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Dominant modifier DFNM1 suppresses recessive deafness DFNB26
Author: Saima Riazuddin
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"letter nature genetics ? volume 26 ? december 2000 431 Dominant modifier DFNM1 suppresses recessive deafness DFNB26 Saima Riazuddin 1,2 , Caley M. Castelein 3 , Zubair M. Ahmed 1,2 , Anil K. Lalwani 3 , Mary A. Mastroianni 4 , Sadaf Naz 2 , Tenesha N. Smith 1 , Nikki A. Liburd 1 , Thomas B. Friedman 1 , Andrew J. Griffith 1,4 , Sheikh Riazuddin 2 & Edward R. Wilcox 1 1 Laboratory of Molecular Genetics, NIDCD/NIH, Rockville, Maryland, USA. 2 Center of Excellence in Molecular Biology, Punjab University, Lahore, Pakistan. 3 Laboratory of Molecular Otology, Department of Otolaryngology, University of California, San Francisco, California, USA. 4 Neuro-Otology Branch, NIDCD, NIH, Bethesda, Maryland, USA. Correspondence should be addressed to E.R.W. (e-mail: wilcoxe@nidcd.nih.gov). More than 50% of severe childhood deafness is genetically determined, approximately 70% of which occurs without other abnormalities and is thus termed nonsyndromic 1,2 . So far, 30 nonsyndromic recessive deafness loci have been mapped and the defective genes at 6 loci, DFNB1, DFNB2, DFNB3, DFNB4, DFNB9 and DNFB21, have been identified, encoding connexin- 26 (ref. 3), myosin VIIA (ref. 4), myosin XV (ref. 5), pendrin 6 , otoferlin 7 and ?-tectorin 8 , respectively. Here we map a new recessive nonsyndromic deafness locus, DFNB26, to a 1.5-cM interval of chromosome 4q31 in a consanguineous Pakistani family. A maximum lod score of 8.10 at ?=0 was obtained with D4S1610 when only the 8 affected individuals in this family were included in the calculation. There are seven unaffected family members who are also homozygous for the DFNB26- linked haplotype and thus are non-penetrant. A dominant modifier, DFNM1, that suppresses deafness in the 7 nonpene- trant individuals was mapped to a 5.6-cM region on chromo- some 1q24 with a lod score of 4.31 at ?=0 for D1S2815. Large consanguineous families are a powerful resource for mapping and identifying additional deafness loci and genes that modify deafness phenotypes. We identified 141 members of a large consanguineous Pakistani family (PK2) segregating deaf- ness, and our study of this family was approved by Institutional Review Boards at the National Institutes of Health and at the Center of Excellence in Molecular Biology (Lahore, Pakistan). After obtaining written informed consent, medical and family histories and pure-tone audiograms were collected from a subset of study participants. Physical examinations were performed by one of the co-authors (A.J.G.). The pedigrees shown (Figs 1 and 2) include only the PK2 sibships segregating profound, congeni- tal, nonsyndromic, sensorineural hearing loss. After exclusion of linkage to loci known to cause nonsyn- dromic deafness, we carried out a genome-wide scan using 358 microsatellite markers (ABI Prism-linkage v1) on affected and unaffected members of the family. We found evidence of linkage with marker D4S1625 on chromosome 4q31. Haplotype analyses Fig. 1 DFNB26-linked haplotypes of PK2 family members on chromosome 4q31. Black symbols, affected individuals; green symbols, normal-hearing, nonpene- trant individuals homozygous for the DFNB26-linked haplotype. The grey-shaded haplotype is linked to DFNB26. Deaf individuals from the five sibships of family PK2 are all offspring of consanguineous matings. Individual VI:32 married into the PK2 family, but she may also be distantly related to PK2 because she carries the DFNB26-linked haplotype and comes from the same caste. Individuals VI:8 and VI:11 are homozygous for alleles of 11 markers of the DFNB26-linked haplotype, except D4S1625. Heterozygosity for the marker D4S1625 in two individuals suggests the occurrence of a de novo mutation in the father V:15. Allele B of D4S1625 is present in individual VI:5 and is also segregating in his offspring (data not shown; ref. 30). Seventy-two individuals of family PK2 are not shown, as none of them were homozygous for the DFNB26-linked haplotype and none had meiotic recombinations that helped to refine the DFNB26 interval. � 2000 Nature America Inc. ? http://genetics.nature.com � 2000 Nature America Inc. ? http://g enetics.nature .com letter 432 nature genetics ? volume 26 ? december 2000 of additional markers refined DFNB26 to a 1.5-cM interval defined by recombinations with D4S424 and D4S2998 in individ- uals VI:2, VI:3 and VII:4. All of the eight affected individuals were homozygous for the DFNB26-linked markers on chromo- some 4q31 (Fig. 1). A maximum lod score of 8.10 at ?