Background: Hyperphenylalaninemia (HPA) is an autosomal recessive disorder that results from a deficiency in the phenylalanine hydroxylase enzyme (PAH) or from a flaw in the genes that are responsible for the biosynthesis or regeneration of the cofactor tetrahydrobiopterin (BH4), including GCH1, SR, QDPR, PTS, and PCD. Identification of disease-causing variants in these genes can help physicians and clinical geneticists in differential diagnosis, appropriate prescription drugs, and saving time and cost. This study attempted to identify these genes’ most prevalent disease-causing variants in Iranian HPA patients. Summary: This study was performed under the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Before it started, the flow work and inclusion/exclusion criteria were published as a protocol in PROSPERO (CRD42021273705). We conducted a comprehensive search on December 10, 2022, on international online databases, including Web of Science, Scopus, EMBASE, Science Direct, PubMed/Medline, Google Scholar, SID, ISC, and Magiran search engine, to find pertinent publications. Some studies were chosen based on inclusion and exclusion criteria. Altogether, 1,243 Iranian patients from 13 articles were considered. In total, we identified 129 distinct disease-causing variants in PAH (20 novel variants), 29 in QDPR (17 novel variants), 15 in PTS (seven novel variants), and one novel variant in PCD. Twenty disease-causing variants for PAH, 18 for QDPR, and 8 for PTS are included in the genes’ proposed genetic diagnostic panels. These panels include more than 75% of the documented disease-causing variants in the Iranian population. Key Messages: The findings of this research illustrated the spectrum of disease-causing variants in the PAH, QDPR, PTS, and PCD genes identified in Iranian HPA patients. Common disease-causing variants of these genes may be chosen as a preliminary diagnostic panel for early diagnosis and lowering therapy costs.

© 2023 The Author(s). Published by S. Karger AG, Basel

Introduction

Hyperphenylalaninemia (HPA) is an autosomal recessive disorder that is caused by a deficit in the phenylalanine hydroxylase enzyme (PAH) (98% of cases) or by a fault in the genes involved in the regeneration or biosynthesis of its cofactor tetrahydrobiopterin (BH4) (2% of cases) such as GCH1, SR, QDPR, PTS, and PCD (GCH1 is inherited in both autosomal recessive and autosomal dominant forms) [1–3]. There is no precise estimation of the prevalence of HPA worldwide; however, it is assumed to be one in 10,000 births [4]; in Iran, this is close to one in every 5,000 births [5–9].

Differential diagnosis should be made since patients with BH4 deficiency showed more severe progressive neurologic impairment [10, 11]. HPA is detected using the plasma phenylalanine (Phe) test, and Phe levels exceeding 120 mol/L (>2 mg/dL) are often utilized as a cut-off [12, 13].

Restricting Phe intake is the most frequent and efficient therapy for PKU. This specific diet should be begun as soon as a patient is diagnosed and continued for the rest of their lives [14, 15]. It should be noted that some BH4-deficient individuals do not benefit from this therapy.

Patients are divided into subsets based on the results of biochemical tests and measurements of the levels of tyrosine, BH4, and Phe in the urine. If the levels of tyrosine and BH4 are normal and the levels of Phe are above 20 mg/dL, between 10 and 20 mg/dL, and between 2 and 10 mg/dL, patients are categorized as having severe or classical PKU, mild PKU, or mild HPA [1, 16, 17].

Due to the extensive implementation of newborn screening (NBS) programs, few instances of classical PKU are recorded in industrialized nations. NBS is the most effective population-based public health screening program for identifying and treating PKU patients. A low-Phe diet and early diagnosis may help keep individuals healthy [18]. In Iran, NBS started as a pilot in a few areas in 2006 and became a countrywide program in 2011 [19]. PKU patients can be easily identified using heel blood sampling 24 h after birth [20]. Diagnosis is made on a dried blood spot using methods such as fluorometric and colorimetric methods, enzymatic methods, high-performance liquid chromatography, and tandem mass spectrometry [21–25]. Although these methods help in the early diagnosis of patients, none of them identifies disease-causing variants; therefore, they cannot be used for prenatal diagnosis and detecting causative variants in families or populations.

In Iran, several studies have been performed to identify disease-causing variants in HPA-causing genes. However, since the sample sizes of these studies were small, they could not be used alone for introducing common disease-causing variants in the population. A systematic review is a remedy that may deal with this issue. This research used a larger sample size to estimate the prevalence of each disease-causing variant in the Iranian population in the PAH, QDPR, PTS, and PCD genes. Finally, by combining information from various studies, this study can more accurately predict disease-causing variants in our population and lead to better diagnosis, classification, and treatment.

