Autozygosity Mapping by Genome-wide Single Nucleotide Polymorphism Array Identifies a Novel Homozygous HR Mutation in a Consanguineous Family with Universal Hereditary Hair Loss

Sirous Zeinali,Leila Youssefian,Hassan Vahidnezhad,Amir Hossein Saeidian,Soheila Sotoudeh,Hamideh Bagherian,Jouni Uitto,*

1Department of Molecular Medicine,Biotechnology Research Center,Pasteur Institute of Iran,Tehran,Iran;2Kawsar Human Genetics Research Center,Tehran,Iran;3Department of Dermatology and Cutaneous Biology,Sidney Kimmel Medical College,and Jefferson Institute of Molecular Medicine,Thomas Jefferson University,Philadelphia,PA 19107,USA;4Department of Medical Genetics,School of Medicine,Tehran University of Medical Sciences,Tehran,Iran;5Department of Dermatology,Children’s Medical Center,Center of Excellence,Tehran University of Medical Sciences,Tehran,Iran.

Abstract

Keywords:familial hypotrichosis,hair loss,homozygosity mapping,mutation detection,hairless gene mutations

Introduction

Hypotrichosis,lack or sparse hair on the scalp and other parts of the body,can be either an isolated finding without other dermatological or extracutaneous manifestations or can be syndromic associated,for example,with retinal degeneration,intellectual disability,and hearing impairment.1The hereditary forms of hypotrichosis,inherited either in an autosomal dominant or autosomal recessive mode,have been mapped to different chromosomal regions(HYPT1-13),and in many cases the corresponding genes have been identified.In particular,the autosomal recessive forms of hair loss have been shown to result from mutations in five genes,including HR,DSG4,LIPH,LPAR6/P2RY5,and DSC3.The autosomal dominant forms have been associated with mutations in APCDD1,CDSN,KRT74,U2HR,EPS8L3,SNRPE,and RPL21.1Mutation analysis of these genes has primarily relied on systematic Sanger sequencing of the exons and flanking intronic sequences,an arduous,time consuming and costly approach considering the number of the candidate genes.

Homozygosity mapping,and specifically identity-bydecent-guided autozygosity mapping,has been successful in identifying candidate genes in a number of genetic disorders in consanguineous families.The initial studies were based on the presence of restriction fragment length polymorphisms reflecting single nucleotide substitutions in the genome or on short tandem repeat microsatellite markers.2-3More recently,high-density single nucleotide polymorphism(SNP)based arrays have allowed genomewide mapping of the homozygosity blocks at high resolution which has facilitated the identification of candidate genes in these families.In this study,we have utilized genome-wide SNP-based autozygosity mapping to identify candidate genes in a consanguineous family with two affected individuals with isolated hereditary hypotrichosis.

Material and methods

Patient

A consanguineous Iranian family with ancestry of Azerbaijani Turks with two affected individuals,8months and 11years of age,with profound hypotrichosis was subjected to mutation analysis by a SNP-based array(Fig.1A).A strategy consisting of identification of homozygosity blocks in the affected individuals,followed by an overlay of the genomic positions of 11 genes previously shown to harbor causative mutations in the heritable forms of hypotrichosis,was utilized to identify the gene responsible for the clinical phenotype in this family.

This study was approved by the Institutional Review Board of the Pasteur Institute of Iran,and the parents of the patients gave written informed consent to participate in the research.

Genome-wide SNP-based autozygosity mapping

DNA was isolated from the two affected individuals in the family and their parents,who were first cousins,by salting out method.Genome-wide SNP-based autozygosity mapping was performed by Ilumina Infinium Exon-24 BeadChip(550,000 SNP markers)using 200 ng of genomic DNA.The SNP genotyping data were analyzed by using PLINK(http://pngu.mgh.harvard.edu/-purcel/plink/ibdibs.shtml#homo),and several runs of homozygosity(ROH)of≥2 Mb were identified in both patients(Fig.1B).Genomic positions of the 11 candidate genes previously shown to be affiliated with hypotrichosis phenotypes,were aligned with the homozygosity blocks to identify candidate genes.The identified gene was sequenced by amplification of exons and flanking intronic sequences by Sanger sequencing with AB3730;Applied Biosystems(Walthman,MA,USA).

Figure 1.Clinical features,autozygosity mapping,and identification of a homozygous HR mutation in a family with isolated hereditary hypotrichosis.(A)The proband,an 8-month old male(center panel),showed considerable hair loss already during the early post-natal period(left panel).He had an 11-year old sister with complete loss of scalp hair(right panel).(B)Autozygosity mapping identified a number of homozygosity blocks of≥2 Mb in size in both patients(P1 and P2)(blue vertical blocks),and alignment of the positions of 11 candidate genes(red lines)revealed co-alignment of HR with homozygosity blocks in both patients but not in their parents.(C)Sanger sequencing of the HR gene identified a homozygous deletion mutation in both patients,while the parents were heterozygous carriers consistent with autosomal recessive inheritance.