=0 was obtained for D4S1610 when only affected individuals were included in the calculation, whereas a lod score of 6.53 was obtained at ?=0.07 for D4S1610 when 60 unaffected PK2 indi- viduals were included in the calculation (Table 1). We found that seven unaffected individuals (VI:8, VI:11, VI:18, VII:2, VII:6. VII:10 and VII:15) from five different sibships within the family were homozygous for the DFNB26-linked hap- lotype (Fig. 1). This observation indicated either the existence of a modifier gene suppressing the development of DFNB26 deaf- ness or that the DFNB26 is located elsewhere. We therefore per- formed a second genome-wide linkage analysis of both affected and nonpenetrant individuals using 348 markers from the Weber 8 panel (Research Genetics). We found no additional regions of homozygosity linked to DFNB26 deafness or to the nonpene- trance trait in the seven unaffected individuals (Fig. 3). We next tested a model for a dominant modifier of DFNB26, and found that the nonpenetrance trait linked to markers on chromosome 1q24. Haplotype analysis of the markers linked to this locus (DFNM1, for deafness, nonsyndromic, modifier 1) demonstrated meiotic recombinations with markers D1S2658 and D1S2790 in individuals VII:15 and VI:18, which reduced the critical interval to 5.6 cM (Fig. 2). A lod score of 4.31 at ?=0 with D1S2850 was obtained for DFNM1 by assigning phenotypic status as affected for the nonpenetrant DFNB26 homozygotes, the deaf as unaffected, and all others as unknown (Table 2). The DFNM1-linked haplotype was not inherited by any deaf individuals. As further support for the correct genetic map localization of DFNB26 and DFNM1, we used a multipoint linkage analysis to exclude other regions of the genome. We excluded 71.5% of the genome with a lod score of less than or equal to ?2 and 27.4% with a lod score of less than or equal to ?1. We calculated the lod scores under a model of recessive inheritance with 100% penetrance and a mutant allele frequency of 0.0001. We also excluded the common mitochondrial mutations that cause deafness in this family by sequencing mitochondrial DNA for the 12S rRNA mutation A1555G, and tRNA ser mutations A7445G and 7472insC (refs 9?11). Audiometric and otoacoustic emissions test- ing revealed no differences between family members with normal hearing and nonpenetrant DFNB26 homozygotes (ranging in age from 18 to 51 years). The map location of DFNM1 is within the 22-cM DFNA7 interval 12 , indicating that the DFNM1 suppressor phenotype and DFNA7 deafness may be two phenotypic variants of the same gene. Within the DFNM1 interval, PMX1 (paired mesoderm homeobox) is a potential candidate gene that is expressed in the cochlea 13 . There are no reported cochlear ESTs within the 1.5-cM interval of DFNB26, and there are no known deafness loci located within the predicted region of conserved linkage on mouse chro- mosome 8. Candidate genes identified within the DFNB26 inter- val encode a ribosomal protein (RPS2; ref. 14), GRB2-associated binding protein-1 (GAB1; ref. 15), SWI/SNF-related, matrix- associated, actin-dependent regulator of chromatin (SMARCA5; ref. 16), and two ESTs (AJ243229 and DKFZp432C2112) with no sequence similarity to known genes. We are carrying out muta- tional analyses of these genes from PK2 individuals. Table 1 ? Maximum 2-point lod scores for DFNB26 on chromosome 4q31 Affected individuals only Unaffected and affected individuals Marker Lod ? Lod ? D4S1576 1.41 0.08 2.34 0.09 D4S1579 0.82 0.07 0.51 0.15 D4S1644 1.97 0.06 1.16 0.08 D4S424 3.31 0.03 2.70 0.10 D4S1625 6.20 0 5.87 0.03 D4S1604 3.62 0 3.89 0 D4S2981 7.59 0 6.10 0.07 D4S1610 8.10 0 6.53 0.07 D4S2998 2.40 0.04 2.31 0.10 D4S3014 2.53 0.04 1.95 0.08 Fig. 2 DFNM1-linked haplotypes of PK2 family members on chromosome 1q24. Green symbols, nonpenetrant individuals homozygous for the DFNB26-linked haplotype; black symbols, phenotypically affected individuals. The green-shaded haplotype is linked to DFNM1-mediated nonpenetrance for DFNB26 deafness. � 2000 Nature America Inc. ? http://genetics.nature.com � 2000 Nature America Inc. ? http://g enetics.nature .com The existence of modifier genes has been known for almost 100 years. In 1919, Bridges reported Drosophila melanogaster genes