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From a large collection of families with autosomal recessive non-syndromic hearing impairment (NSHI) from Pakistan, linkage has been established for two unrelated consanguineous families to 19p13.2. This new locus was assigned the name DFNB68. A 10 cM genome scan and additional fine mapping were carried out using microsatellite marker loci. Linkage was established for both families to DFNB68 with maximum multipoint LOD scores of 4.8 and 4.6. The overlap of the homozygous regions between the two families was bounded by D19S586 and D19S584, which limits the locus interval to 1.9 cM and contains 1.4 Mb. The genes CTL2, KEAP1 and CDKN2D were screened but were negative for functional sequence variants.
The extreme genetic heterogeneity for hearing impairment (HI) testifies to the inherent complexity of the mammalian inner ear. As more HI genes are identified, the elucidation of the function of the proteins that these genes encode contributes greatly to the understanding of cochlear mechanisms and their role in disease causation. Currently, 37 genes have been identified for non-syndromic hearing impairment (NSHI; Van Camp and Smith, 2005); however, for more than 60% of the mapped NSHI loci a gene has yet to be identified. Many of these loci with an unidentified gene were mapped in single families, some of which are consanguineous. The size of the mapped interval plus the number of candidate genes within the interval, if large, may make the task of finding the causative gene daunting. The identification of linkage of multiple NSHI families to a single locus validates the linkage finding and can make gene identification more attainable due to a smaller physical interval for the NSHI locus.
The DFNB68 locus is within the chromosome 19 region for DFNB15. The DFNB15 locus interval was previously mapped in a consanguineous Indian family to two chromosomal locations, 19p13.3–p13.1 and 3q21.3–q25.2. Linkage to both of these regions was based on a non-significant LOD score of 2.8 with a genetic region of ≥32 cM intervals on both chromosomes 3 and 19 (Chen et al. 1997). Presented here are two Pakistani families with autosomal recessive NSHI that exclusively maps to chromosome 19p13.2, with highly significant multipoint LOD scores of 4.8 and 4.6. The overlapping region of homozygosity for these two families is 1.9 cM. Since it is not clear whether the DFNB15 locus is on chromosome 3 or 19, the NSHI locus segregating in these two Pakistani families has been designated as novel NSHI locus DFNB68.
Prior to onset the study was approved by the Institutional Review Boards of the Quaid-I-Azam University and Baylor College of Medicine and Affiliated Hospitals. Informed consent was secured from all individuals who agreed to participate in the research. Family 4100 resides in the southern part of Punjab province, while family 4154 hails from Sind province. For both kindreds, family members rarely marry outside the community, and consequently, consanguineous unions are common. The pedigree drawings (Fig. 1) provided convincing evidence of autosomal recessive mode of inheritance in both families, and consanguineous loops accounted for all the affected persons being homozygous for the mutant allele. Clinical findings in these families are consistent with the diagnosis of autosomal recessive NSHI. All affected individuals have a history of prelingual profound HI and communicate by sign language. Affected individuals underwent examination for 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, and were all negative for findings. No gross vestibular involvement was noted from the clinical history and physical examination.
Peripheral blood samples were taken from ten members in family 4100 including five hearing-impaired individuals, and eleven members from family 4154, six of whom had HI. Genomic DNA was extracted from whole blood following a standard protocol (Grimberg et al. 1999), quantified by spectrophotometric reading at optical density 260, and diluted to 40 ηg/μl for amplification by polymerase chain reaction (PCR). Genome scans were carried out at the Center for Inherited Disease Research (CIDR) and at the National Heart, Lung and Blood Institute (NHLBI) Mammalian Genotyping Service (Marshfield, WI, USA). An average of 399 fluorescently labeled, short tandem repeat markers were genotyped across the 22 autosomes and X and Y chromosomes at approximately 10 cM apart. For fine mapping, microsatellite markers were PCR amplified according to standard procedure in a total volume of 25 μl with 40 ηg genomic DNA, 240 ηM of primer (Invitrogen Corp., Carlsbad, CA, USA), 200 μM dNTP, and 1 unit of Taq DNA Polymerase (Fermentas Life Sciences, Burlington, ON, Canada) in GeneAmp® PCR System 9700 (Applied Biosystems, Applera Corp., Foster City, CA, USA). PCR products were resolved on 8% non-denaturing polyacrylamide gel and genotypes were assigned by visual inspection.
