hypoparathyroidism - deafness - renal dysplasia ( hdr ) syndrome ( mim 146255 ) , also known as barakat syndrome , is a rare autosomal dominant disorder named from a triad of hypoparathyroidism , sensorineural deafness , and renal dysplasia . sensorineural deafness could be the most common clinical feature , while hypoparathyroidism and renal dysplasia were described by various expressions and even could be asymptomatic , making a timely diagnosis of hdr syndrome more important . gata binding protein 3 ( gata3 ) , a gene belonging to the family of zinc finger transcription factors and binding to the [ a / t ] gata [ a / g ] consensus sequence , is the only reported gene responsible for this unusual developmental disease . located on chromosome 10p15 , gata3 contains two n - terminal transactivating domains ( ta1 and ta2 ) and two c - terminal zinc finger domains ( znf1 and znf2 ) , as shown in figure 1 . to date , more than fifty gata3 mutations related with both sporadic and familial hdr syndrome have been reported , and gata3 haploinsufficiency has been considered as the underlying mechanism . compared with sporadic cases , familial cases provide us the opportunity to explore the inheritance pattern and to consider the possible genetic anticipation in patients with hdr . structure map of gata3 gene : gata3 contains 6 exons and the arrow denotes the mutation identified in family 7121 located within exon4 ; gata3 : gata binding protein 3 . n : n - terminus ; ta : transactivating domains ; znf : zinc fingers domains ; c : c - terminus . in the present study , we identified a nonsense mutation in gata3 in a hearing impaired chinese family with various clinical features of hdr syndrome by using targeted capture and next - generation sequencing ( ngs ) . in addition , auditory phenotype in familial hdr syndrome associated with gata3 mutation was analyzed by reviewing previous literatures . a 7-year - old boy ( proband ) came from chinese family 7121 , a three - generation family with a segregating autosomal dominant hearing loss ( hl ) as shown in figure 2 , and four family members were recruited and gave written consent . this study was approved by the ethics committee of chinese people 's liberation army general hospital . , immittance testing , pure tone audiometric examination , and speech audiometry were performed on the three affected members to evaluate the auditory conditions . the diagnosis of sensorineural hearing impairment was made according to the world health organization criteria available at http://www.who.int/. the degrees of hl were categorized as mild ( 2640 db hl ) , moderate ( 4160 db hl ) , severe ( 6180 db hl ) , and profound hl ( > 80 db hl ) . a computed tomography ( ct ) scan for the temporal bone of both ears was also performed on the proband . biochemical laboratory tests included serum calcium , magnesium , phosphorus , and intact parathyroid hormone ( ipth ) levels , plasma creatinine , and carbamide levels , whereas urinalysis , renal ultrasound , and nuclear examinations were applied to detect the renal anomalies . genomic dna was extracted from peripheral blood sample from the three affected members and one unaffected member . after the examination of dna quality , beijing genomics institute built the dna libraries by following the illumina 's protocol , and then 307 deafness - related genes [ supplementary table 1 ] including exons , splicing sites , and their flanking introns were captured by using a custom probe and sequenced by illumina hiseq2000 ( illumina , san diego , ca , usa ) , which had been previously described . targeted captured genes list the paired - end reads generated by sequencing were aligned to ncbi37/hg19 assembly by the burrows - wheeler alignment tool ( version 0.7.10 , http://bio-bwa.sourceforge.net/ ) , and variant calling was performed by genome analysis toolkit ( version 3.3 - 0 , https://software.broadinstitute.org/gatk/index.php ) . variants with allele frequencies higher than 5% in the 1000 genomes project and the local database were excluded . moreover , sift ( http://sift.jcvi.org/ ) and polyphen2 ( http://genetics.bwh.harvard.edu/pph2/ ) softwares were used to evaluate the pathogenic possibility of missense variants . sanger sequencing was performed to establish the co - segregation of the candidate gene mutations with the phenotype in the family members . a three - dimensional structure of gata3 was built by swiss - model ( http://swissmodel.expasy.org/workspace/ ) and then visualized by swiss - pdbviewer ( version 4.1 , http://spdbv.vital-it.ch/ ) . then , a comprehensive inter- and intra - family comparison of clinical deafness characteristics was performed . a 7-year - old boy ( proband ) came