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. 2007 Dec 27;29(2):306–314. doi: 10.1002/humu.20622

Construction of a multiplex allele‐specific PCR‐based universal array (ASPUA) and its application to hearing loss screening†

Cai‐Xia Li 1,‡, Qian Pan 2,§, Yong‐Gang Guo 1, Yan Li 3, Hua‐Fang Gao 3, Di Zhang 3, Hao Hu 2, Wan‐Li Xing 1,4,5, Keith Mitchelson 1,3,5, Kun Xia 2, Pu Dai 6,✉, Jing Cheng 1,4,5,✉
PMCID: PMC7165727  PMID: 18161878

Abstract

We demonstrate a new method, using a universal array approach termed multiplex allele‐specific PCR‐based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele‐specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele‐specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin‐coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost‐effective, and easily‐used, and is especially appropriate for mutation detection in clinical genetic diagnostics. Hum Mutat 29(2), 306–314, 2008. © 2007 Wiley‐Liss, Inc.

Keywords: hereditary hearing loss, mutation detection, universal array, allele‐specific PCR

Supporting information

The Supplementary Material referred to in this article can be accessed at http://www.interscience.wiley.com/jpages/1059-7794/suppmat .

Supporting Information file humu20622‐FINALSupp_Mat_humu‐2007‐0097_07_10.pdf

Click here for additional data file. (86KB, pdf)
†

Communicated by Ann‐Christine Syvänen

Contributor Information

Pu Dai, Email: daipu301@yahoo.com.

Jing Cheng, Email: jcheng@tsinghua.edu.cn.

