New loci ORIGINAL ARTICLE Exome sequencing identified a missense mutation of EPS8L3 in Marie Unna hereditary hypotrichosis Xin Zhang,1,2 Bi-Rong Guo,1,2 Li-Qiong Cai,1,2 Tao Jiang,3 Liang-Dan Sun,1,2 Yong Cui,1,2 Jing-Chu Hu,3 Jun Zhu,1,2 Gang Chen,2 Xian-Fa Tang,1,2 Guang-Qing Sun,3 Hua-Yang Tang,1,2 Yuan Liu,1,2 Min Li,1,2 Qi-Bin Li,3 Hui Cheng,1,2 Min Gao,1,2 Ping Li,1,2 Xu Yang,3 Xian-Bo Zuo,2 Xiao-Dong Zheng,2 Pei-Guang Wang,1,2 Jian Wang,3 Jun Wang,3 Jian-Jun Liu,2 Sen Yang,1,2 Ying-Rui Li,3 Xue-Jun Zhang1,2 1Department of Dermatology, ABSTRACT papules of head and neck. Individuals with HHS Institute of Dermatology, Background Marie Unna hereditary hypotrichosis typically show normal hair at birth, but hair loss No. 1 Hospital, Anhui Medical University, Hefei, Anhui, China (MUHH) is an autosomal dominant disorder and thinning of the hair shaft start without charac- 2State Key Laboratory characterised by coarse, wiry, twisted hair developed in teristic hair shaft anomalies during early childhood Incubation Base of early childhood and subsequent progressive hair loss. and progress with age. The affected individuals of Dermatology, Ministry of MUHH is a genetically heterogeneous disorder. No gene HHS are affected scalp hair and/or body hair. The National Science and in 1p21.1–1q21.3 region responsible for MUHH has been individuals with localised autosomal recessive Technology, Hefei, Anhui, China fi 3Beijing Genomics Institute- identi ed. hypotrichosis have hypotrichosis simplex with Shenzhen, Shenzhen, China Methods Exome sequencing was performed on two shortened length of the hair shaft, skin fragility/ affected subjects, who had normal vertex hair and ectodermal dysplasia and woolly hair with palmo- Correspondence to modest alopecia, and one unaffected individual from a plantar keratoderma and cardiomyopathy syn- Xue-Jun Zhang, Department of Dermatology, four-generation MUHH family of which our previous drome, and associated with absence or scarcity of Institute of Dermatology, linkage study mapped the MUHH locus on chromosome eyebrows, eyelashes and pubic hair. No. 1 Hospital, Anhui Medical 1p21.1–1q21.3. MUHH is a rare autosomal dominant congenital University, 69 Meishan Road, Results We identified a missense mutation in EPS8L3 hair disorder which was first described by the Hefei, Anhui 230032, China; (NM_024526.3: exon2: c.22G->A:p.Ala8Thr) within German dermatologist Marie Unna.1 MUHH is dis- [email protected]; – fi Ying-Rui Li, Beijing Genomics 1p21.1 1q21.3. Sanger sequencing con rmed the tinguished from other forms of hypotrichosis by Institute-Shenzhen, Shenzhen, cosegregation of this mutation with the disease the presence of a twisting hair; affected individuals China; phenotype in the family by demonstrating the presence of this disease have normal, sparse or absent hair at [email protected] of the heterozygous mutation in all the eight affected and birth, then develop to coarse, twisted and wiry hair XZ, B-RG, L-QC and TJ absence in all the seven unaffected individuals. This during childhood and progress during puberty to an contributed equally to mutation was found to be absent in 676 unrelated almost complete alopecia. Eyebrows, eyelashes and this work. healthy controls and 781 patients of other disease from body hair are also markedly diminished or absent. another unpublished project of our group. No other ectodermal abnormalities are observed. So Received 23 June 2012 Conclusions Taken together, our results suggest that Revised 31 August 2012 far, two linkage loci for MUHH have been mapped EPS8L3 2 – 3 Accepted 13 September 2012 is a causative gene for MUHH, which was to chromosome 8p21 and 1p21.1 1q21.3. After Published Online First helpful for advancing us on understanding of the that, an international collaboration discovered that 25 October 2012 pathogenesis of MUHH. Our study also has further MUHH is caused by heterozygous mutations in demonstrated the effectiveness of combining exome the second 50-untranslated region of the HR gene sequencing with linkage information for identifying (U2HR) recently.4 The MUHH pedigree in this Mendelian disease genes. study was reported previously by Yan et al5 and Yang et al.3 The former study found that affected individuals had little or no scalp hair at birth, wiry INTRODUCTION and irregular hair on the scalp that had been diffi- Hereditary hypotrichosis is a kind of inherited hair cult to manage in childhood, and their forehead disorder which can grossly be characterised into and parietal hair were bald or sparse in puberty. two major groups according to the absence (non- Eyebrows and eyelashes had always been thin. syndromic hereditary hypotrichosis) or presence Axillary and pubic hair failed to develop. But the (syndromic hereditary hypotrichosis) of extra- affected individuals have modest scarring alopecia cutaneous features. Alopecia universalis congenita/ and normal vertex hair. The sequent study had alopecia alopecia congenita with papular lesions