Objective: We provide the proof of principle that exome sequencing of only two affected siblings born to first-cousin parents is capable of directly identifying a single candidate gene for an autosomal recessive disorder. This strategy, which we call EX-HOM (EXome HOMozygosity), combines in a single step the capacity of exome sequencing to identify all the coding variants present in a genome with the property of homozygosity mapping to limit the search for candidate genes to specific chromosomal regions. Methods: We sequenced the exomes of two siblings born to first-cousin parents affected with dysmyelinating leukodystrophy and spastic paraparesis caused by a mutation in FA2H. We used exome sequencing data to identify homozygous regions shared by the two affected siblings (EX-HOM regions), compared them with the regions of maximum LOD score obtained with SNP genotyping, and selected the candidate variants within. Results: We identified regions of shared homozygosity (>1 Mb) accounting for about 290 Mb, containing only 3 candidate variants. Among these, the FA2H mutation remained the only plausible one. Conclusion: In single consanguineous pedigrees with a few affected sibs, EX-HOM can be a one-step approach to identify the candidate genetic defect, bypassing obstacles such as genetic heterogeneity and the need for large pedigrees.
© 2011 S. Karger AG, Basel
- Exome sequencing
- Homozygosity mapping
- Autosomal recessive disorders
- Ozçelik T, Kanaan M, Avraham KB, Yannoukakos D, Mégarbané A, Tadmouri GO, Middleton L, Romeo G, King MC, Levy-Lahad E: Collaborative genomics for human health and cooperation in the Mediterranean region. Nat Genet 2010;42:641–645.
- Bilgüvar K, Oztürk AK, Louvi A, Kwan KY, Choi M, Tatli B, Yalnizoğlu D, Tüysüz B, Cağlayan AO, Gökben S, Kaymakçalan H, Barak T, Bakircioğlu M, Yasuno K, Ho W, Sanders S, Zhu Y, Yilmaz S, Dinçer A, Johnson MH, Bronen RA, Koçer N, Per H, Mane S, Pamir MN, Yalçinkaya C, Kumandaş S, Topçu M, Ozmen M, Sestan N, Lifton RP, State MW, Günel M: Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations. Nature 2010;9:207–210.
- Funari VA, Krakow D, Nevarez L, Chen Z, Funari TL, Vatanavicharn N, Wilcox WR, Rimoin DL, Nelson SF, Cohn DH: BMPER mutation in diaphanospondylodysostosis identified by ancestral autozygosity mapping and targeted high-throughput sequencing. Am J Hum Genet 2010;87:532–537.
- Kalay E, Yigit G, Aslan Y, Brown KE, Pohl E, Bicknell LS, Kayserili H, Li Y, Tüysüz B, Nürnberg G, Kiess W, Koegl M, Baessmann I, Buruk K, Toraman B, Kayipmaz S, Kul S, Ikbal M, Turner DJ, Taylor MS, Aerts J, Scott C, Milstein K, Dollfus H, Wieczorek D, Brunner HG, Hurles M, Jackson AP, Rauch A, Nürnberg P, Karagüzel A, Wollnik B: CEP152 is a genome maintenance protein disrupted in Seckel syndrome. Nat Genet 2011;43:23–26.
- Becker J, Semler O, Gilissen C, Li Y, Bolz HJ, Giunta C, Bergmann C, Rohrbach M, Koerber F, Zimmermann K, de Vries P, Wirth B, Schoenau E, Wollnik B, Veltman JA, Hoischen A, Netzer C: Exome sequencing identifies truncating mutations in human SERPINF1 in autosomal-recessive osteogenesis imperfecta. Am J Hum Genet 2011;88:362–371.
- Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C, Garimella KV, Fisher S, Abreu J, Barry AJ, Fennell T, Banks E, Ambrogio L, Cibulskis K, Kernytsky A, Gonzalez E, Rudzicz N, Engert JC, DePristo MA, Daly MJ, Cohen JC, Hobbs HH, Altshuler D, Schonfeld G, Gabriel SB, Yue P, Kathiresan S: Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 2010;363:2220.
- Krawitz PM, Schweiger MR, Rödelsperger C, Marcelis C, Kölsch U, Meisel C, Stephani F, Kinoshita T, Murakami Y, Bauer S, Isau M, Fischer A, Dahl A, Kerick M, Hecht J, Köhler S, Jäger M, Grünhagen J, de Condor BJ, Doelken S, Brunner HG, Meinecke P, Passarge E, Thompson MD, Cole DE, Horn D, Roscioli T, Mundlos S, Robinson PN: Identity-by-descent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome. Nat Genet 2010;42:827–829.
- Hoischen A, van Bon BW, Gilissen C, Arts P, van Lier B, Steehouwer M, de Vries P, de Reuver R, Wieskamp N, Mortier G, Devriendt K, Amorim MZ, Revencu N, Kidd A, Barbosa M, Turner A, Smith J, Oley C, Henderson A, Hayes IM, Thompson EM, Brunner HG, de Vries BB, Veltman JA: De novo mutations of SETBP1 cause Schinzel-Giedion syndrome. Nat Genet 2010;42:483–485.
- Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA, Shendure J, Bamshad MJ: Exome sequencing identifies the cause of a Mendelian disorder. Nat Genet 2010;42:30–35.
- Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloğlu A, Ozen S, Sanjad S, Nelson-Williams C, Farhi A, Mane S, Lifton RP: Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci 2009;106:19096–19101.
- Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, Bamshad M, Nickerson DA, Shendure J: Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009;10:272–276.
