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Abstract Autosomal recessive nonsyndromic hearing impairment (ARNSHI) is the most frequent form of prelingual hereditary hearing loss in humans. Between 75 and 80% of all nonsyndromic deafness is inherited in an autosomal recessive pattern. Using linkage analysis, we have mapped a novel gene responsible for this form of nonsyndromic hearing impairment, DFNB65, in a consanguineous family from the Azad Jammu and Kashmir regions, which border Pakistan and India. A maximum multipoint LOD score of 3.3 was obtained at marker D20S840. The three-unit support interval is contained between markers D20S902 and D20S430, while the region of homozygosity is flanked by markers D20S480 and D20S430. The novel locus maps to a 10.5-cM region on chromosome 20q13.2–q13.32 and corresponds to a physical map distance of 4.3 Mb. DFNB65 represents the first ARNSHI locus to map to chromosome 20.
It is estimated that 1–2 per 1,000 newborns are affected with profound hearing impairment (HI) . For congenital HI, roughly half of the cases are expected to be genetic. Of the genetic cases of HI, 30% are known to be syndromic, with the remaining cases displaying nonsyndromic hearing impairment (NSHI). For NSHI, 70–75% of the cases are due to autosomal recessive (AR) inheritance, 15–20% display autosomal dominant inheritance, 2–3% of cases show X-linked inheritance, and less than 1% of cases are due to mitochondrial inheritance . Thus far, for ARNSHI more than 60 loci have been mapped and 21 genes identified . This extreme heterogeneity reflects involvement of different molecular mechanisms within the auditory system that malfunction to cause HI. In the current study, we investigated a consanguineous family segregating ARNSHI and mapped the gene responsible for the defect to chromosome 20q13.2–q13.32.
Before the start of the study, approval was obtained from the Quaid-I-Azam University and the Baylor College of Medicine Institutional Review Boards. Informed consent was obtained from all family members who agreed to participate in the study. Family 4020 (Fig. 1), which is from the city of Rawlakot in Azad Jammu and Kashmir region that borders Pakistan and India, provided convincing evidence of autosomal recessive mode of inheritance. All affected individuals have a history of prelingual profound HI and use sign language for communication. In addition, all available medical records of the affected individuals were scrutinized for evidence of childhood illness. Each HI individual underwent a physical and ophthalmologic examination. Defects in ear morphology, dysmorphic facial features, eye disorders including night blindness and tunnel vision, limb deformities, mental retardation, and other clinical features that could indicate that HI was syndromic were not identified. The clinical history and physical examination of affected individuals indicated no vestibular involvement.
Figure 2 exhibits the audiometric profile of hearing-impaired individuals IV-2 and IV-3, aged 17 and 15 respectively, who have profound HI at all frequencies in the two ears. The audiograms also display a flat configuration.
Peripheral blood samples were obtained from six family members, four of whom were hearing-impaired. Genomic DNA was extracted from whole blood following a standard protocol , quantified by spectrophotometric reading at optical density 260, and diluted to 40 ηg/μl for polymerase chain reaction (PCR) amplification. A genome scan was carried out on six DNA samples at the Center for Inherited Disease Research (CIDR). A total of 405 fluorescently labeled short tandem repeat markers were genotyped. These markers are spaced at approximately 10 cM apart and are located on 22 autosomes and X and Y chromosomes. After the completion of the genome scan, two additional unaffected (IV-1 and IV-5) family members were ascertained and their DNA samples were used for fine mapping of the DFNB65 locus.
For fine mapping, microsatellite markers were amplified by PCR according to standard procedure in a total volume of 25 μl with 40 ηg genomic DNA, 0.3 μl of primer (Invitrogen Corp., Carlsbad, CA, USA), 200 μM deoxyribonucleotide triphosphate and 1 unit of Taq DNA Polymerase (Fermentas Life Sciences, Burlington, ON, Canada) in a thermal cycler (Whatman Biometra, Goettingen, Germany). PCR products were resolved on 8% nondenaturing polyacrylamide gel.
The National Center for Biotechnology Information Build 34 sequence-based physical map was used to determine the order of the genome scan markers and fine mapping markers . Genetic map distances according to the Rutgers combined linkage-physical map of the human genome  were used to carry out the multipoint linkage analysis for the fine map and genome scan markers. For those genome scan markers where no genetic map position was available, interpolation was performed to place these markers on the Rutgers combined linkage-physical map. PEDCHECK  was used to identify Mendelian inconsistencies, while the MERLIN  program was utilized to detect potential genotyping errors that did not produce a Mendelian inconsistency. Haplotypes were constructed using SIMWALK2 [9, 10]. Two-point linkage analysis was carried out using the MLINK program of the FASTLINK computer package , and multipoint linkage analysis was performed using ALLEGRO . An autosomal recessive mode of inheritance with complete penetrance and a disease allele frequency of 0.001 were assumed. Marker allele frequencies were estimated from the founders and the reconstructed genotypes of founders from this family and 32 additional Pakistani families that underwent a genome scan at the same time at CIDR. Equal allele frequencies were used for fine mapping markers because it was not possible to estimate allele frequencies from the founders, because these markers were only genotyped in this family. Because false positive results can be obtained when analyzing the data using too low of an allele frequency for the allele segregating with the disease locus , a sensitivity analysis was carried out for the multipoint linkage analysis by varying the allele frequency of the allele that is segregating with the disease locus from 0.2 to 0.7 for the fine mapping markers.
