JIIMS final.cdr 15 ORIGINAL ARTICLE ABSTRACT Objective: DNA analysis for the Genetic Mapping of Candidates of Deafness Genes in Pakistani Families. Study Design:It was a cross sectional study. Place and Duration of the Study: Department of Biochemistry/Molecular Biology, Quaid I Azam University, Islamabad Pakistan. The Clinical examination, biochemical tests, interpretation of results and preparation of results completed in approximately one year, 2006 2007. Materials and Methods: Study was conducted on two Pakistani families. Subjects (Families) selected for the study: Two Pakistani families labeled as family A and B were selected for the study. Family A comprises of three normal and three affected (Deaf) individuals. Family B comprises of two normal and four affected (Deaf) individuals. The blood samples were immediately dispatched to Molecular genetic laboratory, Quaid I Azam University, Islamabad for analysis 2006 2007. Results: In family A, linkage was established to DFNB47 locus on the chromosome 2p25.1-p24.3. In family B, linkage to DFNB1 locus was excluded first by genotyping polymorphic microsatellite markers linked to the candidate region and then by sequencing GJB2 gene Conclusion: The genetic mapping of candidates of deafness genes brings greater understanding of molecular basis of deafness and would modify the preventive and curative methods. Key words: DNF, DNA, GJB, PCR and Electrophoresis X-Chromosomal recessive, or maternal 7trait. X-Chromosomal dominant and Y linked transmission are rare. Syndromic hearing impairment is associated with malformation of the external ear or other organs with medical problems involving other organ systems. More than 70% of the hereditary 8hearing loss is non syndromic. Of the 30,000 50,000 human genes, 1% i.e. 300 500 genes, are estimated to be necessary 9for hearing. Gap junctions are clusters of intercellular channels, vital of intercellular communication. The following connexins expressed in the auditory system have been implicated in hereditary deafness, GJB2, 11, 14, 16GJB3, GJB6 and GJA1. Mutation in the Alpha tectorin gene on chromosome 11q has been found in families with both autosomal dominant and autosomal recessive having 15prelingual hearing loss. Mutations in the Trans membrane inner ear (TMIE), Trans membrane channel like 1 (TMC1), MY06 Introduction Hearing impairment is the most common 1sensory disorder worldwide. It is clinically and genetically very heterogeneous and auditory genes are discovered at very rapid p a c e . G e n e t i c f a c t o r s a re p ro b a b l y responsible for more than 50% of the cases of 2early onset H1. Where as in most of the late onset H1 a combination of genetic as well as 3environmental factors is involved. Studies of the epidemiology of hearing impairment have suggested that approximately 1 in 1000 to 1 in 2000 children show a profound 4, 6hearing loss at birth or in early childhood. Most frequently hearing impairment, is classified as syndromic or non syndromic, or according to its transmission via as autosomal dominant, autosomal recessive, ------------------------------------------------- Genetic Mapping of Candidates of Deafness Genes in Pakistani Families Irum Afshan, Mubin Mustafa, Nasim Ilyas, Usman Nawaz, Kashif Rahim, Saleem Murtaza Correspondence: Dr. Irum Afshan M.Sc, M.Phil Biochemistry QAU Ph.D Scholar, Biochemistry NUST, Islamabad 15 16 gene, MY015 gene, transcription regulators, POU3F4, POU4F3, ICERE-1, COCH, KCNQ4, COL11A2 and mitochondrial genes (12 SrRNA gene) have been found to be involved in different types of deafness in 17, 30many studies. A cross sectional study was conducted on two Pakistani families at Department of Biochemistry/Molecular Biology, Quaid I Azam University, Islamabad Pakistan. The Clinical examination, biochemical tests, interpretation of results and preparation of thesis completed in approximately one year 2006-2007. Families Studied Two families labeled as family A and B were selected for the study. Family A comprises of three normal and