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Molecular Diagnosis & Therapy

, Volume 22, Issue 3, pp 353–359 | Cite as

A Study of Triplet-Primed PCR for Identification of CAG Repeat Expansion in the HTT Gene in a Cohort of 503 Indian Cases with Huntington’s Disease Symptoms

  • Pratiksha Chheda
  • Milind Chanekar
  • Yogita Salunkhe
  • Tavisha Dama
  • Anurita Pais
  • Shailesh Pande
  • Rajesh Bendre
  • Nilesh Shah
short communication

Abstract

Background

Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder with an average age at onset of 40 years. It is a polyglutamine (polyQ) disorder that is caused by an increase in the number of CAG repeats in the huntingtin (HTT) gene. Genetic tests that accurately determine the number of CAG repeats are performed for confirmation of diagnosis, predictive testing of persons at genetic risk for inheriting HD, and prenatal testing. The aim of our study was to evaluate efficacy of triplet-primed polymerase chain reaction (TP-PCR) for routine diagnosis of HD in suspected cases from India.

Methods

We evaluated a combination of CAG flanking PCR and triplet-primed PCR for estimation of CAG repeats in 503 cases with clinical suspicion of HD.

Results

There were 250 cases (49.7%) that showed the presence of expanded alleles, with 241 (47.9%) being fully penetrant alleles and nine (1.8%) in the reduced penetrance category. There were seven juvenile cases with an age of onset of < 20 years, with the longest allele comprising 106 CAG repeats found in an 8-year-old male patient. The results demonstrated an inverse (R = − 0.67) relationship between CAG length and age at clinical onset.

Conclusion

Our study on pan-Indian cases is one of the largest studies reported so far in India and focuses on the most accurate and comprehensive molecular diagnostic evaluation of HD.

Notes

Compliance with Ethical Standards

Conflict of interest

All the authors were employed by Metropolis Healthcare Ltd. PC, MC, YS, TD, AP, SP, RB and NS have no conflicts of interest to disclose.

Funding

No funding supported this study.

Ethics approval and consent to participate

The study procedures were approved by Conscience Independent Ethics Committee (CIEC). Informed consent was obtained from all participating individuals.

