Use of Linkage Analysis, Genome-Wide Association Studies, and Next-Generation Sequencing in the Identification of Disease-Causing Mutations

  • Eric Londin
  • Priyanka Yadav
  • Saul Surrey
  • Larry J. Kricka
  • Paolo Fortina
Part of the Methods in Molecular Biology book series (MIMB, volume 1015)


For the past two decades, linkage analysis and genome-wide analysis have greatly advanced our knowledge of the human genome. But despite these successes the genetic architecture of diseases remains unknown. More recently, the availability of next-generation sequencing has dramatically increased our capability for determining DNA sequences that range from large portions of one individual’s genome to targeted regions of many genomes in a cohort of interest. In this review, we highlight the successes and shortcomings that have been achieved using genome-wide association studies (GWAS) to identify the variants contributing to disease. We further review the methods and use of new technologies, based on next-generation sequencing, that are becoming increasingly used to expand our knowledge of the causes of genetic disease.

Key words

Linkage analysis Genome-wide association study Massively parallel sequencing NGS-applications Pharmacogenomics 



This work was supported the Kimmel Cancer Center and the Computational Medicine Center at Thomas Jefferson University Jefferson Medical College.


  1. 1.
    Hindorff LA et al (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci USA 106:9362–9367PubMedCrossRefGoogle Scholar
  2. 2.
    Rommens JM et al (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059–1065PubMedCrossRefGoogle Scholar
  3. 3.
    Riordan JR et al (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066–1073PubMedCrossRefGoogle Scholar
  4. 4.
    Kerem B et al (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073–1080PubMedCrossRefGoogle Scholar
  5. 5.
    Risch N, Merikangas K (1996) The future of genetic studies of complex human diseases. Science 273:1516–1517PubMedCrossRefGoogle Scholar
  6. 6.
    Altmuller J et al (2001) Genomewide scans of complex human diseases: true linkage is hard to find. Am J Hum Genet 69:936–950PubMedCrossRefGoogle Scholar
  7. 7.
    Consortium IH (2005) A haplotype map of the human genome. Nature 437:1299–1320CrossRefGoogle Scholar
  8. 8.
    Manolio TA et al (2009) Finding the missing heritability of complex diseases. Nature 461:747–753PubMedCrossRefGoogle Scholar
  9. 9.
    Klein RJ et al (2005) Complement factor H polymorphism in age-related macular degeneration. Science 308:385–389PubMedCrossRefGoogle Scholar
  10. 10.
    Barrett JC et al (2008) Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 40:955–962PubMedCrossRefGoogle Scholar
  11. 11.
    Lango AH et al (2010) Hundreds of variants clustered in genomic loci and biological pathways affect human height. Nature 467:832–838CrossRefGoogle Scholar
  12. 12.
    Maller J et al (2006) Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration. Nat Genet 38:1055–1059PubMedCrossRefGoogle Scholar
  13. 13.
    Jakobsdottir J et al (2009) Interpretation of genetic association studies: markers with replicated highly significant odds ratios may be poor classifiers. PLoS Genet 5:e1000337PubMedCrossRefGoogle Scholar
  14. 14.
    Rose SP (2006) Commentary: heritability estimates–long past their sell-by date. Int J Epidemiol 35:525–527PubMedCrossRefGoogle Scholar
  15. 15.
    Genomes Project Consortium, Abecasis GR et al (2010) A map of human genome variation from population-scale sequencing. Nature 467:1061–1073PubMedCrossRefGoogle Scholar
  16. 16.