three affected (Deaf) individuals. Family B comprises of two normal and four affected (Deaf) individuals. After detailed discussion with the elders of these families, genetic pedigrees were 37drawn by following standard method. Mode of inheritance was inferred through pedigree analysis. Blood Sampling Blood samples from both normal as well as affected individuals including their parents were collected by 10 cc syringes (08×38 mm 21G×11/2) in standard potassium EDTA tubes. The blood samples were immediately dispatched to Molecular genetic laboratory, Quaid I Azam University, Islamabad for analysis 2006-2007. Extraction and Purification of Genomic DNA from Blood Genomic DNA was extracted from blood by phenol / chloroform method. DNA Dilution and Micro Pipetting Polymerase Chain Reaction (PCR) Materials and Methods PCR was performed using gene Amp PCR System 2400 and 9600 thermo cycler (Perkin Elimer USA). Agarose gel Electrophoresis Agarose gel Electrophoresis was carried out to analyze the amplified DNA samples. After Electrophoresis amplified product was detected by placing the gel on UV Trans illuminators (Life Technology, USA). Polyacrylamide gel Electrophoresis Gel was photographed by using Digital Camera DC 120 (Kodak, USA). Genotyping and Primer Database Analysis M i c ro s a t e l l i t e m a r k e r s m a p p e d b y Cooperative Human Linkage Centre (CHLC), were obtained from research genetics, Inc. (USA). The cytogenetic location of these markers as well as the length of the amplified product was obtained from genome data base homepage (www.gdb.org) and Marshfield Medical Center(www.marshmed.org.gov/genetics) Linkage studies L i n k a g e s t u d i e s w e r e p e r f o r m e d , Automated Genetic Analyzer ABI Prism 310 (Applied Bio System, USA). In the present study family A was first tested for mapping to several known loci by using polymorphic microsatellite markers from their candidate linkage intervals. The family A was found to be linked to DFNB47 locus on chromosomal region 2p25.1-p24.3. Two loci for ARNSH1 have previously been 2localized to chromosome. In family BDNFB1 and several other loci were tested for linkage. Electropherograms obtained by genotyping the microsatellite linked to the candidate linkage gene interval revealed that the affected individuals were heterozygous for different combinations of Results 16 17 parental alleles, thus indicating exclusion of family B from linkage to DFNB1 and several other known autosomal recessive non syndromic hearing loss loci. Linkages to DFNB1 locus were also excluded by sequencing the coding region of exon 2 of GJB2 gene. The novel locus harboring the disease gene in family B can be located by a genome wide search by using polymorphic markers spaced at 10 cM apart on all the autosomes. To date 23 known genes lie in the 5.3 Mb- region that contains DFNB 47. One of the genes in this region, KCNFI, is a strong candidate for DFNB47. This gene codes for p o t a s s i u m v o l t a g e - g a t e d c h a n n e l . Potassium ion channels are a diverse family of plasma member's proteins that play an essential role in various cellular processes, including maintenance of membrane 31potential and cell signaling. KCNQ4 is a voltage gated K+ channel gene expressed in the cochlea. Voltage-gated K+ channel genes have been shown to be responsible for various hereditary diseases. For instance, mutation in the KVLQTI gene (a voltage- gated K+ channel gene) result in Jervell and Lange-Nielsen syndrome (JLNS) and Long QT syndrome, which are inherited AR disease, with congenital HI being one of 32their characteristics. JLNS can also result from mutations in another voltage-gated K+ channel gene, KCNEI. Another good candidate gene is inhibitor of DNA binding 2 (ID2), which is a member of the ID family genes that promotes cell proliferation. In embryonic mouse, ID2 expression was detected in the vestibular and acoustic ganglia, and also in the epithelium of the otic vesicle and Discussion 33surrounding mesenchyme . Other genes that are