PEDCHECK (O’Connell and Weeks 1998) was used to identify Mendelian inconsistencies while the MERLIN (Abecasis et al. 2002) program was utilized to detect potential genotyping errors that did not produce a Mendelian inconsistency. For both genome scan and fine-mapping markers, two-point linkage analysis was carried out with the MLINK program of the FAST-LINK computer package (Cottingham et al. 1993) and multipoint linkage analysis was performed using ALLEGRO (Gudbjartsson et al. 2002). An autosomal recessive mode of inheritance with complete penetrance and a disease allele frequency of 0.001 were assumed. Genome scan marker allele frequencies were estimated from the founders and reconstructed genotypes of founders from these pedigrees and additional pedigrees that underwent a genome scan at the same time. For the fine-mapping markers, it was not possible to estimate allele frequencies from the founders because these markers were only genotyped in these two families, so equal allele frequencies were applied initially. Since false positive results can be obtained when analyzing the data using too low of an allele frequency for an allele segregating with the disease locus (Freimer et al. 1993), 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 0.6 for the fine-mapping markers. In order to determine the order of fine-mapping and genome scan markers, the physical position of each marker was determined from the National Center for Biotechnology Information (NCBI) Build 34 sequence-based physical map (International Human Genome Sequence Consortium 2001). Genetic map distances were then derived from the Rutgers combined linkage-physical map of the human genome (Kong et al. 2004), either directly or by interpolation. After linkage analyses, haplotypes were constructed via SIMWALK2 (Weeks et al. 1995; Sobel and Lange 1996).
Using Primer3 software (Rozen and Skaletsky 2000), primers were designed for the exons of the following genes: MYO1F (MIM 601480; NM_012335); KEAP1 (MIM 606016; NM_012289); SLC44A2/CTL2 (MIM 606106; NM_020428); and CDKN2D (MIM 600927; NM_001800). From each family, DNA from one unaffected and two hearing-impaired individuals were diluted to 5 μg/η l, amplified by PCR under standard conditions, and purified with ExoSAP-IT® (USB Corp., Cleveland, OH, USA). Sequencing was performed with the BigDye® Terminator v3.1 Cycle Sequencing Kit together with an Applied Biosystems 3700 DNA Analyzer (Applera Corp., Foster City). Sequence variants were identified via Sequencher™ Version 4.1.4 software (Gene Codes Corp., Ann Arbor, MI, USA).
Two-point linkage analysis of the genome scan markers generated a maximum LOD score at θ= 0 of 2.2 for family 4100 and 3.3 for family 4154; both LOD scores were derived at marker D19S586. For the multipoint analysis, the maximum LOD score was obtained also at genome scan marker D19S586 for both families, with a score of 3.0 for family 4100 and 3.2 for family 4154.
In order to fine map the region, 23 additional markers from the Marshfield genetic map (Broman et al. 1998) plus five markers from the Rutgers combined linkage-physical map (Kong et al. 2004) were selected and genotyped in both families. The results of the two-point linkage analyses are presented in Table 1, which shows the LOD scores that were derived at each marker at θ= 0. The highest two-point LOD score for family 4100 was 2.3 at markers D19S583 ~ D19S581 and D19S558, and for family 4154 it was 3.3 at D19S586. Note that some of the markers were uninformative for one of the two families (Table 1); these markers were removed while performing multipoint linkage analyses and haplotype reconstruction for the family for which the markers were uninformative.
For family 4100, two peaks in the multipoint LOD scores can be seen: one with a maximum LOD score of 4.8 at D19S581, and a second peak with a value of 4.7 at marker D19S558. For the first peak, the three-unit support interval was delimited by markers D19S884 and D19S906. The second peak was bordered by D19S914 and D19S432. When sensitivity analysis was carried out by varying the frequencies of the fine-mapping marker alleles which segregate with the disease phenotype between 0.2 and 0.6, the maximum LOD score fluctuated from 5.1 to 3.2 but remained at the same markers D19S581 and D19S558.