that by themselves produce little or no effect, yet modify the eye colour of the sex-linked mutant eosin (ref. 17). There are well-documented examples of intrafamilial variable expressivity for deafness in humans, which are usually attributed to environ- mental factors or genetic background effects due to modifier genes 18 . A nuclear-encoded modifier seems to cause deafness in association with the mitochondrial mutation A1555G in the absence of exposure to aminoglycosides 19 . The mouse provides sev- eral examples of interactions of genetic modifiers with deafness genes. The mdfw locus modifies the hearing loss phenotype in dfw/+ heterozygotes 20 , whereas a dominant allele of moth1 protects tubby mice from hearing loss 21 . Modifier genes can therefore act to suppress or enhance the mutant phenotype 22?27 , and DFNM1 would thus be classified as a suppressor of DFNB26 deafness. The genetics of nonpenetrance of DFNB26 deafness seems to be understood. It is possible that DFNM1 will prove to be a more general suppressor of a specific class of mutant alleles of a variety of different genes. The elucidation of the underlying mechanism of suppression, however, awaits the identification of the DFNB26 and DFNM1 genes. Functional analysis of DFNB26 and DFNM1 should provide new insights into the molecular mechanisms of auditory function and facilitate the rational design of therapies for hearing loss. Methods Genotyping. We extracted genomic DNA from blood according to a stan- dard protocol 28 and performed genotypic analysis of polymorphic markers to exclude linkage to known DFNB loci. We performed a genome-wide linkage analysis by genotype analyses of 674 microsatellite markers, with a resolution of less than 10 cM. PCR products were separated and detected on 4.25% acrylamide gels with an ABI 377 DNA sequencer, and alleles were assigned with Genotyper software (v 2.0 Applied Biosystems). Fig. 3 Representative pure- tone audiograms for PK2 family members. Pure-tone response thresholds are shown for right ears (a) and left ears (b) of unaffected individuals VII:11 (solid line) and VII:3 (dashed line), non- penetrant individuals VII:10 (open square) and VII:2 (open circle), and affected individuals VII:14 (filled square) and VII:4 (filled cir- cle). Unaffected individuals, including nonpenetrant individuals, had pure-tone audiometric thresholds at approximately 30 dB HL (Figs 1 and 2). These slightly elevated thresholds may be due to ambient noise present during audiological testing, which was not performed in a soundproof booth. Some individu- als may also have slightly elevated thresholds due to prior exposure to aminoglycosides or noise, which are common in this population. Deaf individuals who are homozygous for the linked DFNB26 haplotype, but do not carry the DFNM1 modifier, have severe to profound congenital sensorineural hearing loss. letter nature genetics ? volume 26 ? december 2000 433 Table 2 ? Maximum 2-point lod scores for DFNM1 on chromosome 1q24 Marker Lod ? D1S2635 1.59 0.07 D1S484 2.45 0.05 D1S1679 2.38 0.05 D1S2878 2.41 0.06 D1S318 2.42 0.06 D1S2658 2.64 0.06 D1S1165 4.18 0 D1S2815 4.31 0 D1S210 1.84 0 D1S2790 1.21 0.06 D1S218 0 0.09 a b Statistical analysis. We calculated lod scores using FASTLINK (v 4.1p; ref. 29) and modelled deafness as an autosomal recessive trait. We carried out computations of two-point lod scores with MLINK on the full pedigree as described in Figs 1 and 2 and analysed multipoint exclusion mapping with LINKMAP on a simplified pedigree containing only three loops. To calcu- late the initial lod score for the DFNB26 locus, we ascribed all deaf indi- viduals as affected and all others as unknown. For the DFNM1 locus, a dominant model was assumed and subjects with normal hearing homozy- gous for the DFNB26-linked haplotype were assigned as affected, deaf individuals homozygous for DFNB26-linked haplotype were assigned as unaffected, and all others were assigned as unknown. We determined marker allele frequencies by genotype analysis of genomic DNA from 90 unrelated Pakistani subjects. Note added in proof: Three additional nonsyndromic recessive deafness loci, DFNB10/B8, DFNB12 and DFNB29, were recently identified, encod- ing transmembrane serine protease-3 (Hereditary Hearing Loss Home Page, http://dnalab-www.uia.ac.be/dnalab/hhh/), cadherin-23 (ref. 31) and claudin-14 (Hereditary Hearing Loss Home Page, http://dnalab- www.uia.ac.be/dnalab/hhh/), respectively. Acknowledgements