Mutational landscape of phenylketonuria in Iran

Abstract To date more than 1000 different variants in the PAH gene have been identified in patients with phenylketonuria (PKU). In Iran, several studies have been performed to investigate the genetics bases of the PKU in different parts of the country. In this study, we have analysed and present an update of the mutational landscape of the PAH gene as well as the population genetics and frequencies of detected variants for each cohort. Published articles on PKU mutations in Iran were identified through a comprehensive PubMed, Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: “phenylketonuria”, “hyperphenylalaninemia”, and “PKU” in combination with “Iran”, “Iranian population”, “mutation analysis”, and “Molecular genetics”. Among the literature‐related to genetics of PKU, 18 studies were on the PKU mutations. According to these studies, in different populations of Iran 1497 patients were included for mutation detection that resulted in detection of 129 different mutations. Results of genetic analysis of the different cohorts of Iranian PKU patients show that the most prevalent mutation in Iran is the pathogenic splice variant c.1066‐11G > A, occurring in 19.54% of alleles in the cohort. Four other common mutations were p.Arg261Gln, p.Pro281Leu, c.168 + 5G > C and p.Arg243Ter (8.18%, 6.45%, 5.88% and 3.7%, respectively). One notable feature of the studied populations is its high rate of consanguineous marriages. Considering this feature, determining the prevalent PKU mutations could be advantageous for designing screening and diagnostic panels in Iran.


| INTRODUC TI ON
Phenylketonuria (PKU; OMIM #261600) is an autosomal recessive inborn error of phenylalanine metabolism caused by wide range of mutations of the phenylalanine hydroxylase (PAH) gene located on chromosome 12q22-24.1 coding for the hepatic enzyme PAH that converts phenylalanine into tyrosine in the presence of molecular oxygen and catalytic amounts of tetrahydrobiopterin (BH4). 1 PKU as epitome of human biochemical genetics 2 can historically claim some 'firsts' in this era: the first in which a toxic agent, high phenylalanine concentration in blood, found to cause intellectual disability 3 ; the first for which the biochemical background was understood; the first for which successful treatment was made available 4 ; the first to be controlled by dietary management; the first to be detected by newborn screening 5 ; and also PKU is a primary application for human gene therapy. PKU is therefore considered as a paradigm for monogenic metabolic disorders. 6 In 98% of PKU patients, defects of the PAH enzyme are due to mutations in the PAH gene. about 1%-2% of cases, mutations occur in the genes that encode enzymes for biosynthesis or regeneration of BH4, an obligatory cofactor required for the activity of PAH. 1 The human PAH gene spans 90 kb (about 171 kb with flanking regions) and contains 13 exons which encodes for a polypeptide of 452 amino acids. 7 Over some four decades of research, several studies have listed the PAH gene variants occurring in all exons, but most commonly in exons 3, 6, 7 and 11. 8 To date more than 1723 variants in the PAH gene have been recorded in BIOPKU (http://www. biopku.org) database. 9 The huge number of PAH mutations and the wide variable distribution of frequent mutations between geographical regions and ethnic groups makes PKU a genetic disease with striking degree of allelic heterogeneity. 10 Clinical manifestations of PKU are related to the toxic accumulation of phenylalanine in the blood and brain and based on the degree of PAH deficiency in a spectrum of disorders including classic PKU, mild PKU and mild hyperphenylalaninaemia (HPA). Classic PKU is a result of complete or near-complete deficiency of PAH activity and if the disease left untreated and/or late-diagnosed, the neurotoxic effects of elevated phenylalanine and its by-products can lead to irreversible severe neurodevelopmental delay and progressive intellectual disability accompanied by several additional symptoms, which may include eczematous rashes, seizures, autism, hyperactivity and motor deficits at an early age. Developmental problems as well as psychiatric symptoms often become apparent as the child grows. Mild PKU and mild HPA are associated with lower risk of neurological, and cognitive abnormalities in the absence of treatment. 11 PKU diagnosis is achieved soon after birth through newborn screening programs using the Guthrie card blood spot obtained from a heel prick. Early identification and treatment prevent the neurotoxic effects of elevated phenylalanine and its metabolites. 12 Changing PKU from a disease of severe intellectual disability to a disease with almost normal development, phenylalanine restricted dietary is the main strategy for the standard treatment of the disease. 13 The novel therapeutic approaches including BH4, large neutral amino acids, phenylalanine ammonia lyase, chaperone treatment may in part replace dietary restriction. 13 Among these therapeutic modalities, supplementation of BH4, is the only alternative therapy that currently have been approved. This treatment is beneficial only in a proportion (at least 20%-30%) of PKU patients (so-called BH4responsive) especially in milder PKU phenotypes. 14 One of the potential modifiers of BH4 treatment effectiveness is PAH genotype and although this modifier cannot predict BH4 responsiveness with 100% accuracy, specific mutations resulting residual PAH activity is strongly associated with the BH4-responsive phenotype. 15 PKU is one of the most common inherited disorders in Iran with an incidence of roughly 1:4698. 16 As PKU has autosomal recessive inheritance, consanguineous marriage is an important risk factor; thus, high disease incidence is expectable for countries with a high rate of consanguineous marriages. 16 The genetic basis of PKU in the Iranian population has been investigated in studies done separately among different Iranian ethnic groups. Herein, we have tried to combine data from these studies and present an update of the mutational landscape of the PKU in Iran.