References

  1. 1.
    Margolis RL, Ross CA. Diagnosis of Huntington disease. Clin Chem. 2003;49:1726–32.CrossRefPubMedGoogle Scholar
  2. 2.
    Moily NS, Kota LN, Anjanappa RM, Venugopal S, Vaidyanathan R, Pal P, et al. Trinucleotide repeats and haplotypes at the huntingtin locus in an Indian sample overlaps with European haplogroup a. PLoS Curr. 2014.  https://doi.org/10.1371/currents.hd.a3ad1a381ab1eed117675145318c9a80.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Squitieri F, Gellera C, Cannella M, Mariotti C, Cislaghi G, Rubinsztein DC, et al. Homozygosity for CAG mutation in Huntington disease is associated with a more severe clinical course. Brain. 2003.  https://doi.org/10.1093/brain/awg077946-955F.PubMedGoogle Scholar
  4. 4.
    Govert F, Schneider SA. Huntington’s disease and Huntington’s disease-like syndromes: an overview. Curr Opin Neurol. 2013;4:420–7.  https://doi.org/10.1097/WCO.0b013e3283632d90.CrossRefGoogle Scholar
  5. 5.
    Schneide SA, Walker RH, Bhatia KP. The Huntington’s disease-like syndromes: what to consider in patients with a negative Huntington’s disease gene test. Nat Clin Pract Neurol. 2007;3:517–25.  https://doi.org/10.1038/ncpneuro0606.CrossRefGoogle Scholar
  6. 6.
    Bean L, Bayrak-Toydemir P. American College of Medical Genetics and Genomics Standards and Guidelines for Clinical Genetics Laboratories, 2014 edition: technical standards and guidelines for Huntington disease. Genet Med. 2014;16:e2.  https://doi.org/10.1038/gim.2014.146.CrossRefPubMedGoogle Scholar
  7. 7.
    Raskin S, Allan N, Teive HA, Cardoso F, Haddad MS, Levi G, et al. Huntington disease: DNA analysis in Brazilian population. Arq Neuropsiquiatr. 2000;58:977–85.CrossRefPubMedGoogle Scholar
  8. 8.
    Rubinsztein David C. Lessons from animal models of Huntington’s disease. Trends Genet. 2002;18:202–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Ridley RM, Frith CD, Crow TJ, Conneally PM. Anticipation in Huntington’s disease is inherited through the male line but may originate in the female. J Med Genet. 1988;25:589–95.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Potter NT, Spector EB, Prior TW. Technical standards and guidelines for Huntington disease testing. Genet Med. 2004;6:61–5.CrossRefPubMedGoogle Scholar
  11. 11.
    Falk M, Vojtis Kova M, Lukas Z, Kroupova I, Froster U. Simple procedure for automatic detection of unstable alleles in the myotonic dystrophy and Huntington’s disease loci. Genetic Testing. 2006;10:85–97.CrossRefPubMedGoogle Scholar
  12. 12.
    Zhao M, Lee CG, Hai-Yang Law HY, Chong SS. Enhanced detection and sizing of the HTT CAG repeat expansion in Huntington disease using an improved triplet-primed PCR assay. Neurodegener Dis. 2016;16:348–51.  https://doi.org/10.1159/000444714.CrossRefPubMedGoogle Scholar
  13. 13.
    Todorov T, Todorova A, Georgieva B, Mitev V. A unified rapid PCR method for detection of normal and expanded trinucleotide alleles of CAG repeats in Huntington chorea and CGG repeats in fragile X syndrome. Mol Biotechnol. 2010;45:150–4.CrossRefPubMedGoogle Scholar
  14. 14.
    Toth T, Findlay I, Nagy B, Papp Z. Accurate sizing of (CAG)n repeats causing Huntington disease by fluorescent PCR. Clin Chem. 1997;43:2422–3.PubMedGoogle Scholar
  15. 15.
    Bates G, Tabrizi S, Jones L. Huntington’s disease. 4th ed. Oxford: Oxford University Press; 2014.Google Scholar
  16. 16.
    Teo CR, Wang W, Law HY, Lee CG, Chong SS. Single-step scalable-throughput molecular screening for Huntington disease. Clin Chem. 2008;54:964–72.CrossRefPubMedGoogle Scholar
  17. 17.
    Wexler NS, Lorimer J, Porter J, Gomez F, Moskowitz C, Shackell E, et al. Venezuelan kindreds reveal that genetic and environmental factors modulate Huntington’s disease age of onset. Proc Natl Acad Sci. 2004;101:3498–503.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Semaka A, Creighton S, Warby S, Hayden MR. Predictive testing for Huntington disease: interpretation and significance of intermediate alleles. Clin Genet. 2006;70:283–94.CrossRefPubMedGoogle Scholar
  19. 19.
    Losekoot M, van Belzen MJ, Seneca S, Bauer P, Stenhouse SA, Barton DE. European Molecular Genetic Quality Network (EMQN). EMQN/CMGS best practice guidelines for the molecular genetic testing of Huntington disease. Eur J Hum Genet. 2013;21:480–6.  https://doi.org/10.1038/ejhg.2012.200.CrossRefPubMedGoogle Scholar
  20. 20.
    Quarrell OW, Rigby AS, Barron L, Crow Y, Dalton A. Reduced penetrance alleles for Huntington’s disease: a multi-centre direct observational study. J Med Genet. 2007;44(3):e68.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kay C, Collins JA, Miedzybrodzka Z, Madore SJ, Gordon ES, Gerry N, et al. Huntington disease reduced penetrance alleles occur at high frequency in the general population. Neurology. 2016;87:282–8.  https://doi.org/10.1212/WNL.0000000000002858 Epub 2016 Jun 22.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Sequeiros J, Ramos EM, Cerqueira J, Costa MC, Sousa A, Pinto-Basto J, Alonso I, et al. Large normal and reduced penetrance alleles in Huntington disease: instability in families and frequency at the laboratory, at the clinic and in the population. Clin Genet. 2010;78:381–7.  https://doi.org/10.1111/j.1399-0004.2010.01388.x.CrossRefPubMedGoogle Scholar
  23. 23.
    Sheth J, Shah S, Patel H, Bhavsar R, Bhatt K, Sheth F. A study on triplet repeat expansion disorders in Western Indian population. Hereditary Genet. 2015;4:141.  https://doi.org/10.4172/2161-1041.1000141.CrossRefGoogle Scholar
  24. 24.
    Pramanik S, Basu P, Gangopadhaya PK, Sinha KK, Jha DK, Sinha S, et al. Analysis of CAG and CCG repeats in Huntingtin gene among HD patients and normal populations of India. Eur J Hum Genet. 2000;8:678–82.CrossRefPubMedGoogle Scholar
  25. 25.
    Cloud LJ, Rosenblatt A, Margolis RL, Ross CA, Pillai JA. Seizures in juvenile Huntington’s disease: frequency and characterization in a multicenter cohort. Mov Disord. 2012;27:1797–800.  https://doi.org/10.1002/mds.25237 (Epub 2012 Nov 2).CrossRefPubMedGoogle Scholar
  26. 26.
    Jama M, Millson A, Miller CE, Lyon E. Triplet repeat primed PCR simplifies testing for Huntington disease. J Mol Diagn. 2013;15:255–62.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Molecular PathologyMetropolis Healthcare LtdMumbaiIndia
  2. 2.Genetics DepartmentMetropolis Healthcare LtdMumbaiIndia

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