Volume 27, Issue 1 (1-2023)                   IBJ 2023, 27(1): 46-57 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Namdar Aligoodarzi P, Rostami G, Kazemi Nezhad S R, Hamid M. Multiplex Snapshot Minisequencing for the Detection of Common PAH Gene Mutations in Iranian Patients with Phenylketonuria. IBJ 2023; 27 (1) :46-57
URL: http://ibj.pasteur.ac.ir/article-1-3856-en.html
Background: Phenylketonuria is a common inborn defect of amino acid metabolism in the world. This failure is caused by an autosomal recessive insufficiency of the hepatic enzyme hyperphenylalaninemia (PAH), which catalyzes the irreversible hydroxylation of phenylalanine to tyrosine. More than 1,040 different disease-causing mutations have already been identified in the PAH gene. The most prominent complication of Phenylketonuria, if not diagnosed and treated, is severe mental retardation. Hence, early diagnosis and initiation of nutritional therapy are the most significant measures in preventing this mental disorder. Given these data, we developed a simple and rapid molecular test to detect the most frequent PAH mutations.
 Methods: Multiplex assay was developed based on the SNaPshot minisequencing approach to simultaneously perform genotyping of the 10 mutations at the PAH gene. We optimized detection of these mutations in one multiplex PCR, followed by 10 single-nucleotide extension reactions. DNA sequencing assay was also used to verify genotyping results obtained by SNaPshot minisequencing.
Result: All 10 genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. Sequencing results of these frequent mutations showed that by using this method, a 100% detection rate could be achieved in the Iranian population.
Conclusion: SNaPshot minisequencing can be useful as a secondary test in neonatal screening for HPA in neonates with a positive screening test, and it is also suitable for carrier screening. The assay can be easily applied for accurate and time- and cost-efficient genotyping of the selected SNPs in various population.
Type of Study: Full Length | Subject: Molecular Genetics & Genomics

1. Christ SE. Asbjorn folling and the discovery of phenylketonuria. Journal of the history of the neurosciences 2003; 12(1): 44-54. [DOI:10.1076/jhin.]
2. Zare Karizi S, Hosseini Mazinani S, Khazaei Koohpar Z, Seifati S, Shahsavan Behboodi B, Akbari M, Koochmeshgi J. Mutation spectrum of phenylketonuria in Iranian population. Molecular genetics and metabolism 2011; 102(1): 29-32. [DOI:10.1016/j.ymgme.2010.09.001]
3. Elhawary NA, Aljahdali IA, Abumansour IS, Elhawary EN, Gaboon N, Dandini M, Madkhali A, Alosaimi W, Alzahrani A, Aljohani F, Melibary EM, Kensara OA. Genetic etiology and clinical challenges of phenylketonuria. Human genomics 2022; 22: 1-17. [DOI:10.1186/s40246-022-00398-9]
4. Muntau AC, Gersting SW. Phenylketonuria as a model for protein misfolding diseases and for the development of next generation orphan drugs for patients with inborn errors of metabolism. Journal of inherited metabolic disease 2010; 33(6): 649-658. [DOI:10.1007/s10545-010-9185-4]
5. Blau N, Harding C, Burlina A, Longo N, Bosch AM. Phenylketonuria. Nature reviews. Disease primers 2021; 0123456789: 1-19.
6. Williams RA, Mamotte CD, Burnett JR. Phenylketonuria: an inborn error of phenylalanine metabolism. The Clinical biochemist. Reviews 2008; 29(1): 31.
7. Pronina N, Giannattasio S, Lattanzio P, Lugovska R, Vevere P, Kornejeva A. The molecular basis of phenylketonuria in Latvia. Human mutation 2003; 21(4): 398-399. [DOI:10.1002/humu.9114]
8. Tolve M, Artiola C, Pasquali A, Giovanniello T, D'Amici S, Angeloni A, Pizzuti A , Carducci C, Leuzzi D, Carducci C. Molecular analysis of PKU-associated PAH mutations: a fast and simple genotyping test. Methods and protocols 2018; 1(3): 30. [DOI:10.3390/mps1030030]
