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Identifying Variable Number of Tandem Repeat Alleles in Phenylalanine Hydroxylase Gene in Patients with Phenylketonuria

10.30699/jambs.28.129.198 Background & Objective: Phenylketonuria (PKU) is a metabolic disorder that is caused by mutations in the phenylalanine hydroxylase (PAH) gene. The multiplicity of mutations in the PAH gene of PKU leads to the cases, in which the pathogenic mutation cannot be detected. In these cases, the variable number of tandem repeat (VNTR), which is the polymorphic marker associated with the PAH gene, is used to identify PKU carriers. The present study was conducted to investigate the allele frequency of this marker in PKU population of Golestan Province (north of Iran).


Introduction
Phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism with an autosomal recessive inheritance pattern (1,2), which was first described by Folling in 1934 (3). PKU is the result of phenylalanine hydroxylase (PAH) deficiency that catalyzes the irreversible conversion of phenylalanine into tyrosine in the presence of the tetrahydrobiopterin (BH4) cofactor (4). PAH deficiency results in the abnormally elevated serum phenylalanine levels of more than 120 μmol/L, which causes irreversible mental retardation in untreated patients (5). The PAH gene is located on the long (q) arm of chromosome 12 in q22-q24.1 regions. This gene contains 13 exons and 12 introns (6). PKU has the prevalence of 1 per 10,000 births in Caucasians, but is considerably higher in the eastern Mediterranean region. In fact, the highest prevalence of PKU is reported in this region of the world: 1 per 4,000 in Turkey and 1 per 3627 in Iran. This high prevalence can be attributed primarily to the high rate of consanguineous marriage in the region (3). Another study also reported the highest prevalence in Turkey and the lowest prevalence in the UAE (7). Based on pre-treatment blood phenylalanine (Phe) levels, hyperphenylalaninemia and PKU are clinically divided into four groups: HPA (2-10 mg/dl), mild PKU (10-15mg/dl), moderate PKU (15-20mg/dl), and classic PKU (>20mg/dl) (8). PKU is often caused by mutations in the PAH gene (4,9). The PAH locus database lists over 800 PAH mutations including missense and nonsense mutations, small and large deletions, as well as small insertions and splicing defects. This gene is located on chromosome 12 of humans. The cDNA sequence contains 13 exons and encodes 452 amino acids (4). The PAH gene has various polymorphic markers such as intragenic short tandem repeats (STRs) located in intron 3, intragenic variable number of tandem repeats (VNTR) located in the 3′ untranslated regions (3′-UTR), and several restriction fragment length polymorphisms (RFLPs), which are highly associated with the PAH region (1). Multiallelic VNTR polymorphism has an AT-rich repeat unit of 30 bp and several alleles of this polymorphism have been identified and reported in the world (3). Polymorphic markers that are strongly linked to the PAH gene facilitate the prenatal diagnosis and identification of carriers and are also important in detecting the haplotypes in the PAH gene and tracking the geographical origin of the mutations (10). Many studies have been performed regarding the tandem repeat sequences of PAH VNTR gene thus far. In these studies, polymorphism of this sequence, its association with a variety of mutations in the PAH gene, ability of this sequence in identifying PKU carriers, and use of the VNTR sequence alongside repeat STR sequences in forensic identification have also been considered (1,3,5). Overall, polymorphic systems have a high degree of heterogeneity and are highly informative for the prenatal diagnosis and carrier screenings in most of the populations (11). According to studies conducted in Iran, the high heterozygosity rate of the VNTR has provided a 66% information rate for the Iranian population (3). Given the high prevalence of PKU and consanguineous marriages in the Iranian population (12), the present study was conducted to identify PAH VNTR alleles in PKU patients in Golestan Province, located in the north of Iran.

Patients
This study was approved by Ethics Committee of Golestan University of Medical Sciences and Health Services with the confirmation code: IR.GOUMS.REC.1394.204. The present study was a cross-sectional and descriptive work, in which 26 nonrelative PKU patients were identified from different parts of Golestan Province within a one-year period (2016). These patients were identified based on their records at Taleqani Hospital in Gorgan. Only one patient was included from each family and the patients with BH4 deficiency were excluded. The pre-treatment Phe levels were determined to be 4.5-250 mg/dl in 24 patients and their levels were not available in two patients (7.7%). Then, the patients and their families were invited to participate in this study. After justifying the participants, consent forms and questionnaires were completed by the patients or their families (in case where the patient was a child or had mental retardation). First, 2-5 mL blood samples were collected from each patient and, to prevent blood coagulation, Falcons 15 mL containing 300 µL of 0.5 M EDTA was used as the anticoagulant.

DNA Extraction
High pure PCR template preparation kit (Roche, Germany) was used for DNA extraction according to the manufacturer's instructions. After DNA extraction and before PCR reaction, the obtained DNA was analyzed by a Nano Spectrophotometer (Thermo Scientific NanoDrop, 2000C, USA) to evaluate the quantity and quality of nucleic acid.