On the other hand, for family 4154, the maximum LOD score after multipoint linkage analysis was 4.6 at markers D19S432 ~ D19S714 ~ D19S252, with the three-unit support interval between markers D19S1034 and D19S199. When sensitivity analyses were performed at marker allele frequencies 0.2–0.6, the maximum multipoint LOD score was maintained at 4.5–4.6, not only at markers D19S432 ~ D19S714 ~ D19S252 but also at markers D19S581 ~ D19S584.
The three-unit support interval for family 4154 contains 10.8 Mb according to the human reference sequence and has a length of 21.8 cM based on the Rutgers combined linkage-physical map. In comparison, the 3-unit support interval for family 4100 is smaller, with 8.2 cM/3.7 Mb for the first peak and 5.8 cM/2.7 Mb for the second peak, both of which are completely contained within the interval for family 4154.
To further delineate the boundaries of the DFNB68 locus, haplotypes were reconstructed for each family (Fig. 1). In four hearing-impaired siblings from family 4100, the region of homozygosity is bounded by markers D19S884 and D19S432 (Fig. 1a), and therefore spans the two 3-unit support intervals (includes 7.5 Mb and is 14.3 cM long). The region of homozygosity in family 4154 is the same as the 3-unit support interval, and is delimited at the proximal end by D19S1034 in all affected family members, and at the distal end by marker D19S199 due to a historic recombination event in individual V-6 (Fig. 1b). Upon closer inspection, however, the unaffected individuals from both families are also homozygous for several of the markers within the region of homozygosity. If we take into consideration solely those markers that are homozygous only in the hearing-impaired but not among unaffected relatives, the true region of homozygosity in family 4100 is bounded by D19S586 and D19S906, while in family 4154 the interval is bordered by D19S905 and D19S584. The overlap between these two regions from the two families is flanked by markers D19S586 and D19S584, which can now be assigned as the limits of the DFNB68 locus. The genetic interval for the DFNB68 locus is 1.9 cM and contains 1.4 Mb.
Within the 1.4 Mb region of homozygosity, there are 40 known genes, of which four encode hypothetical proteins (Build 34 of the human reference sequence as seen from the University of California Santa Cruz Genome Browser). Five genes were found within published inner ear databases (The Hearing Research Group at Brigham and Women’s Hospital 2002; Holme et al. 2002), namely, DNMT1 (MIM 126375), ICAM1 (MIM 147840), KEAP1, CTL2, and SMARCA4 (MIM 603254) (Fig. 2). The genes KEAP1 and CTL2 were screened by sequencing in the two families, but no functional sequence variants were identified. The CDKN2D gene, which is homologous to hearing loss gene Ink4d in mice (Chen et al. 2003), was also sequenced; however no functional variants were found in the coding exons of CDKN2D.
The DFNB15 locus was previously mapped to two regions, 3q21.3–q25.2 and 19p13.3–p13.1 (Chen et al. 1997). However, it has not been established which one of these regions actually holds the causative HI gene, or if there is digenic inheritance for the family involved. One caveat was that the maximum LOD score that could ever be derived for the DFNB15 family was less than 3.0, and analyses of both regions on chromosomes 3 and 19 both resulted in a LOD score of 2.8. The new locus described here is contained within the DFNB15 interval on chromosome 19 (Fig. 2). Additionally, for the two families reported here, there is no evidence of linkage to chromosome 3q or to any other chromosome, discounting any possibility of digenic inheritance. Nevertheless, this does not bolster or refute the hypothesis that an HI gene in the 19p13 region caused the HI in the DFNB15 family. Due to the lack of evidence that the HI locus in this article is the same as DFNB15, this novel locus on 19p13.2 was assigned as DFNB68. Only one candidate gene, MYO1F, a member of the unconventional myosin family (which includes HI genes MYO1A (MIM 601478), MYO6 (MIM 600970), MYO7A (MIM 276903), and MYO15 (MIM 602666)), was reported to have been sequenced in the DFNB15 family, but no mutation was found (Chen et al. 2001). Likewise, the MYO1F gene had no functional sequence variants in families 4100 and 4154. This is not surprising given that MYO1F lies outside the 1.9 cM region of DFNB68 (Fig. 2).
