon 10 genomic fragment were subjected to automated DNA sequencing after purification. The results showed the cosegregation of mutation c.1897delC with the disease phenotype.3. RFLP analysisBesides the normal 284bp fragment, a 250bp fragment and a 34bp fragment were seen in all affected individuals from the family but were not detected in all unaffected family members or unrelated normal controls. Therefore, this heterozygous mutation could not be a polymorphism and it is the causal mutation for the phenotype of HME in this Chinese family.ConclusionA new heterozygous mutation c.1897de1C (p.Leu633TyrfsX10) in the EXT1 gene was identified as the pathogenic mutation of HME in this Chinese family.Paper 2 Mutation Identification in Three Acne Inversa FamiliesIntroductionAcne inversa (AI), also known as hidradenitis suppurativa (HS), is a chronic relapsing inflammatory disease characterized by recurrent draining sinuses and abscesses. Inflamed lesions are in the apocrine gland-bearing area of the body, most commonly in the axillae, perineum, and inframammary regions. The exact incidence of AI is uncertain, but the literature suggests from 1 in 100 to 1 in 600. AI is now considered a disease of follicular occlusion rather than an inflammatory or infectious process of the apocrine glands. Now, the etiology is predominantly associated with genetic factors. The pattern of transmission in familial cases is consistent with autosomal dominant inheritance (OMIM %142690). At present, three loci for this disorder were reported and localized to chromosome 1,6,19, but no gene is responsible for it.On the basis of the preliminary work of Department of Medical Genetics in Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, we recruited three AI pedigrees for research. Both traditional DNA sequencing and next-generation DNA sequencing were used to identify gene mutations.Materials and Methods1. SubjectsTwo Chinese and one English AI families collected by Department of Medical Genetics in Sciences Chinese Academy of Medical Sciences were recruited.2. Traditional DNA sequencing of the candidate genesWe selected 25 relatively-known genes as candidates. PCR was performed to amplify the exons of these genes, and then the amplicons were sequenced by traditional DNA sequencing.3. Next-generation DNA sequencing of exomesOn the basis of the preliminary haplotyping analysis work of Department of Medical Genetics and one lab in United Kingdom, we used Roche Sequence Capture 385K Array to capture all exomes in chr19:20916746-50538495. Then next-generation DNA sequencing was used to sequence the exomes.4. IGV 1.4.2 was used to read the files of Roche 454AllDiffsReg.primary_target_region.gff3, 454HCDiffsReg.primary_target_region.gff3 and 454AlignmentCoverage.igv. UltraEdit was used to read the files of 454AllDiffs.txt and 454HCDiffs.txt. At the same time, when we found the variants, we did blat on UCSC to find the predicted damaging variants.5. To confirm the next-generation DNA sequencing results, the predicted damaging variants were resequenced by traditional DNA sequencing.8% denaturing polyacrylamide gel electrophoresis or PCR-RFLP or HRM were used to confirm whether the vatients cosegregate with the disease phenotype.Results1. Traditional DNA sequencing of the candidate genes'exonsIn 25 candidategenes, no significant mutation was found, except about 100 known SNP and one new SNP c.502C>G in FLT3LG gene.2. Next-generation DNA sequencing of exomesIn total, there were 33979 unkown varients in these three patients. We found 125 predicted damaging variants. The HRM results showed the cosegregation of a mutation 'HS19'c.483delC with the disease phenotype in AI Family 2.Conclusion'HS19'gene is considered to be associated with AI.