Why Next-Generation Sequencing Studies May Fail: Challenges and Solutions for Gene Identification in the Presence of Familial Locus Heterogeneity. R. L. P. Santos-Cortez1, A. U. Rehman2, M. C. Drummond2, M. Shahzad3,4, K. Lee1, R. J. Morell2, M. Ansar1,5, A. Jan5, X. Wang1, A. Aziz5, S. Riazuddin3,6, J. D. Smith7, G. T. Wang1, Z. M. Ahmed4,6, K. Gul3, A. E. Shearer8, R. J. H. Smith8, J. Shendure7, M. J. Bamshad7, D. A. Nickerson7, J. Hinnant9, S. N. Khan3, R. A. Fisher10, W. Ahmad5, K. H. Friderici10,11, S. Riazuddin3,12,13, T. B. Friedman2, E. S. Wilch11, S. M. Leal1, University of Washington Center for Mendelian Genomics 1) Center for Statistical Genetics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA; 2) Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, USA; 3) National Center of Excellence in Molecular Biology, University of the Punjab, Lahore 54590, Pakistan; 4) Division of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA; 5) Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan; 6) Laboratory of Molecular Genetics, Division of Pediatric Otolaryngology Head and Neck Surgery, Cincinnati Childrens Hospital Medical Center, Cincinnati 45229, Ohio, USA; 7) Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA; 8) Molecular Otolaryngology & Renal Research Labs, Department of OtolaryngologyHead & Neck Surgery, University of Iowa, Iowa City, Iowa 52242, USA; 9) Department of Religious Studies, Michigan State University, East Lansing, Michigan 48824, USA; 10) Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan 48824, USA; 11) Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA; 12) Allama Iqbal Medical College-Jinnah Hospital Complex, University of Health Sciences, Lahore 54550, Pakistan; 13) University of Lahore, Lahore 54000, Pakistan.

   Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fail to identify causal variants, with estimates for success rates of uncovering the pathologic variant underlying disease etiology being as low as 25 percent (Yang et al. 2013). An important reason for such failures is familial locus heterogeneity, where causal variants in two or more genes within a single pedigree underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous mutations in two different hearing impairment genes and a large European-American pedigree in which hearing impairment is caused by pathogenic variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single known hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3 percent (95 percent confidence interval: 11.9 to 19.9 percent) of the families in our collection where we have identified at least one variant in a previously published hearing impairment gene which only segregates with hearing impairment phenotype in a subset of affected pedigree members. These families have been evaluated by screening genes which commonly underlie nonsyndromic hearing impairment, whole-genome linkage analysis and NGS. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees) which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from the linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby aid in gene identification. If the analysis is performed using SNP genotyping arrays, before sequencing the results can be used to aid in the selection of pedigree members for NGS. It is demonstrated how the novel applications of linkage analysis and homozygosity mapping can increase the success rate of gene identification for families with locus heterogeneity.

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