MethodStudy Protocol

This study was performed under the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [26]. Before it started, the flow work and inclusion/exclusion criteria were published as a protocol in PROSPERO (CRD42021273705). This protocol is available at: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021273705.

The current study was conducted using performed studies in which none of the authors had ever researched humans or animals. Thus, this research did not need the Ethical Committee’s approval. The two authors carried out all phases of the research separately, and disagreements were settled by talking to other group members.

Search Strategy

We performed a comprehensive search on December 10, 2022, across many worldwide web databases, including Web of Science, Scopus, EMBASE, Science Direct, PubMed/Medline, Google Scholar, SID, ISC, and Magiran, to find relevant publications. The search was conducted repeatedly while drafting the paper to ensure the research was current when submitted. The search disregarded time, language, and journal filters. To find all relevant articles, several terms were employed. The keywords were combined using “AND” and “OR.” For example, the applied search strategy in PubMed was as follows (“PAH” (All Fields) OR (“phenylalanine hydroxylase” (MeSH Terms) OR (“phenylalanine” (All Fields) AND “hydroxylase” (All Fields)) OR “phenylalanine hydroxylase” (All Fields)) OR (“prev chronic dis” (Journal) OR “pcd” (All Fields)) OR (“pterin-4” (All Fields) AND “alpha-carbinolamine” (All Fields) AND (“hydro lyases” (MeSH Terms) OR “hydro lyases” (All Fields) OR “dehydratase” (All Fields) OR “dehydratases” (All Fields))) OR (“6 pyruvoyltetrahydropterin synthase” (Supplementary Concept) OR “6 pyruvoyltetrahydropterin synthase” (All Fields) OR “ptps” (All Fields) OR “protein tyrosine phosphatases” (MeSH Terms) OR (“protein” (All Fields) AND “tyrosine” (All Fields) AND “phosphatases” (All Fields)) OR “protein tyrosine phosphatases” (All Fields)) OR “PTS” (All Fields) OR (“6 pyruvoyltetrahydropterin synthase” (Supplementary Concept) OR “6 pyruvoyltetrahydropterin synthase” (All Fields) OR “6 pyruvoyltetrahydropterin synthase” (All Fields)) OR “QDPR” (All Fields) OR “DHPR” (All Fields) OR ((“quinoid” (All Fields) OR “quinoidal” (All Fields) OR “quinoidization” (All Fields) OR “quinoids” (All Fields)) AND (“dihydropteridine reductase” (MeSH Terms) OR (“dihydropteridine” (All Fields) AND “reductase” (All Fields)) OR “dihydropteridine reductase” (All Fields)))) AND (“HPA” (All Fields) OR “hyperphenylalanemia” (All Fields) OR (“hyperphenylalaninemias” (All Fields) OR “phenylketonurias” (MeSH Terms) OR “phenylketonurias” (All Fields) OR “hyperphenylalaninaemia” (All Fields) OR “hyperphenylalaninaemias” (All Fields) OR “hyperphenylalaninemia” (All Fields)) OR “PKU” (All Fields) OR (“phenylketonurias” (MeSH Terms) OR “phenylketonurias” (All Fields) OR “phenylketonuria” (All Fields))) AND “Iran” (Affiliation). The reference part of the articles was also examined to ensure all relevant articles were read.

Inclusion and Exclusion Criteria

The inclusion and exclusion criteria were determined using PICO [27]. We included peer-reviewed studies that genetically analyzed all the PAH, QDPR, PTS, and PCD genes coding regions in HPA patients. On the other hand, exclusion criteria contained duplicate studies, non-Iranian studies, irrelevant studies, patients with other diseases, studies with non-sequencing approaches, studies that examined only some exons or variants (incomplete studies), case reports, review articles, and letters.

Study Selection

All of the identified papers’ titles and abstracts were first examined. The entire content of the articles was then reviewed for compliance with inclusion and exclusion criteria. Finally, disagreements were settled in the presence of other authors.

Definitions

Serum Phe levels are used to identify HPA patients. Affected people are divided into three categories, as previously mentioned: severe or classical PKU (Phe levels >20 mg/dL), mild PKU (Phe levels 10–20 mg/dL), and mild HPA (Phe levels 2–10 mg/dL) [1]. The genes contributing to the etiology of HPA include PAH, QDPR, PTS, and PCD[28].

Data Extraction

Data extraction was done in an Excel file that included the author’s name, email address of the first or corresponding author, publication’s year, province, study’s year, sample size, data collection method, diagnostic approach, and frequency of each variant.