9. Blau N, Yue W, Perez B. PAHvdb. 2006-2017; Available at: http://www.biopku.org/pah/.
10. Li N, He C, Li J, Tao J, Liu Z, Zhang C, Yuan Y, Jiang H, Zhu J, Deng Y, Guo Y, Li Q, Yu P, Wang Y. Analysis of the genotype-phenotype correlation in patients with phenylketonuria in mainland China. Scientific reports 2018; 8(1): 1-7. [DOI:10.1038/s41598-018-29640-y]
11. Brosco JP, Paul DB. The political history of PKU: reflections on 50 years of newborn screening. Pediatrics 2013; 132(6): 987. [DOI:10.1542/peds.2013-1441]
12. Zschocke J, Haverkamp T, Møller LB. Clinical utility gene card for: Phenylketonuria. European journal of human genetics 2012; 20(2): 248. [DOI:10.1038/ejhg.2011.172]
13. Fateh A, Aghasadeghi M, Siadat SD, Vaziri F, Sadeghi F, Fateh R, Keyvani H , Tasbiti AH, Yari S, Ataei Pirkooh A, Monavari SA. Comparison of three different methods for detection of IL28 rs12979860 polymorphisms as a predictor of treatment outcome in patients with hepatitis C virus. Osong public health and research perspectives 2016; 7(2): 83-89. [DOI:10.1016/j.phrp.2015.11.004]
14. Fanis P, Kousiappa I, Phylactides M, Kleanthous M. Genotyping of BCL11A and HBS1L-MYB SNPs associated with fetal haemoglobin levels: a SNaPshot minisequencing approach. Osong public health and research perspectives 2014; 15(1): 1-12. [DOI:10.1186/1471-2164-15-108]
15. Jian Y, Li M. A narrative review of single-nucleotide polymorphism detection methods and their application in studies of Staphylococcus aureus. Journal of Bio-X research. 2021; 4(1): 1-9. [DOI:10.1097/JBR.0000000000000071]
16. Ganji F, Naseri H, Rostampour N, Sedighi M, Lotfizadeh M. Assessing the phenylketonuria screening program in newborns, Iran 2015-2016. Acta medica Iranica 2018; 56(1): 49-55.
17. Biglari A, Saffari F, Rashvand Z, Alizadeh S, Najafipour R, Sahmani M. Mutations of the phenylalanine hydroxylase gene in Iranian patients with phenylketonuria. Springerplus 2015; 4(1): 1-5. [DOI:10.1186/s40064-015-1309-8]
18. Moghadam MR, Shojaei A, Babaei V, Rohani F, Ghazi F. Mutation analysis of phenylalanine hydroxylase gene in Iranian patients with phenylketonuria. Medical journal of the Islamic Republic of Iran 2018; 32: 21. [DOI:10.14196/mjiri.32.21]
19. Hamzehloei T, Hosseini S, Vakili R, Mojarad M. Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran. Gene 2012; 506(1): 230-232. [DOI:10.1016/j.gene.2012.06.043]
20. Alibakhshi R, Mohammadi A, Salari N, Khamooshian S, Kazeminia M, Moradi K. Spectrum of PAH gene mutations in 1547 phenylketonuria patients from Iran: a comprehensive systematic review. Metabolic brain disease 2021; 36(5): 767-780. [DOI:10.1007/s11011-021-00698-4]
21. Esfahani MS, Vallian S. A comprehensive study of phenylalanine hydroxylase gene mutations in the Iranian phenylketonuria patients. European journal of medical genetics 2019; 62(9): 1-6. [DOI:10.1016/j.ejmg.2018.10.011]
22. Mojibi N, Ghazanfari Sarabi S, Hashemi Soteh SMBH. The Prevalence and incidence of congenital phenylketonuria in 59 countries: A systematic review. Journal of pediatrics review 2021; 9(2): 83-96. [DOI:10.32598/jpr.9.2.826.2]
23. Bagheri M, Rad IA, Jazani NH, Zarrin R, Ghazavi A. Mutation analysis of the phenylalanine hydroxylase gene in Azerbaijani population, a report from West Azerbaijan province of Iran. Iranian journal of basic medical sciences 2015; 18(7): 649-653.
24. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic acids research 1988; 16(3): 1215. [DOI:10.1093/nar/16.3.1215]
25. Fiorentino F, Magli M, Podini D, Ferraretti A, Nuccitelli A, Vitale N, Baldi M, Gianaroli L. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Molecular human reproduction 2003; 9(7): 399-410. [DOI:10.1093/molehr/gag046]
26. Sanchez JJ, Borsting C, Hallenberg C, Buchard A, Hernandez A, Morling N. Multiplex PCR and minisequencing of SNPs-a model with 35 Y chromosome SNPs. Forensic science international 2003; 137(1): 74-84. [DOI:10.1016/S0379-0738(03)00299-8]
27. Lindblad Toh K, Winchester E, Daly MJ, Wang DG, Hirschhorn JN, Laviolette JP, Reich D E, Robinson E, Sklar P, Shah N, Thomas D, Fan J B, Gingeras T, Warrington J, Patil N, Hudson T J, Lander ES. Large scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nature genetics 2000; 24(4): 381-386. [DOI:10.1038/74215]
28. Wang W, Kham SKY, Yeo G, Quah T, Chong SS. Multiplex Minisequencing Screen for Common Southeast Asian and Indian Thalassemia Mutations. Clinical chemistry 2003; 218: 209-218. [DOI:10.1373/49.2.209]
29. Moradi K, Alibakhshi R, Ghadiri K, Khatami SR, Galehdari H. Molecular analysis of exons 6 and 7 of phenylalanine hydroxylase gene mutations in Phenylketonuria patients in Western Iran. Indian journal of human genetics 2012; 18(3): 290-293. [DOI:10.4103/0971-6866.107978]
30. Alibakhshi R, Moradi K, Mohebbi Z, Ghadiri K. Mutation analysis of PAH gene in patients with PKU in western Iran and its association with polymorphisms: identification of four novel mutations. Metabolic brain disease 2014; 29(1): 131-138. [DOI:10.1007/s11011-013-9432-0]
31. Vallian S, Barahimi E, Moeini H. Phenylketonuria in Iranian population: a study in institutions for mentally retarded in Isfahan. Mutation research 2003; 526(1-2): 45-52. [DOI:10.1016/S0027-5107(03)00015-0]
32. Bagheri M, Rad IA, Jazani NH, Zarrin R, Ghazavi A. Association between PAH mutations and VNTR alleles in the West Azerbaijani PKU patients. Maedica 2014; 9(3): 242.