    Venter JC et al (2001) The sequence of the human genome. Science 291:1304–1351PubMedCrossRefGoogle Scholar
  17. 17.
    Lander ES et al (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921PubMedCrossRefGoogle Scholar
  18. 18.
    Schloss JA (2008) How to get genomes at one ten-thousandth the cost. Nat Biotechnol 26:1113–1115PubMedCrossRefGoogle Scholar
  19. 19.
    Hert DG, Fredlake CP, Barron AE (2008) Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non-electrophoresis methods. Electrophoresis 29:4618–4626PubMedCrossRefGoogle Scholar
  20. 20.
    Pareek CS, Smoczynski R, Tretyn A (2011) Sequencing technologies and genome sequencing. J Appl Genet 52:413–435PubMedCrossRefGoogle Scholar
  21. 21.
    Metzker ML (2010) Sequencing technologies—the next generation. Nat Rev Genet 11:31–46PubMedCrossRefGoogle Scholar
  22. 22.
    Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402PubMedCrossRefGoogle Scholar
  23. 23.
    Zheng Z et al (2010) Titration-free massively parallel pyrosequencing using trace amounts of starting material. Nucleic Acids Res 38:e137PubMedCrossRefGoogle Scholar
  24. 24.
    Margulies M et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380PubMedGoogle Scholar
  25. 25.
    Depristo MA et al (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491–498PubMedCrossRefGoogle Scholar
  26. 26.
    Ng SB et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272–276PubMedCrossRefGoogle Scholar
  27. 27.
    Ng SB et al (2009) Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 42:30–35PubMedCrossRefGoogle Scholar
  28. 28.
    Hoischen A et al (2010) De novo mutations of SETBP1 cause Schinzel-Giedion syndrome. Nat Genet 42:483–485PubMedCrossRefGoogle Scholar
  29. 29.
    Bansal V et al (2010) Statistical analysis strategies for association studies involving rare variants. Nat Rev Genet 11:773–785PubMedCrossRefGoogle Scholar
  30. 30.
    Nalls MA et al (2011) Imputation of sequence variants for identification of genetic risks for Parkinson’s disease: a meta-analysis of genome-wide association studies. Lancet 377:641–649PubMedCrossRefGoogle Scholar
  31. 31.
    Su Z et al (2011) Next-generation sequencing and its applications in molecular diagnostics. Expert Rev Mol Diagn 11:333–343PubMedGoogle Scholar
  32. 32.
    Marian AJ (2011) Medical DNA sequencing. Curr Opin Cardiol 26:175–180PubMedCrossRefGoogle Scholar
  33. 33.
    Diamandis EP (2009) Next-generation sequencing: a new revolution in molecular diagnostics? Clin Chem 55:2088–2092PubMedCrossRefGoogle Scholar
  34. 34.
    Choi M et al (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA 106:19096–19101PubMedCrossRefGoogle Scholar
  35. 35.
    Maxmen A (2011) Exome sequencing deciphers rare diseases. Cell 144:635–637PubMedCrossRefGoogle Scholar
  36. 36.
    St HC et al (2011) NT5E mutations and arterial calcifications. N Engl J Med 364:432–442CrossRefGoogle Scholar
  37. 37.
    Daly AK (2010) Genome-wide association studies in pharmacogenomics. Nat Rev Genet 11:241–246PubMedCrossRefGoogle Scholar
  38. 38.
    Dewey FE et al (2011) Phased whole-genome genetic risk in a family quartet using a major allele reference sequence. PLoS Genet 7:e1002280PubMedCrossRefGoogle Scholar
  39. 39.
    Trapnell C, Salzberg SL (2009) How to map billions of short reads onto genomes. Nat Biotechnol 27:455–457PubMedCrossRefGoogle Scholar
  40. 40.
    Nielsen R et al (2011) Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet 12:443–451PubMedCrossRefGoogle Scholar
  41. 41.
    Hinchcliffe M, Webster P (2011) In silico analysis of the exome for gene discovery. Methods Mol Biol 760:109–128PubMedCrossRefGoogle Scholar
  42. 42.