We thank the PK2 family members for their participation, which is supported by the National Institute on Deafness and Other Communication Disorders, Intramural Research Project Z01DC00035; B.U. Amjad for help in initiating this study; R. Morell, D. Drayna, J. Bork, T. Ben-Yosef and K. Kurima for comments; and S. Khan and B. Ploplis for technical support. University Grants Commission supported part of this study in Pakistan. This study used the computational capabilities of the Biowoulf Center at the CIT, NIH. Received 12 September; accepted 18 October 2000. � 2000 Nature America Inc. ? http://genetics.nature.com � 2000 Nature America Inc. ? http://g enetics.nature .com letter 434 nature genetics ? volume 26 ? december 2000 1. Morton, N.E. Genetic epidemiology of hearing impairment. Ann. NY Acad. Sci. 630, 16?31 (1991). 2. Willems, P.J. Genetic causes of hearing loss. N. Engl. J. Med. 342, 1101?1109 (2000). 3. Kelsell, D.P. et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387, 80?83 (1997). 4. Liu, X.Z. et al. Mutations in the myosin VIIA gene cause non-syndromic recessive deafness. Nature Genet. 16, 188?190 (1997). 5. Wang, A. et al. Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3. Science 280, 1447?1451 (1998). 6. Li, X.C. et al. A mutation in PDS causes non-syndromic recessive deafness. Nature Genet. 18, 215?217 (1998). 7. Yasunaga, S. et al. A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nature Genet. 21, 363?369 (1999). 8. Mustapha, M. et al. An ?-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21. Hum. Mol. Genet. 8, 409?412 (1999). 9. Prezant, T.R. et al. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nature Genet. 4, 289?294 (1993). 10 Tiranti, V. et al. Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene. Hum. Mol. Genet. 4, 1421?1427 (1995). 11. Verhoeven, K. et al. 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Cloning and mapping of SMARCA5 encoding hSNF2H, a novel human homologue of Drosophila ISWI. Cytogenet. Cell. Genet. 81, 191?193 (1998). 17. Bridges, C.B. Specific modifiers of eosin eye color in Drosophila melanogaster. J. Exp. Zool. 28, 337?384 (1919). 18. Friedman, T.B. et al. Modifier genes of hereditary hearing loss. Curr. Opin. Neurobiol. 10, 487?493 (2000). 19. Bykhovskaya, Y. et al. Candidate locus for a nuclear modifier gene for maternally inherited deafness. Am. J. Hum. Genet. 66, 1905?1910 (2000). 20. Noben-Trauth, K., Zheng, Q.Y., Johnson, K.R. & Nishina, P.M. mdfw: a deafness susceptibility locus that interacts with deaf waddler (dfw). Genomics 44, 266?272 (1997). 21. Ikeda, A. et al. Genetic modification of hearing in tubby mice: evidence for the existence of a major gene (moth1) which protects tubby mice from hearing loss. Hum. Mol. Genet. 8, 1761?1767 (1999). 22. Bejjani, B.A. et al. Multiple CYP1B1 mutations and incomplete penetrance in an inbred population segregating primary congenital glaucoma suggest frequent de novo events and a dominant modifier locus. Hum. Mol. Genet. 9, 367?374 (2000). 23. Cheng, N.C. et al. A human modifier of methylation for class I HLA genes (MEMO- 1) maps to chromosomal bands 1p35?36.1. Hum. Mol. Genet. 5, 309?317 (1996). 24. Marin, M.C. et al. A common polymorphism acts as an intragenic modifier of mutant p53 behavior. Nature Genet. 25, 47?54 (2000). 25. Wu, X. et al. Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc. Natl Acad. Sci. USA 97, 4233?4238 (2000). 26. Zielenski, J. et al. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13. Nature Genet. 22, 128?129 (1999). 27. Sertie, A.L., Sousa, A.V., Steman, S., Pavanello, R.C. & Passos-Bueno, M.R. Linkage analysis in a large Brazilian family with van der Woude syndrome suggests the existence of a susceptibility locus for cleft palate at 17p11.2?11.1. Am. J. Hum. Genet. 65, 433?440 (1999). 28. Grimberg, J. et al. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 17, 8390 (1989). 29. Schaffer, A.A., Gupta, S.K., Shriram, K. & Cottingham, R.W. Jr Avoiding recomputation in linkage analysis. Hum. Hered. 44, 225?237 (1994). 30. Weber, J.L. & Wong, C. Mutation of human short tandem repeats. Hum. Mol. Genet. 2, 1123?1128 (1993). 31. Bork, J.M. et al. Usher syndrome 1D (USH1D) and nonsyndromic recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23. Am. J. Hum. Genet. (in press). � 2000 Nature America Inc. ? http://genetics.nature.com � 2000 Nature America Inc. ? http://g enetics.nature .com "
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