from chinese family 7121 , a three - generation family with a segregating autosomal dominant hearing loss ( hl ) as shown in figure 2 , and four family members were recruited and gave written consent . this study was approved by the ethics committee of chinese people 's liberation army general hospital . their medical histories were collected by a questionnaire . physical examination , otoscopy , immittance testing , pure tone audiometric examination , and speech audiometry were performed on the three affected members to evaluate the auditory conditions . the diagnosis of sensorineural hearing impairment was made according to the world health organization criteria available at http://www.who.int/. the degrees of hl were categorized as mild ( 2640 db hl ) , moderate ( 4160 db hl ) , severe ( 6180 db hl ) , and profound hl ( > 80 db hl ) . a computed tomography ( ct ) scan for the temporal bone of both ears was also performed on the proband . biochemical laboratory tests included serum calcium , magnesium , phosphorus , and intact parathyroid hormone ( ipth ) levels , plasma creatinine , and carbamide levels , whereas urinalysis , renal ultrasound , and nuclear examinations were applied to detect the renal anomalies . genomic dna was extracted from peripheral blood sample from the three affected members and one unaffected member . after the examination of dna quality , beijing genomics institute built the dna libraries by following the illumina 's protocol , and then 307 deafness - related genes [ supplementary table 1 ] including exons , splicing sites , and their flanking introns were captured by using a custom probe and sequenced by illumina hiseq2000 ( illumina , san diego , ca , usa ) , which had been previously described . targeted captured genes list the paired - end reads generated by sequencing were aligned to ncbi37/hg19 assembly by the burrows - wheeler alignment tool ( version 0.7.10 , http://bio-bwa.sourceforge.net/ ) , and variant calling was performed by genome analysis toolkit ( version 3.3 - 0 , https://software.broadinstitute.org/gatk/index.php ) . variants with allele frequencies higher than 5% in the 1000 genomes project and the local database were excluded . moreover , sift ( http://sift.jcvi.org/ ) and polyphen2 ( http://genetics.bwh.harvard.edu/pph2/ ) softwares were used to evaluate the pathogenic possibility of missense variants . sanger sequencing was performed to establish the co - segregation of the candidate gene mutations with the phenotype in the family members . a three - dimensional structure of gata3 was built by swiss - model ( http://swissmodel.expasy.org/workspace/ ) and then visualized by swiss - pdbviewer ( version 4.1 , http://spdbv.vital-it.ch/ ) . literature review was performed by searching embase and pubmed databases . the genotypes and auditory phenotypes of these familial hdr syndrome cases then , a comprehensive inter- and intra - family comparison of clinical deafness characteristics was performed . all the three hearing - impaired family members were identified to carry out the same gata3 mutation . the heterozygous c.826c > t ( nm_002051.2 ) is a nonsense mutation located within exon4 that resulted in a premature termination codon ( r276 * ) predicted to lead to gata3 haploinsufficiency [ figures 1 and 2 ] . co - segregation of this mutation with the disease was confirmed by using sanger sequencing as shown in figure 3 . the normal member among the siblings did not have the mutation , while the other three affected members were carrying the same nonsense mutation . moreover , the absence of this mutation in the 1000 genomes project and 1751 ethnicity - matched normal hearing individuals further supported the pathogenicity . sanger sequencing results and the co - segregation of the mutation with the phenotype in the family members with hypoparathyroidism - deafness - renal syndrome . as shown in table 1 and figure 4 , the three affected members in family 7121 had early - onset sensorineural deafness . the average hearing thresholds in the better ears of proband and his mother ( ii2 ) were 56 and 45 db hl , respectively , belonging to moderate hl according to the grades of hearing impairment from the world health organization . however , grandmother of proband ( i2 ) had profound hearing impairment with the average hearing threshold of 85 db hl . the proband and his mother ( ii2 ) could communicate without any difficulty because their hearing disturbances were not severe . pure - tone audiograms of the three affected family members with gata3 mutation p.r276 * : blue represents left ear , red represents right ear . hl : hearing loss ; gata3 : gata binding protein . the results of biochemistry tests are summarized in