    Blaby-Haas CE, de Crecy-Lagard V (2011) Mining high-throughput experimental data to link gene and function. Trends Biotechnol 29:174–182PubMedCrossRefGoogle Scholar
  43. 43.
    Nothnagel M et al (2011) Technology-specific error signatures in the 1000 Genomes Project data. Hum Genet 130:505–516PubMedCrossRefGoogle Scholar
  44. 44.
    Al Badr W et al (2011) Exome capture and massively parallel sequencing identifies a novel HPSE2 mutation in a Saudi Arabian child with Ochoa (urofacial) syndrome. J Pediatr Urol 7:569–573PubMedCrossRefGoogle Scholar
  45. 45.
    Alvarado DM et al (2011) Exome sequencing identifies an MYH3 mutation in a family with distal arthrogryposis type 1. J Bone Joint Surg Am 93:1045–1050PubMedCrossRefGoogle Scholar
  46. 46.
    Barak T et al (2011) Recessive LAMC3 mutations cause malformations of occipital cortical development. Nat Genet 43:590–594PubMedCrossRefGoogle Scholar
  47. 47.
    Becker J et al (2011) Exome sequencing identifies truncating mutations in human SERPINF1 in autosomal-recessive osteogenesis imperfecta. Am J Hum Genet 88:362–371PubMedCrossRefGoogle Scholar
  48. 48.
    Bolze A et al (2010) Whole-exome-sequencing-based discovery of human FADD deficiency. Am J Hum Genet 87:873–881PubMedCrossRefGoogle Scholar
  49. 49.
    Caliskan M et al (2011) Exome sequencing reveals a novel mutation for autosomal recessive non-syndromic mental retardation in the TECR gene on chromosome 19p13. Hum Mol Genet 20:1285–1289PubMedCrossRefGoogle Scholar
  50. 50.
    de Greef JC et al (2011) Mutations in ZBTB24 are associated with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2. Am J Hum Genet 88:796–804PubMedCrossRefGoogle Scholar
  51. 51.
    Erlich Y et al (2011) Exome sequencing and disease-network analysis of a single family implicate a mutation in KIF1A in hereditary spastic paraparesis. Genome Res 21:658–664PubMedCrossRefGoogle Scholar
  52. 52.
    Gilissen C et al (2010) Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 87:418–423PubMedCrossRefGoogle Scholar
  53. 53.
    Glazov EA et al (2011) Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia. PLoS Genet 7:e1002027PubMedCrossRefGoogle Scholar
  54. 54.
    Gotz A et al (2011) Exome sequencing identifies mitochondrial alanyl-tRNA synthetase mutations in infantile mitochondrial cardiomyopathy. Am J Hum Genet 88:635–642PubMedCrossRefGoogle Scholar
  55. 55.
    Greif PA et al (2011) Somatic mutations in acute promyelocytic leukemia (APL) identified by exome sequencing. Leukemia 25:1519–1522PubMedCrossRefGoogle Scholar
  56. 56.
    Johnson JO et al (2010) Exome sequencing reveals VCP mutations as a cause of familial ALS. Neuron 68:857–864PubMedCrossRefGoogle Scholar
  57. 57.
    Li Y et al (2010) Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants. Nat Genet 42:969–972PubMedCrossRefGoogle Scholar
  58. 58.
    Liu Y et al (2011) Confirmation by exome sequencing of the pathogenic role of NCSTN mutations in acne inversa (hidradenitis suppurativa). J Invest Dermatol 131:1570–1572PubMedCrossRefGoogle Scholar
  59. 59.
    Mondal K et al (2011) Targeted sequencing of the human X chromosome exome. Genomics 98:260–265PubMedCrossRefGoogle Scholar
  60. 60.
    Montenegro G et al (2011) Exome sequencing allows for rapid gene identification in a Charcot-Marie-Tooth family. Ann Neurol 69:464–470PubMedCrossRefGoogle Scholar
  61. 61.
    Ng SB et al (2010) Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 42:790–793PubMedCrossRefGoogle Scholar
  62. 62.
    O’Roak BJ et al (2011) Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet 43:585–589PubMedCrossRefGoogle Scholar
  63. 63.