33. Cernomaz A T, Macovei II, Pavel I, Grigoriu C, Marinca M, Baty F, Peter S, Zonda R, Brutsche M, Grigoriu BD. Comparison of next generation sequencing, SNaPshot assay and real-time polymerase chain reaction for lung adenocarcinoma EGFR mutation assessment. BMC pulmonary medicine 2016; 16(1):1-7. [DOI:10.1186/s12890-016-0250-0]
34. Ralf A, Oven M Van, Montiel D, Knij P De, Beek K Van Der, Wootton S, Lagacé R, Kayser M. Forensic Science International : Genetics Forensic Y-SNP analysis beyond SNaPshot : High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing. Forensic science international. genetics 2019; 41: 93-106. [DOI:10.1016/j.fsigen.2019.04.001]
35. Mehta B, Daniel R, Phillips C, McNevin D. Forensically relevant SNaPshot assays for human DNA SNP analysis: a review. International journal of legal medicine 2017; 131(1): 21-37. [DOI:10.1007/s00414-016-1490-5]
36. Lai G, Zhang W, Tang H, Zhao T, Wei L, Tao Y, Wang Z, Huang A. A SNaPshot assay for the rapid and simple detection of hepatitis B virus genotypes. Molecular medicine reports 2014; 10(3): 1245-1251. [DOI:10.3892/mmr.2014.2372]
37. Arpan A, Salil V, Harsh P, Nm P. Single base primer extension assay (SNaPshot) for rapid detection of human immunodeficiency virus 1 drug resistance mutations. journal of molecular biomarkers and diagnosis 2016; 7(2): 2-5. [DOI:10.4172/2155-9929.1000271]
38. Samples R. Genetics and genome research A comparison of SNaPshot minisequencing and HRM analysis in mtSNP clinMed. Journal of genetics and genome research 2015; 2: 1-5. [DOI:10.23937/2378-3648/1410021]
39. Fanis P, Kousiappa I, Phylactides M, Kleanthous M. Minisequencing assay for the detection of 12 BCL11A single nucleotide polymorphisms. BMC genomics 2014; 15: 108. [DOI:10.1186/1471-2164-15-108]
40. Hashim HO, Al Shuhaib MBS. Exploring the potential and limitations of PCR-RFLP and PCR-SSCP for SNP detection: A review. Journal of applied biotechnology reports 2019; 6(4): 137-144. [DOI:10.29252/JABR.06.04.02]
41. Gundorova P, Stepanova AA, Kuznetsova IA, Kutsev SI, Polyakov A V. Genotypes of 2579 patients with phenylketonuria reveal a high rate of BH4 non-responders in Russia. PLoS one 2019; 14(1): 1-12. [DOI:10.1371/journal.pone.0211048]
42. Shaykholeslam Esfahani M, Shaykholeslam Esfahani E, Vallian S. A novel compound-primed multiplex ARMS-PCR (CPMAP) for simultaneous detection of common PAH gene mutations. Metabolic brain disease 2018; 33(4): 1165-1173. [DOI:10.1007/s11011-018-0210-x]
43. Tresbach RH, Sperb-Ludwig F, Ligabue-Braun R, Tonon T, de Oliveira Cardoso MT, Heredia RS, Teresa Alves da Silva Rosa M, Cátia Martins B, Oliveira Poubel M, Carlos Santana da Silva L, Maillot F, Doederlein Schwartz V. Phenylketonuria diagnosis by massive parallel sequencing and genotype-phenotype association in brazilian patients. Genes (Basel) 2021; 12(1): 1-12. [DOI:10.3390/genes12010020]
44. Zarinkoob M, Khazaei Koohpar Z. Mutation analysis of exon 5 of PAH gene in phenylketonuria patients from Golestan Province, Iran. Journal of Shahrekord university of medical sciences. 2022; 24(1): 15-19. [DOI:10.34172/jsums.2022.03]
45. Jafarzadeh Esfehani R, Vojdani S, Hashemian S, Mirinezhad M, Pourafshar M, Forouzanfar N, Zargari S, Ehsan Jaripour M, Sadr Nabavi A. Genetic variants of the phenylalanine hydroxylase gene in patients with phenylketonuria in the northeast of Iran. Journal of pediatric endocrinology and metabolism 2020; 33(3): 355-359. [DOI:10.1515/jpem-2019-0351]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2023 CC BY-NC 4.0 | Iranian Biomedical Journal

Designed & Developed by : Yektaweb