Two point linkage analysis of the genome scan markers generated maximum LOD scores at θ=0 of 1.2 and 1.9 with markers D20S480 and D20S451, respectively. A maximum multipoint LOD score of 1.4 was obtained at D20S451. Figure 3 is a genome-wide representation of the multipoint LOD scores for the 22 autosomes. Only on chromosome 20 was a LOD score greater than 1.0 achieved.
To fine-map this region on chromosome 20, 33 additional markers located in the vicinity of D20S480 and D20S451 were selected from the Marshfield genetic map . Sixteen of the markers were uninformative, although all markers that were selected had a reported heterozygosity of >0.7. Of the 17 fine mapping markers that are informative for linkage, eight markers (including D20S428, D20S877, D20S606, and D20S902) lie between genome scan markers D20S481 and D20S480, while seven markers (D20S840, D20S876, D20S1085, D20S832, D20S100, D20S102, and D20S430) are between D20S480 and genome scan marker D20S451. In addition, D20S164 and D20S171, two of the fine mapping markers, are distal to the genome scan marker D20S451. Analysis of the genome scan and fine mapping marker genotypes within this region with PEDCHECK and MERLIN did not elucidate any genotyping errors. Table 1 summarizes the two-point LOD scores obtained after fine mapping. The maximum two-point LOD score of 2.4 was obtained with five markers (D20S840, D20S1085, D20S832, D20S100 and D20S102) at θ=0. Multipoint linkage analysis resulted in a maximum LOD score of 3.3 at marker D20S840 (Fig. 4). The three-unit support interval was between markers D20S902 and D20S430, spanning 11.7 cM according to the Rutgers combined linkage-physical map of the human genome  and corresponding to a physical map distance of 4.4 Mb (Table 1).
Haplotypes were constructed to determine the critical recombination events in the family (Fig. 1). All hearing-impaired individuals were homozygous for markers D20S902 and D20S480, but unaffected individual IV-1 is also homozygous for the same markers. Thus, the centromeric boundary of the interval is assigned between markers D20S480 and D20S840. The telomeric boundary of the interval corresponds to a recombination event between markers D20S102 and D20S430, which occurred in unaffected individual IV-1. The region of homozygosity that is flanked by markers D20S480 and D20S430 contains 4.3 Mb and is 10.5-cM long according to the Rutgers combined linkage-physical map of the human genome (Table 1). The region of homozygosity is therefore narrower than the three-unit support interval and most likely includes the gene for DFNB65.
When the marker allele frequencies for the alleles segregating with HI were varied for the fine mapping markers between 0.2 and 0.4, the maximum multipoint LOD score remained to be 3.3 at marker D20S840. When the allele frequencies were increased, the maximum LOD score still occurred at marker D20S840 but decreased to 3.2 and 3.1 at allele frequencies of 0.6 and 0.7, respectively.
The linkage data presented here suggest that a novel gene for ARNSHI is located on chromosome 20q13.2–q13.32. This is the first HI locus identified on human chromosome 20. Many of the known nonsyndromic HI loci have been identified in single large families in which linkage can be established independently. However, due to the limited number of meiosis within each family, the genetic interval for the HI locus is often large, which makes gene identification difficult.
The DFNB65 interval spans 4.3 Mb and contains 17 known genes (Table 2). Of these, bone morphogenetic protein 7(BMP7, MIM 112267) has extensive expression in the otic placode of embryonic chick, which eventually becomes restricted to cochlear supporting cells over time . This makes the BMP7 gene the best candidate of the known genes in the DFNB65 locus. However, sequencing of DNA for the BMP7 gene from one unaffected and two hearing-impaired individuals from family 4020 resulted in no functional sequence variants within the promoter and exonic regions.
Other genes within the interval that are included in expression databases for the inner ear [19, 20] are breast carcinoma amplified sequence 1 (BCAS1, MIM 602967) and prefoldin 4 (PFDN4, MIM 604898). These two genes have been implicated in several soft tissue cancers (e.g., breast, prostate, colon), which may indicate the oncogenic potential of these genes, although their specific functions remain unknown.
We wish to thank the family members for their invaluable participation and cooperation. This research was funded by the National Institutes of Health—National Institute of Deafness and other Communication Disorders grant R01-DC03594-06A1 and the Higher Education Commission, Islamabad, Pakistan. Genotyping services were provided by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, Contract Number N01-HG-65403.
Aamira Tariq received her MSc and MPhil degrees in Biochemistry/Molecular Biology from Quaidi-Azam University, Islamabad, Pakistan. She is presently serving as a Research Associate at the COMSATS Institute of Biosciences, Islamabad, Pakistan. Her area of teaching and research is molecular genetics of human diseases. She has been teaching courses and supervising research of students working in the area of human genetic disorders.
Suzanne M. Leal received her M.S. in biostatistics and Ph.D. in epidemiology from Columbia University in New York City, USA. She is currently an Associate Professor in the Department of Molecular and Human Genetics at Baylor College of Medicine. Her research interests include gene mapping of complex and Mendelian traits, the genetics of nonsyndromic hearing impairment, and the study of methodological problems in statistical genetics.
Aamira Tariq, Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan.
Regie Lyn P. Santos, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Alkek N1619.01, Houston, TX 77030, USA.
Mohammad Nasim Khan, Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan.
Kwanghyuk Lee, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Alkek N1619.01, Houston, TX 77030, USA.
Muhammad Jawad Hassan, Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan.
Wasim Ahmad, Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan.
Suzanne M. Leal, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Alkek N1619.01, Houston, TX 77030, USA.