The first or corresponding author was contacted on studies with insufficient information, and the issue was often remedied. In the absence of a response, the authors chose other research that was more in-depth.

ResultsSearch Results and the Features of Studies Included the Systematic Review

Figure 1 depicts the flowchart of the chosen studies. A total of 40 articles were found after thorough searches across several databases and references. Following this, 14 unrelated publications were eliminated by going through the titles and abstracts; the search result had no duplicate reports. Following a comprehensive text examination, 13 articles were left out because they did not meet the qualifying requirements. In the end, there were 13 publications involving 1,243 Iranian patients (Fig. 1). In some studies, more than one gene had been examined [19]. Included articles are listed in detail in Table 1.

Fig. 1.Table 1.

Summary of characteristics of included studies in the systematic review

RefFirst author, published yearYearPlace or ethnicityDesignMethodSample sizeDeficient gene(29)Rastegar Moghadam M, 20182015–2016Azeri, Kurdish, and PersianScreening programSequencing30PAH(30)Jafarzadeh-Esfehani R, 20192012–2018NortheastScreening programSequencing122PAH(31)Alibakhshi R, 20182014–2016Kermanshah, Hamedan, LorestanScreening programSequencing18PAH(32)Shaykholeslam Esfahani M, 2018unknownCenter of countryScreening programSequencing140PAH(33)Biglari A, 2015unknownQazvin and ZanjanScreening programSequencing39PAH(34)Shirzad T, 20182002–2017unknownScreening programSequencing611PAH(35)Razipour M, 2017unknownunknownScreening programSequencing81PAH(36)Alibakhshi R, 20132010–2012KermanshahScreening programSequencing27PAH(37)Bonyadi M, 2010unknownAzeriScreening programSequencing44PAH(28)Foroozani H, 20152008–2015unknownScreening programSequencing24QDPR(19)Khatami S, 20162010–2014Afghan, Arab, Fars, Kurd and AzeriScreening programSequencing76QDPR, PTS, and PCD(38)Khaghani F, 2022unknownKhorasan-RazaviScreening programSequencing21PAH(39)Sadat Fatemi H, 2022UnknownKhorasan-RazaviScreening programSequencing10QDPR and PTSDisease-Causing Variants in PAH

Among the included studies, ten studies examined variants in PAH by sequencing the gene’s whole coding and flanking regions. The subjects in these studies were from various areas and ethnic groups of the country. One thousand one hundred thirty-three patients (2,438 alleles) were examined, and 129 disease-causing variants were identified, 20 of which were not reported while publishing related articles (novel variants). The details of these studies, including study name, disease-causing variant, frequency, and relative frequency percentage, are listed in Table 2.

Table 2.

Identified disease-causing variants of PAH in included studies

Study ref(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)TotalRelative frequencyDisease-causing variantsc.1066−11G>A2727333633417449620.34454471c.782G>A4736613613532108.613617719c.168+5G>C5392140914311867.629204266c.842C>T2845487291731777.260049221c.727C>T2976012143984.019688269c.969+5G>A101264109963.937653815c.143T>C174244125843.445447088c.526C>T1121842326753.076292043c.898G>T2155824722.953240361c.441+5G>T3582632.584085316c.781C>T24208442441.804757998c.838G>A122228123411.681706317c.1199+1G>C111122113401.640689089c.754C>T630361.47662018c.1089G>T630361.47662018c.113_115del1716331.353568499c.1208C>T35221311.271534044c. 843−5T>C*244281.148482363c.1169A>G614111230.943396226c.691T>C44141230.943396226c.473G>A21622220.902378999c.1222C>T616220.902378999c.688G>A2161190.779327317c.1089delG22114190.779327317c.721C>T13122180.73831009c.1114A>T116170.697292863c.671T>C962170.697292863c.926C>A88160.656275636c.728G>A128221160.656275636c.(168+1)_(353−1)del14140.574241181c.165delT122140.574241181c.2T>C*12120.492206727c.592_613del22102120.492206727c.510−1G>A12120.492206727c.755G>A48120.492206727c.1223G>A2422100.410172272c.1049C>A8190.369155045c.441+1G>A6390.369155045c.970−1G>T*6280.328137818c.169–13T>G2680.328137818c.1068C>G4480.328137818c.696A>G5270.287120591c.590_612del1560.246103363c.803A>G660.246103363c.441+1G>C*4260.246103363c.842C>G660.246103363c.913–7A>G32160.246103363c.533A>G14160.246103363c.1139C>T14160.246103363c.1250A>G4260.246103363c.506G>C*22260.246103363c.740G>A21250.205086136c.535T>C22150.205086136c.722G>A4150.205086136