Results

Clinical features of the patients

A consanguineous Iranian family with ancestry of Azerbaijani Turks with two children with profound hypotrichosis were enrolled.Besides hypotrichosis,the two affected individuals with generalized hair loss appeared otherwise healthy and had no evidence of overt extracutaneous manifestations.The parents were clinically normal with normal hair.

Genetic results

Homozygosity mapping with 550,000 SNP markers identified a number of homozygosity blocks in both affected individuals with very little overlap with those found in their clinically unaffected parents who are obligate heterozygous carriers(Fig.1B).Alignment of the 11 candidate genes identified the HR gene as a potential candidate gene for mutations,because it was the only candidate gene whose position overlapped with a homozygosity block in both affected individuals on chromosome 8,while the parents did not show an ROH in this region.Interestingly,the overlapping homozygosity blocks co-aligning with HR locus in the two patients were considerably different in size:In the proband(Patient 2)the ROH consisted of 24.7 Mb while the ROH in Patient 1 was 3.3 Mb(Fig.2A).Polymerase chain reaction amplification of all exons and 50-100bp of flanking intronic sequences of HR,followed by Sanger sequencing,revealed a homozygous single nucleotide deletion,c.381delT,which resulted in shift of the translational reading frame and a premature termination codon 40 amino acids downstream from the mutation,p.Ser127ArgfsTer40(Fig.1C).The parents of the affected individuals were heterozygous for this mutation,confirming the autosomal recessive mode of inheritance.

Discussion

The two affected individuals in the family reflected the progression of the hypotrichosis phenotype with age.The proband,a male of 8months of age was shown to have partial alopecia already during the neonatal period progressing with age(Fig.1A).The 11-year old sister had complete universal hair loss.The parents,obligate heterozygous carriers of this recessive trait,have normal hair.

The HR gene,harboring mutation in this family,has been previously shown to cause hypotrichosis,and 52 distinct mutations in this gene have been previously identified(www.hgmd.cf.ac.uk/).These mutations have been encountered in patients diagnosed with alopecia universalis,atrichia with papular lesions,and hypotrichosis 4.4-6The mutation identified in this family,c.381delT,p.Ser127ArgfsTer40,is previously unreported,and is pathogenic due to predicted truncation of the encoded protein by-85%.

Figure 2.Strategy for mutation detection by autozygosity mapping.(A)The overlapping homozygosity blocks in both patients co-aligned with the HR locus at chromosome 8.(B)Steps to narrow the number of ROHs(≥2 Mb)in the proband to one harboring HR as the candidate gene.ROH:Run of homozygosity.

HR was identified as the candidate gene in this family by homozygosity mapping with a SNP-based panel consisting of 550,000 markers,and co-alignment of its genomic location with overlapping ROHs in both affected individuals,but not in their unaffected parents(Fig.1B),and subsequent sequencing of HR identified a homozygous pathogenic deletion mutation(Fig.1C).Thus,utilization of the homozygosity mapping allowed us to focus the analysis on one gene from potentially 11 candidate genes,which have been previously shown to be associated with hypotrichosis,thus significantly facilitating the mutation detection process(Fig.2B).In addition,this approach is highly cost effective as the cost of homozygosity mapping is low,and the overall sequencing cost is reduced due to necessity to sequence only one gene.

In summary,in this study we have identified the causative mutated gene in a family with two affected siblings with hereditary hypotrichosis taking advantage of the genome-wide high density autozygosity mapping followed by Sanger sequencing.This study illustrates the efficiency of the strategy in disorders with considerable genetic heterogeneity,as illustrated here by hypotrichosis with 11 different candidate genes.Consequently,this approach is applicable not only to hypotrichosis but also to other heritable skin diseases in consanguineous families.7-9In this study,the homozygosity mapping was based on SNP polymorphisms identified by an array specifically designed for this purpose.Similar information can also be gleaned from sequencing data generated by whole-exome sequencing,whole-genome sequencing,or whole-transcriptome sequencing by RNA-Seq,thus integrating homozygosity mapping to the contemporary strategies that are used for mutation detection in heritable Mendelian disorders.10

Acknowledgments

The authors thank our patients and their family members for participation.Sara Afsharaalam and Mohsen Siavashi(Tehran University of Medical Sciences)referred the patients to this study.Cecilia Kim,Jonathan Bradfield,and Hakon Hakonarson(Children’s Hospital of Philadelphia)assisted in homozygosity mapping.