expressed in the inner ear include: (1) cleavage and polyadenylation specific factor 3 2004); (2) tyrosine 3/ tryptophan 5- monooxygenase (YWHAQ), which is also expressed in the spinal cord of patients with 3 5amyotrophic lateral sclerosis. And ornithine decarboxylase 1 (ODCI), the rate limiting enzyme in polyamine synthesis. The recent identification of several deafness genes by molecular genetic studies has enabled the molecular basis of normal and pathological auditory function. In the coming years, further deafness genes are sure to be identified and mouse models for the human disease will be constructed as start in the long process of understanding the pathological processes involved in deafness. The rate of discovery of deafness genes by positional cloning in human will be accelerated by the freely available human genome sequence and by a catalogue of Expressed Sequence Tags (ESTs) within genetic intervals known to contain locus for human hereditary hearing loss. To assist in the identification of deafness genes cDNA library has been synthesized, partially sequenced and many ESTs assigned map 36position. The genetic mapping of candidates of d e a f n e s s g e n e s b r i n g s g r e a t e r understanding of molecular basis of d e a f n e s s a n d w o u l d m o d i f y t h e preventive and curative methods. 1. Berlin-Glindzicz M. Hereditary Deafness and Pheno-typing in Humans. Br Med Bull 2002;63 :73-94. 2. Marzita Ml, Rawlings B, Remington B, Amos Ks, Nance We. Genetic Epidemiological Studies of Conclusion References 17 18 Early-Early On set Deafness in the U.S School- Age Population. Am J Hum Genet 1993;57:629- 35. 3. Cohen Mm, Gorlin Rj. Epidemiology, Etiology, and Genetic Patterns; In Gorlin Rj Toriello Hv, Cohenmm (Ed). Hereditary Hearing Loss and Its Syndromes. Oxford Monographs on Medical Genetics. 1995; p: 9-21. 4. Parving A. Epidemiology of Hearing Loss and Etiological Diagnosis of Hearing Impairment in Childhood. Int J Ped Otorhined 1983; 7:29-38. 5. Newton Ve. Etiology of Bilateral Sensorineural Hearing Loss in Young Children. J Laryngol Otology 1985; 10:1-57. 6. Fortnum H, Davis A. Epidemiology of Permanent Childhood Hearing Impairment in Trent Region. Brit J Aud 1997;31:409-46. 7. Smith An, Brothwick Kj. Molecular Cloning and Characterization of Novel Tissue Specific Isoforms of the Human Vacuolar H (+) ATPase C, G and D Subunits, and Their Evaluation in Autosomal Recessive Distal Renal Tubular Acidosis. Gene 2002; 297: 169-77. 8. Van camp G, coucke P, nalenans W. Localization of gene for non syndromic hearing loss to chromosome 7p15. Mol genet 1995; 4: 2159.63 9. Friedman Bt, Griffith Ja. Human Non syndromic Sensorineural Deafness. Ann Rev Hum Genet. 2003; 4:341-402. 10. Kikuchi T, Kimura Rs, Paul Dl, Adams Jc. Gap Junctions in the Rat Cochlea: Immuno histochemical and ultra structural Analysis. Anat Embryo 1995; 191:101-18. 11. Scott Da, Kraft Ml. Conexant Mutations and Hearing Loss. Nature 1998; 391: 32. 12. Carrasquillo Mm, Zlotogora J, Barges S, Chakravarti A. Two Different Connexin 26 Mutations in an Inbred Kindred Segregating N o n - S y n d r o m i c R e c e s s i v e D e a f n e s s : Implications for Genetic Studies in Isolated Populations. Hum Mol Genet 1997; 6:2163-72. 13. Richard G, White Tw. Functional Defect of C*26 Resulting Form A Heterozygous Missense Mutation in A Family with Dominant Deaf Mutes and Palm planter Kerato derma. Hum Genet 1998; 103: 393-9. 14. Teubner B, Michel V. Connexin 30 (GJB6) deficiency causes severe hearing impairment and lack of end cochlear potential. Hum Mol Genet 2003;12: 13-21 15. Verhoeven K, Van Laer L, Kirschlofer K. Mutation in human alpha tectorin gene cause autosomal dopminant non syndromic hearing impairment. Nat Genet 1998;19: 60-2. 16. Liu Xz, Xia X Adams J, Chen Zy. Mutation In Gja (Connexin 43) Are Associated With Non Syndromic Autosomal Recessive Deafness. Hum Mol Genet 2001; 10: 2945 - 51 17. Mitchem Kl, Hibbard E Beyer La Bosom K Dootz Ga. Mutation Of The Novel Gene Tmie Results In Sensory Cell Defects In The Inner Ear Of Sponner, A Mouse Model Of Human Hearing Loss Dfnb6. Hum Mol Genet 2002; 11:1887-98. 