- Garone C, Pippucci T, Cordelli DM, Zuntini R, Castegnaro G, Marconi C, Graziano C, Marchiani V, Verrotti A, Seri M, Franzoni E: FA2H-related disorders: a novel c.270+3A>T splice-site mutation leads to a complex neurodegenerative phenotype. Dev Med Child Neurol 2011, E-pub ahead of print.
- Li H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 2010;26:589–595.
- McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010;20:1297–1303.
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup: The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009;25:2078–2079.
- Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: a tool set for whole-genome association and population based linkage analyses. Am J Hum Genet 2007;81:559–575.
- McQuillan R, Leutenegger AL, Abdel-Rahman R, Franklin CS, Pericic M, Barac-Lauc L, Smolej-Narancic N, Janicijevic B, Polasek O, Tenesa A, Macleod AK, Farrington SM, Rudan P, Hayward C, Vitart V, Rudan I, Wild SH, Dunlop MG, Wright AF, Campbell H, Wilson JF: Runs of homozygosity in European populations. Am J Hum Genet 2008;83:359–372.
- Woods CG, Cox J, Springell K, Hampshire DJ, Mohamed MD, McKibbin M, Stern R, Raymond FL, Sandford R, Malik Sharif S, Karbani G, Ahmed M, Bond J, Clayton D, Inglehearn CF: Quantification of homozygosity in consanguineous individuals with autosomal recessive disease. Am J Hum Genet 2006;78:889–896.
- Abecasis GR, Cherny SS, Cookson WO, Cardon LR: Merlin – rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 2002;30:97–101.
- Thusberg J, Olatubosun A, Vihinen M: Performance of mutation pathogenicity prediction methods on missense variants. Hum Mutat 2011;32:358–368.
- Li B, Krishnan VG, Mort ME, Xin F, Kamati KK, Cooper DN, Mooney SD, Radivojac P: Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 2009;25:2744–2750.
- Ramensky V, Bork P, Sunyaev S: Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002;30:3894–3900.
- Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR: A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–249.
- Kumar P, Henikoff S, Ng PC: Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009;4:1073–1081.
- Calabrese R, Capriotti E, Fariselli P, Martelli PL, Casadio R: Functional annotations improve the predictive score of human disease-related mutations in proteins. Hum Mutat 2009;30:1237–1244.
- Brunak S, Engelbrecht J, Knudsen S: Prediction of human mRNA donor and acceptor sites from the DNA sequence. J Mol Biol 1991;220:49–65.
- Roux-Rouquie M, Chauvet ML, Munnich A, Frezal J: Human genes involved in chromatin remodeling in transcription initiation, and associated diseases: an overview using the GENATLAS database. Mol Genet Metab 1999;67:261–277.
- Berglund L, Björling E, Oksvold P, Fagerberg L, Asplund A, Szigyarto CA, Persson A, Ottosson J, Wernérus H, Nilsson P, Lundberg E, Sivertsson A, Navani S, Wester K, Kampf C, Hober S, Pontén F, Uhlén M: A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol Cell Proteomics 2008;7:2019–2027.
- Niki T, Takahashi-Niki K, Taira T, Iguchi-Ariga SM, Ariga H: DJBP: a novel DJ-1-binding protein, negatively regulates the androgen receptor by recruiting histone deacetylase complex, and DJ-1 antagonizes this inhibition by abrogation of this complex. Mol Cancer Res 2003;1:247–261.
- Eckhardt M, Yaghootfam A, Fewou SN, Zöller I, Gieselmann V: A mammalian fatty acid hydroxylase responsible for the formation of alpha-hydroxylated galactosylceramide in myelin. Biochem J 2003;388:245–254.
- Zöller I, Meixner M, Hartmann D, Büssow H, Meyer R, Gieselmann V, Eckhardt M: Absence of 2-hydroxylated sphingolipids is compatible with normal neural development but causes late-onset axon and myelin sheath degeneration. J Neurosci 2008;24:9741–9754.
- Walsh T, Shahin H, Elkan-Miller T, Lee MK, Thornton AM, Roeb W, Abu Rayyan A, Loulus S, Avraham KB, King MC, Kanaan M: Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 2010;87:90–94.
- Sirmaci A, Walsh T, Akay H, Spiliopoulos M, Sakalar YB, Hasanefendioğlu-Bayrak A, Duman D, Farooq A, King MC, Tekin M: MASP1 mutations in patients with facial, umbilical, coccygeal, and auditory findings of Carnevale, Malpuech, OSA, and Michels syndromes. Am J Hum Genet 2010;87:679–686.
- Pierce SB, Walsh T, Chisholm KM, Lee MK, Thornton AM, Fiumara A, Opitz JM, Levy-Lahad E, Klevit RE, King MC: Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dysgenesis, hearing loss, and ataxia of Perrault Syndrome. Am J Hum Genet 2010;87:282–288.
- Gilissen C, Arts HH, Hoischen A, Spruijt L, Mans DA, Arts P, van Lier B, Steehouwer M, van Reeuwijk J, Kant SG, Roepman R, Knoers NV, Veltman JA, Brunner HG: Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 2010;87:418–423.
Medical Genetics Unit, Department of Gynecological, Obstetric and Pediatric Sciences
University of Bologna
IT–40138 Bologna (Italy)
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Received: January 24, 2011
Accepted after revision: June 3, 2011
Published online: August 18, 2011
Number of Print Pages : 9
Number of Figures : 3, Number of Tables : 2, Number of References : 35
Additional supplementary material is available online - Number of Parts : 1
Human Heredity (International Journal of Human and Medical Genetics)
Vol. 72, No. 1, Year 2011 (Cover Date: September 2011)
Journal Editor: Devoto M. (Philadelphia, Pa./Rome)
ISSN: 0001-5652 (Print), eISSN: 1423-0062 (Online)
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