REFERENCES

  1. Abe S, Usami S, Shinkawa H, Kelley PM, Kimberling WJ. 2000. Prevalent connexin 26 gene (GJB2) mutations in Japanese. J Med Genet 37:41–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. American College of Medical Genetics (ACMG) . 2002. Genetics evaluation guidelines for the etiologic diagnosis of congenital hearing loss. Genetic evaluation of congenital hearing loss expert panel. ACMG statement. Genet Med 4:162–171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bai RK, Wong LJ. 2004. Detection and quantification of heteroplasmic mutant mitochondrial DNA by real‐time amplification refractory mutation system quantitative PCR analysis: a single‐step approach. Clin Chem 50:996–1001. [DOI] [PubMed] [Google Scholar]
  4. Campbell C, Cucci RA, Prasad S, Green GE, Edeal JB, Galer CE, Karniski LP, Sheffield VC, Smith RJ. 2001. Pendred syndrome, DFNB4, and PDS/SLC26A4: identification of eight novel mutations and possible genotype–phenotype correlations. Hum Mutat 17:403–411. [DOI] [PubMed] [Google Scholar]
  5. Chen J, Iannone MA, Li MS, Taylor JD, Rivers P, Nelsen AJ, Slentz‐Kesler KA, Roses A, Weiner MP. 2000. A microsphere‐based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension. Genome Res 10:549–557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cowie S, Drmanac S, Swanson D, Delgrosso K, Huang S, du Sart D, Drmanac R, Surrey S, Fortina P. 2004. Identification of APC gene mutations in colorectal cancer using universal microarray‐based combinatorial sequencing‐by‐hybridization. Hum Mutat 24:261–271. [DOI] [PubMed] [Google Scholar]
  7. Cremers FP, Kimberling WJ, Kulm M, de Brouwer A, van Wijk E, Te Brinke H, Cremers CW, Hoefsloot LH, Banfi S, Simonelli F, Fleischhauer JC, Berger W, Kelley PM, Haralambous E, Bitner‐Glindzicz M, Webster AR, Saihan Z, De Baere E, Leroy BP, Silvestri G, McKay G, Koenekoop RK, Millan JM, Rosenberg T, Joensuu T, Sankila EM, Weil D, Weston MD, Wissinger B, Kremer H. 2007. Development of a genotyping microarray for Usher syndrome. J Med Genet 44:153–160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dai P, Han DY, Feng B, Kang DY, Liu X, Yuan HJ, Cao JY, Zhang X, Zhai SQ, Yang WY, Wu BL. 2006. Genetic testing for the enlarged vestibular aqueduct syndrome and mutation analysis of the SLC26A4 gene. Chin Arch Otolaryngol Head Neck Surg 13:303–307. [Google Scholar]
  9. Du H, Wu M, Yang W, Yuan G, Sun Y, Lu Y, Zhao S, Du Q, Wang F, Yan S, Pan M, Lu Y, Wang S, Cheng J. 2005. Development of miniaturized competitive immunoassays on a protein chip as a screening tool for drugs. Clin Chem 51:368–375. [DOI] [PubMed] [Google Scholar]
  10. Estivill X, Fortina P, Surrey S, Rabionet R, Melchionda S, D'Agruma L, Mansfield E, Rappaport E, Govea N, Milà M , Zelante L, Gasparini P. 1998. Connexin‐26 mutations in sporadic and inherited sensorineural deafness. Lancet 351:394–398. [DOI] [PubMed] [Google Scholar]
  11. Ferraris A, Rappaport E, Santacroce R, Pollak E, Krantz I, Toth S, Lysholm F, Margaglione M, Restagno G, Dallapiccola B, Surrey S, Fortina P. 2002. Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss. Hum Mutat 20:312–320. [DOI] [PubMed] [Google Scholar]
  12. Friedman TB, Griffith AJ. 2003. Human nonsyndromic sensorineural deafness. Annu Rev Genomics Hum Genet 4:341–402. [DOI] [PubMed] [Google Scholar]
  13. Gardner P, Oitmaa E, Messner A, Hoefsloot L, Metspalu A, Schrijver I. 2006. Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow‐up. Pediatrics 118:985–994. [DOI] [PubMed] [Google Scholar]
  14. Gerry NP, Witowski NE, Day J, Hammer RP, Barany G, Barany F. 1999. Universal DNA microarray method for multiplex detection of low abundance point mutations. J Mol Biol 292:251–262. [DOI] [PubMed] [Google Scholar]
  15. Gómez‐Llorente C, Antúnez A, Blanco S, Suarez A, Gómez‐Capilla JA, Farez‐Vidal ME. 2004. Multiplex analysis of the most common mutations related to hereditary haemochromatosis: two methods combining specific amplification with capillary electrophoresis. Eur J Haematol 72:121–129. [DOI] [PubMed] [Google Scholar]
  16. Guo Y, Guo H, Zhang L, Xie H, Zhao X, Wang F, Li Z, Wang Y, Ma S, Tao J, Wang W, Zhou Y, Yang W, Cheng J. 2005. Genomic analysis of anti‐HBV activity by siRNA and lamivudine in stable HBV‐producing cell. J Virol 79:14392–14403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hardenbol P, Banér J, Jain M, Nilsson M, Namsaraev EA, Kailin‐Neumann GA, Fakhrai‐Rad H, Ronaghi M, Willis TD, Landegren U, Davis RW. 2003. Multiplexed genotyping with sequence‐tagged molecular inversion probes. Nat Biotechnol 21:673–678. [DOI] [PubMed] [Google Scholar]