    O’Sullivan J et al (2011) Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival hyperplasia syndrome. Am J Hum Genet 88:616–620PubMedCrossRefGoogle Scholar
  64. 64.
    Ostergaard P et al (2011) Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype. J Med Genet 48:251–255PubMedCrossRefGoogle Scholar
  65. 65.
    Puente XS et al (2011) Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome. Am J Hum Genet 88:650–656PubMedCrossRefGoogle Scholar
  66. 66.
    Rios J et al (2010) Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia. Hum Mol Genet 19:4313–4318PubMedCrossRefGoogle Scholar
  67. 67.
    Saarinen S et al (2011) Exome sequencing reveals germline NPAT mutation as a candidate risk factor for Hodgkin lymphoma. Blood 118:493–498PubMedCrossRefGoogle Scholar
  68. 68.
    Simpson MA et al (2011) Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. Am J Hum Genet 43:303–305Google Scholar
  69. 69.
    Snape K et al (2011) Mutations in CEP57 cause mosaic variegated aneuploidy syndrome. Am J Hum Genet 43:527–529Google Scholar
  70. 70.
    Szperl AM et al (2011) Exome sequencing in a family segregating for celiac disease. Clin Genet 80:138–147PubMedCrossRefGoogle Scholar
  71. 71.
    Sundaram SK et al (2011) Exome sequencing of a pedigree with tourette syndrome or chronic tic disorder. Ann Neurol 69:901–904PubMedCrossRefGoogle Scholar
  72. 72.
    Timmermann B et al (2010) Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis. PLoS One 5:e15661PubMedCrossRefGoogle Scholar
  73. 73.
    Tsurusaki Y et al (2011) Exome sequencing of two patients in a family with atypical X-linked leukodystrophy. Clin Genet 80:161–166PubMedCrossRefGoogle Scholar
  74. 74.
    Vissers LE et al (2010) A de novo paradigm for mental retardation. Am J Hum Genet 42:1109–1112Google Scholar
  75. 75.
    Vissers LE et al (2011) Chondrodysplasia and abnormal joint development associated with mutations in IMPAD1, encoding the Golgi-resident nucleotide phosphatase, gPAPP. Am J Hum Genet 88:608–615PubMedCrossRefGoogle Scholar
  76. 76.
    Wei X et al (2011) Exome sequencing identifies GRIN2A as frequently mutated in melanoma. Nat Genet 43:442–446PubMedCrossRefGoogle Scholar
  77. 77.
    Worthey EA et al (2011) Making a definitive diagnosis: successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease. Genet Med 13:255–262PubMedCrossRefGoogle Scholar
  78. 78.
    Yamaguchi T et al (2011) Exome resequencing combined with linkage analysis identifies novel PTH1R variants in primary failure of tooth eruption in Japanese. J Bone Miner Res 26(7):1655–1661PubMedCrossRefGoogle Scholar
  79. 79.
    Zhou C et al (2011) Mutation in ribosomal protein L21 underlies hereditary hypotrichosis simplex. Hum Mutat 32:710–714PubMedCrossRefGoogle Scholar
  80. 80.
    Zuchner S et al (2011) Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa. Am J Hum Genet 88:201–206PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Eric Londin
    • 1
  • Priyanka Yadav
    • 2
  • Saul Surrey
    • 3
  • Larry J. Kricka
    • 4
  • Paolo Fortina
    • 5
  1. 1.Computational Medicine CenterThomas Jefferson University Jefferson Medical CollegePhiladelphiaUSA
  2. 2.Cancer Genomics Laboratory, Kimmel Cancer CenterThomas Jefferson University, Jefferson Medical CollegePhiladelphiaUSA
  3. 3.Department of MedicineThomas Jefferson University, Jefferson Medical CollegePhiladelphiaUSA
  4. 4.Department of Pathology and Laboratory MedicineUniversity of Pennsylvania School of MedicinePhiladelphiaUSA
  5. 5.Cancer Genomics Laboratory, Kimmel Cancer Center, Department of Cancer BiologyThomas Jefferson University, Jefferson Medical CollegePhiladelphiaUSA

Personalised recommendations