Although high LOD scores were obtained for the two families at the more proximal marker D19S391 and at distal markers D19S558 and D19S840 (Table 1), it was discovered that at these markers the families were dissimilar in terms of the allele that was homozygous among affected individuals in each family. On the other hand, hearing-impaired individuals in both families are homozygous for the same alleles at contiguous markers from D19S586 to D19S914, though some of these markers were non-informative for each family. Based on the segregation of alleles among hearing impaired and hearing members of each pedigree, the assigned region of homozygosity for each pedigree decreased in length (Fig. 1) and from these smaller intervals the region of overlap was designated as the DFNB68 locus. Currently we are concentrating our search for candidate genes within the 1.4 Mb region of overlap seen in the two families, which is between markers D19S586 and D19S584 (Fig 2). It is not possible to conclude if both families have HI due to the same functional variant, due to different functional variants within the same gene, or even due to two different genes. Nevertheless, the observation of the same haplotypes in both families within the 1.9 cM region of homozygosity suggests that they may segregate the same variant.
Of the 40 genes within the overlapping regions of homozygosity, two candidate genes in the inner ear databases, KEAP1 and CTL2, were selected for sequencing in the DFNB68 families. KEAP1 (Kelch-like Ech-associated protein 1) is a functional repressor of transcription factor NRF2 (MIM 600492), which is a regulator of detoxifying and antioxidant genes. It was discovered that the Kelch repeat domain of the KEAP1 protein associates with the SH3 domain of MYO7A in specialized adhesion junctions in the testis (Velichkova et al. 2002). Moreover, both KEAP1 and MYO7A are expressed in cell bodies of cochlear inner hair cells, and it was proposed that KEAP1 facilitates the association of MYO7A with the actin cytoskeleton of hair cell stereocilia, thus promoting cell adhesion that is important for stereocilia bundle organization. Screened family members of 4100 and 4154 were negative for functional sequence variants in the coding regions of the KEAP1 gene.
The CTL2 (choline-transporter like protein 2) gene encodes a highly conserved transmembrane protein which is the target of antibody-mediated hair cell loss (Nair et al. 2004). The protein is expressed in cochlear and vestibular supporting cells, and is purported to function in these cells as a choline transporter. However, DNA sequencing in families that were linked to DFNB68 revealed no functional variants in the CTL2 gene.
The CDKN2D (cyclic-dependent kinase inhibitor 2D) gene is the human homolog of mouse gene Ink4d, which is required to maintain the inner hair cells in the post-mitotic state (Chen et al. 2003). In Ink4d knockout mice, re-entry of the inner hair cells into the cell cycle resulted in apoptotic cell death and subsequently progressive hearing loss. Although CDKN2D was not identified in the inner ear databases, other kinase inhibitors (CDKN1A, CDKN1B) were also found to be expressed in the inner ear (The Hearing Research Group at Brigham and Women’s Hospital 2002). Members of families 4100 and 4154 did not carry functional variants within the coding exons of CDKN2D.
Three other genes within the DFNB68 interval, DNMT1, ICAM1, and SMARCA4, were found in the inner ear databases. For these genes, there is currently no additional information that is linked to hearing physiology. There is, however, a report of the ICAM1 mRNA expression by cultured spiral ligament fibro-cytes after cytokine (TNF-α) stimulation (Ichimiya et al. 2003), which may suggest a role for ICAM1 in immune-mediated inner ear disease. Based on a positional cloning approach, the genes within the DFNB68 interval will be investigated further for a possible role in HI causation.
We thank the two families who participated in this research. We are also grateful to Xuan Zhang for assistance with the figures. This work was supported by the Higher Education Commission, Pakistan, and National Institutes of Health, National Institute of Deafness and other Communication Disorders grant R01-DC03594. Genotyping services were provided by the CIDR and the NHLBI Mammalian Genotyping Service (Marshfield, WI, USA). CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, Contract Number N01-HG-65403.
Regie Lyn P. Santos, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Alkek Building N1619.01, Houston, TX 77030, USA. Genetic Epidemiology Unit, Department of Epidemiology and Biostatistics, Erasmus Medical Centre, Rotterdam, The Netherlands.
Muhammad Jawad Hassan, Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan.
Shaheen Sikandar, 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 Building N1619.01, Houston, TX 77030, USA.
Ghazanfar Ali, Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan.
Protacio E. Martin, Jr, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Alkek Building N1619.01, Houston, TX 77030, USA.
Michael Angelo L. Wambangco, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Alkek Building N1619.01, Houston, TX 77030, USA.
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 Building N1619.01, Houston, TX 77030, USA.