Polymerase Chain Reaction (PCR)
The PCR was performed on a thermocycler (TC-4000, TECHNE, UK). The primers used in this study were designed in Oligo7 software. The sequence of the forward and reverse primers employed in this reaction was as follows: The primers were synthesized by Bioneer Company (South Korea).
The length of the final PCR product was 484 bp and the final volume of PCR mixture was 50 µL, which was prepared by Taq PCR Mix 2x kit (Biotechrabbit, Germany) and via adding genomic DNA, primer pairs (20 pmol), and sterile water to PCR mixture. The results of modifying PCR conditions for amplification of PAH VNTR region were as follows: initial denaturation temperature of 95°C for 5 minutes and 33 reaction cycles each including: denaturation temperature of 95°C for 15 seconds, primers adhesion temperature of 60°C for 10 seconds, elongation temperature of 72°C for 40 seconds, and final elongation temperature of 72°C for 5 minutes.

Electrophoresis of PCR Products
To ensure the amplification of the desired fragment and its quality and not amplifying the non-specific products, 3 µL of the reaction products (patient samples) and 100 bp marker (100bp DNA Ladder, BR0800201, Biotechrabbit, Germany) were loaded and electrophoresed on the 2% agarose gel.

Sequencing of PCR Products
After viewing a clear DNA band on the gel, the remainder of the PCR product was sent to Bioneer Company. (South Korea) for sequencing. Sequencing was performed by the ABI 3730 DNA Sequencer using the chain termination method.

Applications
Chromas and CLC main workbench v3.5 applications were utilized to analyze the results of the samples sequencing data.

Patient Phenotype
In this study, 26 HPA patients referring to Taleqani Hospital in Gorgan were examined by a pediatric endocrinologist. Patients were divided into five groups according to pretreatment Phe levels (

Quantitative and Qualitative Results of Extracted DNA
Nano Spectrophotometer showed the quantity and quality of DNA extracted from the blood samples of patients to be in the range of 100-1000 ng/μL and absorption ratio of 260/280 in the range of 1.8-2, respectively. The DNA extracted with the mentioned quantity and quality was appropriate for PCR.

Sequencing Results
After PCR reaction, the samples were sequenced to confirm the number of repeats in the VNTR alleles.
The base sequence of the PAH VNTR alleles was determined in Chromas software and CLC main workbench v3.5; this way, the number of repeats in the VNTR alleles was specified ( Table 2).

Discussion
In this study, the VNTR alleles were investigated in the PAH gene in 26 patients with phenylketonuria in Golestan Province for the first time. PCR sequencing was used in this study. The results of analyzing 52 alleles led to identifying 5 VNTR alleles that were related to 3, 7, 8, 9, and 12 copies of the repeat units with frequencies of 15 (28.85%), 15 (28.85%), 9 (17.3%), 10 (19.23%), and 3 (5.77%), respectively. Furthermore, VNTR with 3 and 7 repeat units had the highest frequency in our study. It is noteworthy that VNTR with 3 repeats was observed in all the three ethnic groups of Fars, Turkmen, and Lor, and the highest frequency belonged to Fars ethnic group.
On the other hand, in a study by Razipour et al. (2017) among 81 PKU patients from different parts of Iran, different mutations and mini-haplotypes were identified. Five VNTR alleles with 3, 7, 8, 9, and 12 repeats were reported in their study (1), which was consistent with our findings in Golestan Province. Subsequently, 18 non-relative PKU patients from three provinces of Kermanshah, Hamadan, and Lorestan were investigated in terms of PAH gene mutations and their association with VNTR alleles by Alibakhshi et al. (2018). They detected 11 specific mutations and 4 VNTR alleles with 3, 7, 8, and 9 repeats (4). However, in comparison with Golestan Province, their study did not show VNTR allele with 12 repeats. In addition, another investigation was conducted in 2018 on 25 non-relative PKU patients in Gilan Province, northern Iran, in which 3 VNTR alleles were identified with 3, 7, and 8 repeats while the highest frequency (60%) belonged to VNTR allele with 8 repeats (data were not published). However, Golestan Province (in the present study), which is another province in northern Iran, had more diversity of VNTR alleles and VNTR alleles with 3 and 7 repeats had the highest frequency.
On the other hand, the VNTR alleles identified in our study (in PKU patients of Golestan Province) corresponded to alleles previously described among the European white families (3). VNTR12, which accounts for 4.3% of PKU alleles in European white people, was about 5.77% in our study, whereas the diversity in this polymorphic locus was higher in the samples compared to Chinese PKU families, in which only alleles of VNTR with 3 and 8 repeats were reported (14). The frequency of VNTR3 and VNTR7 on PKU chromosomes in Golestan Province was significantly different from reports of European population; VNTR3 showed lower frequency and VNTR7 showed higher frequency in Golestan Province than the European population. This is partly attributable to the different mutation spectra in these populations. The R408W mutation is the most common mutation that causes PKU in European populations and is associated with VNTR3 allele, especially in the eastern parts of this continent (15). This mutation is very rare in the Iranian population. On the other hand, the Mediterranean IVS10nt546 mutation has been associated with VNTR7, which is the most common mutation in Iran (16,17). This mutation was also highly prevalent in Golestan Province (18,19). Due to the lack of funding for investigating another polymorphic marker (STR) associated with the PAH gene in PKU patients, in the present work, only the VNTR marker was considered in these patients.