table 1 . clinically , they all had no symptom for hypoparathyroidism , but the assessment showed hypocalcemia , lower ipth level , and mild hyperphosphaturia . in contrast , the urinalysis of all the affected members revealed no abnormalities and indicated a normal renal function . however , nephrosonography showed that the proband had left renal agenesis while the other two affected family members had normal bilateral kidneys without any detectable abnormality . the reported familial cases of hdr syndrome were summarized in table 2 by different mutations . a total of 30 families carrying various gata3 abnormalities contained missense / nonsense mutations , small deletions and insertions ( indels ) , splicing , and gross deletions . all the corresponding onset time and laterality of hl remarkably , nine parent - child pairs were proved to have hearing impairment earlier more than a decade or more severe in the younger generation , which was observed in 30% of all familial cases . there was a significant difference in the types of the mutations in these nine familial cases , indicating a high proportion of premature stop mutations as much as 66.7% . review of genotype and auditory phenotypes in familial hypoparathyroidism - deafness - renal syndrome * represents the stop of coding , hearing impairment occurred earlier at least a decade or more severe in parent - child pairs . b : bilateral ; r : right ear ; l : left ear ; nm : not mentioned ; abr : auditory brainstem response ; no : no existence of deafness . all the three hearing - impaired family members were identified to carry out the same gata3 mutation . the heterozygous c.826c > t ( nm_002051.2 ) is a nonsense mutation located within exon4 that resulted in a premature termination codon ( r276 * ) predicted to lead to gata3 haploinsufficiency [ figures 1 and 2 ] . co - segregation of this mutation with the disease was confirmed by using sanger sequencing as shown in figure 3 . the normal member among the siblings did not have the mutation , while the other three affected members were carrying the same nonsense mutation . moreover , the absence of this mutation in the 1000 genomes project and 1751 ethnicity - matched normal hearing individuals further supported the pathogenicity . sanger sequencing results and the co - segregation of the mutation with the phenotype in the family members with hypoparathyroidism - deafness - renal syndrome . as shown in table 1 and figure 4 , the three affected members in family 7121 had early - onset sensorineural deafness . the average hearing thresholds in the better ears of proband and his mother ( ii2 ) were 56 and 45 db hl , respectively , belonging to moderate hl according to the grades of hearing impairment from the world health organization . however , grandmother of proband ( i2 ) had profound hearing impairment with the average hearing threshold of 85 db hl . the proband and his mother ( ii2 ) could communicate without any difficulty because their hearing disturbances were not severe . pure - tone audiograms of the three affected family members with gata3 mutation p.r276 * : blue represents left ear , red represents right ear . hl : hearing loss ; gata3 : gata binding protein . the results of biochemistry tests are summarized in table 1 . clinically , they all had no symptom for hypoparathyroidism , but the assessment showed hypocalcemia , lower ipth level , and mild hyperphosphaturia . in contrast , the urinalysis of all the affected members revealed no abnormalities and indicated a normal renal function . however , nephrosonography showed that the proband had left renal agenesis while the other two affected family members had normal bilateral kidneys without any detectable abnormality . the reported familial cases of hdr syndrome were summarized in table 2 by different mutations . a total of 30 families carrying various gata3 abnormalities contained missense / nonsense mutations , small deletions and insertions ( indels ) , splicing , and gross deletions . all the corresponding onset time and laterality of hl remarkably , nine parent - child pairs were proved to have hearing impairment earlier more than a decade or more severe in the younger generation , which was observed in 30% of all familial cases . there was a significant difference in the types of the mutations in these nine familial cases , indicating a high proportion of premature stop mutations as much as 66.7% . review of genotype and auditory phenotypes in familial hypoparathyroidism - deafness - renal syndrome * represents the stop of coding , hearing impairment occurred earlier at least a decade or more severe in parent - child pairs . b : bilateral ; r : right ear ; l : left ear ; nm : not mentioned ; abr : auditory brainstem response ; no : no existence of deafness . in the present study , a heterozygous gata3 nonsense mutation c.826c > t ( p. r276 * ) was identified in a chinese hdr family 7121 by applying a combination of the target deafness genes capture and ngs . initially , all the affected members from three generations came to consult for their autosomal dominant hearing disturbances . then , hdr syndrome was diagnosed precisely and effectively by using advanced genetic testing technology although there was no symptom of hypocalcemia and renal agenesis . the mutation c.826c > t ( p.r276 * ) reported in the present study had been identified in a family 12/99 by van esch et al . in the year 2000 . the identification of r276 * in the chinese family 7121 further ensured its pathogenic possibility in hdr syndrome : ( 1 ) sanger sequencing confirmed the co - segregation of this mutation with the phenotype in chinese family 7121 , and this specific mutation was absent in both the 1000 genomes and 1751 ethnicity - matched controls ; ( 2 ) the location of mutation c.826c > tin gata3 was visualized clearly and this nonsense mutation resulted in truncating gata3 protein completely by losing both znf1 and znf2 domains [ figures 1 and 5 ] . notably , the two zinc fingers domains were described to be necessary for gata3 protein in binding to dna as well as the stabilization of binding function . further functional studies demonstrated that cochlear wiring and postsynaptic differentiation were disrupted without normal gata3 expression . three - dimensional structure of gata3 wild - type created by swiss - model mutation p.r276 * causing loss of both znf1 and znf2 domains . gata3 : gata binding protein 3 . clinical spectrum of hdr syndrome includes hypoparathyroidism , sensorineural deafness , and renal dysplasia . as previously reported , about 62.3% of the patients had complete clinical triad in hdr syndrome . the patients carrying the same mutation p.r276 * in another european family displayed different clinical phenotypes . contrary to the chinese family 7121 , the two affected members in 12/99 family did not have any renal anomaly . moreover , clinical features of patients with hdr syndrome were variable even in the same chinese family 7121 , which was also observed in other cases . in fact , due to the high heterogeneous expression in individuals , it is not easy for clinicians to make a distinction between human nonsyndromic and syndromic hereditary hl such as hdr syndrome . therefore , ngs technology could be a powerful tool in early diagnosis and appropriate management . genetic anticipation is a biological symptom in successive generation , in which the pedigrees appear to have earlier onset or more severity in the disease tendency . considering the existing ascertainment bias , we insist on the presence of genetic anticipation that only a different decade is significant and reveal that at least 30% of familial cases ( a total of 9 families ) showed the possible genetic anticipation , which might be one of the characteristics of familial hdr syndrome . this information is especially important for assisting with family planning in genetic consulting . to our knowledge , a number of genetic diseases such as charcot - marie - tooth disease , lynch syndrome , and familial essential tremor have been recognized with anticipation in the different mechanisms including trinucleotide repeat expansion , telomeric dysfunction as well as epigenetic factors . regarding the study , gata3 mutation analysis in table 2 reflected a high proportion of premature stop mutations in familial patients with the possible genetic anticipation , which might be associated with the potential mechanism . in conclusion , we have described a three - generation hearing impaired family with gata3 nonsense mutation p.r276 * , which was identified in chinese population for the first time by targeted genes capture and ngs technology . an overview of familial cases revealed a decrease in the age at onset of deafness or more severity between generations in 30% of families , indicating the presence of possible anticipation . further studies are needed to elucidate the molecular mechanisms of this phenomenon in hdr syndrome . supplementary information is linked to the online version of the paper on the chinese medical journal website . this work was supported by grants from the national natural science foundation of china ( no . 2014cb943001 ) , and the china postdoctoral science foundation ( no . 2015m572766 and no . this work was supported by grants from the national natural science foundation of china ( no . 2014cb943001 ) , and the china postdoctoral science foundation ( no . 2015m572766 and no .