identified a locus for MUHH on chromosome (AUC/APL), hereditary hyportrichosis simplex 1p21.1–1q21.3 in this family. (HHS), localised autosomal recessive hypotrichosis For the past decades, the genes underlying and Marie Unna hereditary hypotrichosis (MUHH; Mendelian diseases have been identified through OMIM 146550/612841) are different forms of non- positional cloning, a process of meiotic mapping, syndromic hereditary hypotrichosis. AUC/APL is physical mapping and candidate-gene sequencing. characterised by early-onset, complete hair loss Recently, exome sequencing (also known as tar- including eyebrows, eyelashes, armpit hair, pubic geted exome capture) was demonstrated to be a hair and body hair, and/or small erythematous cheaper but efficient strategy to selectively J Med Genet 2012;49:727–730. doi:10.1136/jmedgenet-2012-101134 727 New loci sequence the whole genome coding regions. It has been widely Genomic DNA was extracted from peripheral blood lympho- used to identify genes for rare monogenic diseases and can also cytes by standard procedures using FlexiGene DNA kits provide molecular identification of Mendelian diseases when (Qiagen, California, USA). This study was approved by Anhui – the clinical diagnosis is uncertain.6 9 Here, we identified a novel Medical Institutional Review Board and conducted according to causative gene for MUHH by combining exome sequencing the Declaration of Helsinki Principles. with the linkage information from our previous study.3 Exome capture library preparation and capture MATERIALS AND METHODS Exome capture was carried out using Agilent SureSelect Clinical sample Human All Exon Kit (in solution), guided by the manufac- turer’s protocols. In brief, the qualified genomic DNA samples A four-generation MUHH family consisting of 21 individuals 5 were randomly fragmented to 200–500 bp in size, followed by was first reported by Yan et al from Anhui province in China end-repair, A-tailing and paired-end index adapter ligation. (figure 1A). There were nine affected individuals including four Ligation-mediated PCR amplified extracted DNA purified by males and five female subjects. After obtaining written the Agencourt AMPure SPRI beads. Final libraries were vali- informed consent from all the participants, EDTA anticoagu- dated by Bioanalyzer analysis (Agilent) and quantitative PCR. lated venous blood samples were collected from eight patients Each captured library was loaded on Hiseq 2000 platform and and seven controls. The clinical features, histological character- paired-end sequencing was performed, with read lengths of istics, light microscopic examination and disease history sup- 5 90 bp, providing at least 50 average depth for each sample. Raw ported the diagnosis of MUHH. Sanger sequencing was image files were processed by Illumina pipeline (V.1.3.4) for performed to exclude U2HR mutation in this family. After base calling with default parameters. that, two affected individuals (II3 and III10) and one unaffected individual (III2) were selected for exome sequencing. An additional 12 members (six cases and six controls) were Next-generation sequencing data analysis evaluated in mutation validation analyses. SOAPaligner 2.2010 11 was used to align the reads to human ref- In addition, the unpublished exome sequencing data of eth- erence hg19 with parameters set to ‘-a -b -D -o -v 2 -r 1 -t -n 4’. nically and geographically matched 1457 subjects including 676 SOAPsnp (V.1.03)12 was used to call consensus genotypes with unrelated controls and 781 unrelated patients of other disease Phred-like quality of at least 20 and at least four coverage depth were used to filter variants. All the 1457 subjects were excluded was considered as the high-confident genotype. SNP (single MUHH by at least two dermatologist examinations. nucleotide polymorphisms) were extracted from consensus Figure 1 (A) The genealogical tree. ‘+’ in pedigree indicates those who are subjected to exome sequencing, and ‘−’ in pedigree indicates those who had undergone Sanger sequencing. (B) Chromatogram of the heterozygous c.22G>A variant resulting in the EPS8L3 Ala8Thr substitution. This figure is only reproduced in colour in the online version. 728 J Med Genet 2012;49:727–730. doi:10.1136/jmedgenet-2012-101134 New loci genotypes files, and those achieved minimum quality score: Table 1 Summary of detected variants across three exomes Phred-like quality≥20, overall depth from 4 to 200, copy Variants II3 III10 III2 number estimate <2 and distance between two adjacent SNPs Total number of variants (SNP+coding 49 574+233 52 420+260 56 353+393 no less than 5. indels) 13 For indels calling, BWA (V.0.5.8) was used to gap align the Synonymous-coding 8148 8369 8345 sequence reads with default parameters to the human reference Missense 7139 7223 7148 14 hg19. GATK IndelRealigner was used to perform local realign- Nonsense 57 67 59 ment of the BWA-aligned reads and GATK IndelGentotyperV2 Read-through 8 5 5 was used to call indels.