| DE S I G N OF THE S TUDY
Published articles on PKU mutations in Iran were identified through a comprehensive electronic search using the databases MEDLINE (via PubMed), Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: "phenylketonuria", "hyperphenylalaninemia", and "PKU" in combination with "Iran", "Iranian population", "mutation analysis", F I G U R E 1 Study selection flow chart. and "Molecular genetics". We gave preference to papers published during 2003-2023 covering the genetics of PKU in different regions of the country. Although the Persian language publications were also accepted, but were replaced by two newly searched English publications with the same number of patients and the same authors, which were more complete than their Persian publications. All original articles that directly reported mutations of PKU in the Iranian populations were included.

| Geographic and ethnic distribution of PAH mutations
Enrolled studies in this systematic review cover multiple Iranian ethnic groups including Persians, Kurds, Azeris, Lurs, Arabs, Baluchis, Turkmen, Gilak, Tabari, Talesh, Qashqai and tribal groups. The first report of PAH mutations in Iranian PKU patients has been performed by Vallian et al. 17 Overall, 17 different common mutations (frequency ≥1%) and more than 100 less common mutations have been reported in multiple Iranian ethnicities that show broad ranges of the PAH gene mutations in different regions of Iran indicating high degree of ethnic heterogeneity for PKU in our population. Table 1 show the common mutations by region and ethnicity found in Iranian patients with PKU and Figure S1. demonstrates common mutations in some of the geographical regions in Iran that characterized by 18 studies.
In some regions two studies have been conducted and we pooled these studies for calculating the allele frequency of detected variants. Other studies recruited the patients from different regions of the country without characterising the exact geographical regions.

| Spectrum and Frequency of PAH mutations in Iran
By the last update (February, 2023), the mutational spectrum included 129 different mutations (Table S1), which were distributed along almost the entire PAH gene sequence and Table 2 provides those mutations that were observed with ≥1% allele frequency.

| Common PAH gene variants
The second most common PAH pathogenic variant in Iran Turkey. 28 This variant has a relatively high allele frequency in some parts of Iran such as Esfahan, 22 Mazandaran and Golestan, 34,35 Guilan, 36 Qazvin and Zanjan, 37 East Azerbaijan 24 and less allele frequency in northeast 27 as well as among Kurds. 38 The distribution of p.Arg261Gln as a CpG mutation is difficult to explain by ancient or recent migration which has a moderate allele frequency in several South European countries and high frequency in Middle East unconnected by known movement of peoples in the past.
Linking to various haplotypes, independent recurrence in two or more founders is possible for p.Arg261Gln. 39 The third most common variant in Iran is p.Pro281Leu with the frequency of 6.45%. This severe pathogenic variant is a C-to-T transition at the second base of codon 281, which is also the last nucle-

| Less common and private variants
Sixty-seven variants were found with frequencies of <1%. Twentyfour variants (18.75%) were observed in only one allele and one family (private variants, Table S2). Representing high variation of mutational spectrum, these variable compound heterozygous variants shows that the number of different variants in Iran like several other populations in the world is usually high, with a few prevalent variants and a large number of less common variants and also private variants. Furthermore, there are substantial differences in the mutational spectrum among Iranian populations indicating cultural, geographical, ethnical and racial diversity in Iran (Table S1).

| Mutational hotspots and arginine mutation
There are 1198 and 48 CpG dinucleotides in the genomic sequence of PAH and its cDNA, respectively. These CpG dinucleotides are potential sites for recurrent mutation. 45 Mutability prediction for the cDNA sequence of PAH has shown that the majority of the predicted hypermutable regions coincide with the 24 CpG dinucleotide sites. 46 Among the 129 PAH variants in this study, 20 are known to be CpG-type alleles; another 2 occur at CpG sites but are not C → T or G → A transitions (Table S3). Taken together, CpG-type alleles accounts for more than 33.56% (854 alleles) of PKU alleles in Iran. Of note, it has been widely assumed that CpG sites could be hypermu-

| BH4 response prediction
According to the genotype, BH4 responsiveness can be predicted for PKU patients. 48 Mutations with residual protein activity of 10% or less were classified as 'severe' with negative BH4 responsiveness.
Mutations with a residual protein activity of more than 10% were classified as 'mild' with a probable responsiveness to BH4. 9  the Persian Gulf is a multi-ethnic country of more than 80 million inhabitants that has a higher incidence of PAH-deficient PKU than in either Turkey, the United States or Europe. 16 Consanguineous marriage, as a social and cultural norm in Iran, has led to an increased incidence of PKU. Moreover, inbreeding within the same ethnic and religious group has also contributed to an increased dis-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest to declare.