Identification By Exome Sequencing Of The Pathogenic Genes And Analysis Of POFUT1 Gene Mutation Of Dowling-Degos Disease

Background Dowling-Degos disease（DDD[MIM 179850]） is an autosomal dominant genodermatosis characterized by reticular pigmented anomaly mainly affecting flexures, such as the neck, axilla and areas below the breasts and groin. In 2006, Betz al. Performed a genomewide linkage analysis of two German families and identified loss-of-function mutations in the keratin 5gene（KRT5）. However, no KRT5 mutations have been identified in several familial and sporadic cases. The same year, Li al.Performed a genome-wide scan in a Chinese family with DDD to map the chromosome locations of the disease-causing gene. They mapped the linkage critical region at 17p13.3. Therefore, additional genetic mutations responsible for this disease should be investigated.Objective To identify the candidate pathogenic genes by genome-wide linkage and exome sequencing analyses in a multiplex Chinese DDD family, in which the KRT5 mutation was excluded.Methods（1） Approximately 200 ng of genomic DNA of each of 10 individuals from the family â…  was used for genotyping by Illumina Human 660W-Quad Bead Chip.Multipoint parametric linkage analyses were performed in Merlin[5] by using the prunedautosomal SNPs（with LD <0.1 in population data） and assuming a dominant inheritance mode with a disease allele frequency of 0.001.（2）We performed exome capture by Genome Analyzerâ…¡x in three affected individuals and two unaffected individual from a family with DDD.（3）Each captured library was loaded on a Hi Seq2000 platform, and paired-end sequencing was performed with read lengths of 100 bp.Variants were called and filtered based on the “best practice variant detection withGATK（v3） [ref]”, that is "QD < 2.0", "MQ < 40.0", "FS > 60.0", "Haplotype Score >13.0", "MQRank Sum <-12.5", "Read Pos Rank Sum <-8.0" for single nucleotide variants（SNV）, and "QD < 2.0", "Read Pos Rank Sum <-20.0", "Inbreeding Coeff <-0.8", "FS > 200.0" for indel.（4）We sequenced the candidate pathogenic gene of DDD in all available individuals in family1.（5）By PCR sequencing methods, we test whether functional mutation occurs in the candidate pathogenic gene in other DDD family and sporadic DDD cases. And then, all the functional mutations in the candidate pathogenic gene were identified in a wide range of normal population.（6）All seven coding exons including intron–exon boundaries of POFUT1 were amplified by polymerase chain reaction（PCR） using published primers（4）. After amplification,products were purified and directly sequenced on ABI 3130 xl Genetic Analyser.Mutations were identified by comparing with the reported DNA reference sequence（Gen Bank accession number: NG₀32110）. All the identified mutations were verified by the subsequent opposite-direction sequencing. Both the patients and control samples were analyzed using the same protocol.Results（1）We firstly performed genome-wide linkage analyses in family â…  to determine the pathogenic locus that co-segregate with the disease. Maximal LOD score of 1.5 was obtained on chromosome 20. Co-segregation of haplotype analyses by Haplopainter[6] mapped the linkage critical region to chr20:17,722,478-36,009,145.（2）By exome sequencing in family â… ,5421 coding indels were identified on average in each individual. 170 were shared by the two patients and absent in the two healthy controls, 98 of which were not in 1000 Genome Phase I data（Oct 2012）. Six out of these 98 indels lied in the linkage critical region, including one in POFUT1（c.246+5del G, 5 bp downstream of the exon-intron boundary）, but none of them were frameshift or splice site.（3）After screening POFUT1 coding sequences by Sanger sequencing in two DDD families and one sporadic case（10 affected and 12 unaffected individuals together）, novel mutation（c.246+5del G） in intron 2 of POFUT1 was present in all affected and absent in all unaffected members in family I,except â…¢8 who is 12 years old and may well be under disease onset age（the average age at onset in Family I is about 20）. This indel was absent in 100 unrelated healthyindividuals of Chinese ethnicity. No mutations of POFUT1 were identified in Familyâ…¡ and the sporadic cases.Conclusion（1）This study further confirmed the discovery of Ming Li, that POFUT1 is another pathogenic gene of DDD.（2）And our result further expands the database of POFUT1 mutations for DDD and contributes to the understanding of DDD genotype/phenotype correlations.