ORIGINAL RESEARCH

Determining the frequency of PAH mutations in Moscow region residents with phenylketonuria using a combination of real-time PCR and next-generation sequencing

Nikiforova AI1, Abramov DD1, Kadochnikova VV1, Zobkova GU1, Ogurtsova KA2, Brjuhanova NO2, Shestopalova EA2, Kochetkova TO3, Shubina ES, Donnikov AE1, Trofimov DYu1
About authors

1 DNA-Technology LLC, Moscow, Russia

2 Morozov Children's Municipal Clinical Hospital, Moscow, Russia

3 Laboratory of Molecular Genetic Methods,
Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Moscow

Correspondence should be addressed: Alena I. Nikiforova
Kashirskoe shosse, d. 24, Moscow, Russia, 115478; ur.ygolonhcet-and@avorofikin

About paper

Contribution of the authors to this work: Nikiforova AI — research planning, next-generation sequencing, data analysis and interpretation, drafting and editing of a manuscript; Abramov DD — research planning, development of the diagnostic panel, data analysis and interpretation, editing of a manuscript; Kadochnikova VV — polymerase chain reaction, data analysis; Zobkova GYu — analysis of literature, data interpretation, editing of a manuscript; Ogurtsova KA, Brjuhanova NO — data collection and analysis; Shestopalova EA — research planning, data collection and analysis, editing of a manuscript; Kochetkova TO — next-generation sequencing, data analysis; Shubina ES — bioinformatic analysis, editing of a manuscript; Donnikov AE — research planning, data interpretation, editing of a manuscript; Trofimov DYu — research planning, conception and design of next-generation sequencing.

Received: 2017-08-07 Accepted: 2017-08-16 Published online: 2017-10-30
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The present study aimed to determine frequencies of mutations in the phenylalanine hydroxylase gene (PAH) in unrelated children (n = 71) diagnosed with phenylketonuria, who presented to Morozovskaya Children’s City Clinical hospital (Moscow) over the period from 2015 to 2016. The patients were tested for the most common PAH mutations using the original real- time PCR-based technique for the identification of nucleotide variants; additionally, next generation sequencing (NGS) was performed on the unidentified genotypes. The original PCR-based technique allowed us to effectively identify 83 % of the pathogenic allelic variants in the sample. Using the combination approach (real-time PCR + NGS), we found mutations in both alleles of PAH in 66 of total 71 patients. Altogether, 26 pathogenic PAH mutations were identified, the most common being p.R408W (47.9 %) and p.R261Q (9.9 %). Frequencies of mutations common for the Russian population, such as IVS10nt546, IVS12+1G>A, p.R158Q, p.Y414C, and IVS4+5G>T, ranged from 4.2 to 2.8 %. Half of the identified variants accounted for the total frequency of < 10 %. Sequencing of PAH revealed a few functional mutations previously unreported for Moscow region residents, including p.D222Terfs, p.R111Ter, p.F161S, p.G188D, p.R270K, p.L311P, p.F55L, p.F55Leufs, IVS1+5G>T, and IVS8-7A>G. It could be reasonable to include mutations p.D222Terfs and p.R111Ter (carrier frequency of 2.1 %) in PCR testing panels. The data obtained in our study can also be used in the development of genetic tests for phenylketonuria.

Keywords: phenylketonuria, phenylalanine hydroxylase gene, PAH, real-time PCR genotyping

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