  18. Hurd CM, Cavanagh G, Schuh A, Ouwehand WH, Metcalfe P. 2002. Genotyping for platelet‐specific antigens: techniques for the detection of single nucleotide polymorphisms. Vox Sang 83:1–12. [DOI] [PubMed] [Google Scholar]
  19. Kong X, Xu Y, Wu X, Liang Y, Wang C, Guo J, Wang Y, Chen M, Wu D, Wang Y, Bi S, Qiu Y, Lu P, Cheng J, Xiao B, Hu L, Gao X, Liu J, Wang Y, Song Y, Zhang L, Suo F, Chen T, Huang Z, Zhao Y, Lu H, Pan C, Tang H. 2005. Proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome. Clin Chem 51:56–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin D, Goldstein JA, Mhatre AN, Lustig LR, Pfister M, Lalwani AK. 2001. Assessment of denaturing high‐performance liquid chromatography (DHPLC) in screening for mutations in connexin 26 (GJB2). Hum Mutat 18:42–51. [DOI] [PubMed] [Google Scholar]
  21. Liu X, Dai P, Huang DL, Yuan HJ, Li WM, Cao JY, Yu F, Zhang RN, Lin HY, Zhu HX, He Y, Yu YJ, Yao K. 2006. [Large scale screening of mtDNA A1555G mutation in China and its significance in prevention of aminoglycoside antibiotic induced deafness]. Zhonghua Yi Xue Za Zhi [Natl Med J China] 86:1318–1322. [Chinese] [PubMed] [Google Scholar]
  22. Lo YMD, Patel P, Newton CR, Markham AF, Fleming KA, Wainscoat JS. 1991. Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications. Nucleic Acids Res 19:3561–3567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morton NE. 1991. Genetic epidemiology of hearing impairment. Ann NY Acad Sci 630:16–31. [DOI] [PubMed] [Google Scholar]
  24. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. 1989. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503–2516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen L, Syvänen AC. 2000. A system for specific, high‐throughput genotyping by allele‐specific primer extension on microarrays. Genome Res 10:1031–1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Prezant TR, Agapian JV, Bohlman MC, Bu XD, Öztas S, Qiu WQ, Arnos KS, Cortopassi GA, Jaber L, Rotter JI, Shohat M, Fischel‐Ghodsian N. 1993. Mitochondrial ribosomal RNA mutation associated with both antibiotic‐induced and non‐syndromic deafness. Nat Genet 4:289–294. [DOI] [PubMed] [Google Scholar]
  27. Rabionet R, Gasparini P, Estivill X. 2000. Molecular genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins. Hum Mutat 16:190–202. [DOI] [PubMed] [Google Scholar]
  28. Roberts R, Sullivan P, Joyce P, Kennedy MA. 2000. Rapid and comprehensive determination of cytochrome P450 CYP2D6 poor metabolizer genotypes by multiplex polymerase chain reaction. Hum Mutat 16:77–85. [DOI] [PubMed] [Google Scholar]
  29. Siemering K, Manji SS, Hutchison WM, Du Sart D, Phelan D, Dahl HH. 2006. Detection of mutations in genes associated with hearing loss using a microarray‐based approach. J Mol Diagn 8:483–489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sobe T, Vreugde S, Shahin H, Berlin M, Davis N, Kanaan M, Yaron Y, Orr‐Urtreger A, Frydman M, Shohat M, Avraham KB. 2000. The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli population. Hum Genet 106:50–57. [DOI] [PubMed] [Google Scholar]
  31. Taton TA, Mirkin CA, Letsinger RL. 2000. Scanometric DNA array detection with nanoparticle probes. Science 289:1757–1760. [DOI] [PubMed] [Google Scholar]
  32. Tsukamoto K, Suzuki H, Harada D, Namba A, Abe S, Usami S. 2003. Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese. Eur J Hum Genet 11:916–922. [DOI] [PubMed] [Google Scholar]
  33. Wang YC, Kung CY, Su MC, Su CC, Hsu HM, Tsai CC, Lin CC, Li SY. 2002. Mutations of Cx26 gene (GJB2) for prelingual deafness in Taiwan. Eur J Hum Genet 10:495–498. [DOI] [PubMed] [Google Scholar]
  34. Xia JH, Liu CY, Tang BS, Pan Q, Huang L, Dai HP, Zhang BR, Xie W, Hu DX, Zheng D, Shi XL, Wang DA, Xia K, Yu KP, Liao XD, Feng Y, Yang YF, Xiao JY, Xie DH, Huang JZ. 1998. Mutations in the gene encoding gap junction protein β‐3 associated with autosomal dominant hearing impairment. Nat Genet 20:370–373. [DOI] [PubMed] [Google Scholar]
  35. Zhang ZW, Zhou YM, Zhang Y, Guo Y, Tao SC, Li Z, Zhang Q, Cheng J. 2005. Sensitive detection of SARS coronavirus RNA by a novel asymmetric multiplex nested RT‐PCR amplification coupled with oligonucleotide microarray hybridization. Methods Mol Med 114:59–78. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

The Supplementary Material referred to in this article can be accessed at http://www.interscience.wiley.com/jpages/1059-7794/suppmat .

Supporting Information file humu20622‐FINALSupp_Mat_humu‐2007‐0097_07_10.pdf

Click here for additional data file. (86KB, pdf)

Articles from Human Mutation are provided here courtesy of Wiley

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