18. Kurima K, Peters Lm, Yang Y, Reazuddin S Ahmed Zm. Dominant And Recessive Deafness Caused By Mutation Of A Novel Gene, Tmci, Required For Cochlear Hair-Cell Function. Nat Genet 2002; 30:277-84. 19. Petersen Mb. Non Syndromic, Autosomal Dominant Deafness. Clin Genet 2002; 62: 1-13. 20. Wang A, Liang Y, Friedell RA. Association of unconventional myosin MYO15 mutations with human non syndromic deafness DFNB3. Science 1998; 280: 1447-51. 21. Cantos R, Cole Lk, Acampora D, Simeone A, Wu Dk. Patterning Of the Mammalian Cochlea. Proc Natl Acad Sci USA 2000; 97:1707-13. 22. Anagnostopoulos Av. A Compendium of Mouse Knockouts with Inner Ear Defects. Trends Genet 2002;18:21-38. 23. De Kok Yj, Van Der Maarel SM, Biter- Glindziez M, Huber I, Monaco Ap, Malcolm S et.al. Association between X- Linked Mixed Deafness And Mutations In The Pou Domain Gene Pou3f4. Science. 1995;267: 685-8. 24. Vahava O, Morell R, Lynch ED. Mutation in transcription factor POU4F3 associated with inherited progressive hearing loss in humans. Science 1998;27: 1950-4. 25. Van Camp G, willens PJ, smith RJ. Non syndromic hearing impairment unparalleled heterogeneticity. Am J Hum Genet 1997;60: 758- 64. 26. Khetarpal U, Schuknecht Hf, Gacek Rr, Holmes Lb. Autosomal Dominant Sensorineural Hearing Loss, Pedigrees, Audiologic Finding, and Temporal Bone Findings Two Kindreds. Arch Otolaryngol Head Neck Surg. 1991; 117: 1032-42. 18 19 27. Kubisch C, Schroeder Bc, Friedrich T, Lutjohann B, E1- Amraoui A. Kcnq4, A Novel Potassium Channel Expressed In Sensory Outer Hair Cells, Is Mutated In Dominant Deafness. Cell 1999;96:437-46. 28. Vikkula M, Mariman EC, Liu VC. Autosomal dominant and recessive osteochondrodysplasias associated with the COL11A2 locus. Cell 1995;80: 431-7. 29. Fischel Ghodsian N. Mitochondrial Deafness Mutations Reviewed. Hum Mutat 1999;13:261- 70. 30. Zhoa H, Li R, Wang Q. Maternally inherited aminoglycoside induced and non syndromic deafness is associated with novel C149T mutation in the mitochondrial 12s rRNA gene in large Chinese family. AM J Hum Genet 2004; 75: 139-52. 31. S u K , K y a w H , F a n P. I s o l a t i o n a n d characterization, and mapping of two human potassium channels. Biochem Biophys res Commun 1997; 24: 675-81. 32. Ilhan A, Tuncer C Komsuoglu Ss, Kali S. Jervell and Lange-Nielsen Syndrome: Neurologic and Cardiologic Evaluation. Pediatr Neurol 1999;21:809-13. 33. Jen Y, Manova K, Benezra R. Each Member Of T h e I d G e n e F a m i l y E x h i b i t s A U n i q u e Expression Pattern In Mouse Gastrulation And Neurogenesis. Dev Dyn 1997;208:92-106. 34. Calzado Ma, Sancho R, Munoz E. Human Immunodefiency Virus Type 1 that Increases The Expression Of Cleavage And Polyadenylation Specificity Factor 73- Kilodalton Subunit Modulating Cellular And Viral Expression. J Viral 2004; 78:6846-54. 35. Malaspina A, Kaushik N De Bellerouche J. A 14- 3.3 M Rnais Up- Regulated in Amyotropic Lateral Sclerosis Spinal Cord. J Neurochem 2000;75:2511-20. 36. Skvorak Ab, Weng Z. Human Cochlear Expressed Sequence Tags Provide Inside into Cochlear Gene Expression and Identify Candidate Gene for Deafness. Hum Mol Genet 1999; 8: 439-52. 37. B e n n e t t R L , S t e l n h a u s K A , U h r i c h . Recommendations for standardized human pedigree nomenclature. Am J Hum Genet 1995; 56: 745-52. 38. MA Tabatabaiefar, F Alasti, Zohour M. Genetic Linkage Analysis of 15 DFNB Loci in a Group of Iranian Families with Autosomal Recessive Hearing Loss. Iranian Journal of Public Health 2011;40 : 34-8. 39. Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 2009; 387: 80-3. 40. H e r e d i t a r y H e a r i n g L o s s h o m e p a g e . Available:http://herditaryhearingloss.org/.Ac ces-sed April 2010. 41. B Sagong, R Park, Kim Y. .Two Novel Missense Mutations in the TECTA Gene in Korean F a m i l i e s w i t h A u t o s o m a l D o m i n a n t Nonsyndromic Hearing Loss Ann Clin Lab Sci Autumn 2010;40: 380-5. 19