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Introduction

Phenylketonuria (PKU; OMIM 261600; https://omim.org/entry/261600) is a disease that belongs to the family of inborn errors of metabolism that is caused by genetic variations in the gene encoding the enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1; OMIM 612349), which is required to break down the amino acid phenylalanine (Phe). PKU, also known as PAH deficiency, is the most common amino acid metabolism disorder that occurs in ~1 out of 24,000 individuals; ~450,000 individuals globally are affected (1-3). PKU is also considered as one of the most common autosomal recessive diseases after thalassemia in the Jordanian population with an incidence of ~1 in 5,263 newborn infants (4). The PAH enzyme catalyzes the conversion of Phe to Tyrosine (Tyr) by using tetrahydrobiopterin (BH4) as a cofactor (5). The defect of this metabolic pathway occurs due to PAH deficiency and leads to accumulation of Phe (hyperphenylalaninemia) and its metabolites. The toxicity of the metabolites leads to multiple organ damage predominantly of the central nervous system (6). If PKU is left untreated, patients will display severe intellectual disability, developmental delay, epilepsy, neurotransmitter depletion, motor deficits and behavioral problems as well as heart problems, eczema and hypopigmentation (3,5,7,8).

The PAH gene is mapped to chromosome 12q22-24.1 and consists of 13 exons and 12 intervening sequences (9). More than 1,000 pathogenic variants in the PAH gene have been reported in ~98% individuals affected by the disease (http://www.biopku.org/home/pah.asp) (10). A small percentage of cases with hyperphenylalaninemia are also caused by variants in genes involved in the BH4 pathway, including QDPR (OMIM 612676), PCBD1 (OMIM 126090) PTS (OMIM: 612719) and GCH1 (OMIM: 600225) or by variants in the DNAJC12 (OMIM 606060) gene (8). The predominant proportion (63%) of the suspected pathogenic alleles in PAH are derived from point mutations that induce missense errors during translation, of which a minority have a significant effect on the PAH enzyme's kinetic properties; yet they possess a greater effect on its functionality by disrupting secondary structures resulting in aggregation, misfolding and degradation of the protein inside the cell (11).

Despite the high prevalence of PKU in Jordan, which affects ~1 individual in 5,263 children, the data on the spectrum of the PAH gene variants remain limited (4). Therefore, the present study aimed to identify the PAH gene variants in a cohort of 77 children diagnosed with PKU in Jordan.

Genetic testing for PKU will enable early diagnosis, treatment and management of patients. It also improves family counseling and reproductive decision-making in the affected families. The identification of PAH variants among Jordanians can also pave the way to more effective screening and diagnostic strategies, which can reduce the incidence of the disease in future generations.

Materials and methods

Subjects and sample collection

A total of 77 children with PKU from 50 families were recruited in the present study, of whom 29 were females (38%) and 48 were males (62%); the age of the participants ranged between 1 month and 30 years old. These patients were diagnosed at the National Center for Diabetes (Amman, Jordan), Endocrinology and Genetics clinics between January 2012 and January 2025. Allelic phenotype values were used to perform genotypic phenotype prediction (classic PKU, mild PKU or mild hyperphenylalaninemia) in 86% of patients. Venous blood samples were collected from all affected children, parents and available family members.

DNA extraction

Genomic DNA from all samples was extracted using the phenol/chloroform method (12). DNA concentration and purity were evaluated using a Nanodrop 2000 C (Thermo Fisher Scientific, Inc.), with an A260/A280 nm ratio of ~1.8.

PAH gene sequencing and variant detection

A total of 13 pairs of primers were designed flanking all exons and exon/intron boundaries of the PAH gene (NM_000277.3) using Primer3 online software (v.0.4.0; https://bioinfo.ut.ee/primer3-0.4.0/) and NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers were tagged with the M13 primers. The primer sequences are shown in Table SI.

PCR amplification was performed on the XP Thermal Cycler (Hangzhou Bori Technology Co., Ltd.). The following conditions were used: Initial denaturation at 95˚C for 5 min followed by 35 cycles of denaturation at 95˚C for 30 sec, annealing at 61˚C for 30 sec, extension at 72˚C for 45 sec and a final elongation at 72˚C for 5 min. The amplified products were purified and sequenced using ABI 3500 genetic analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequenced results were aligned and compared with the reference PAH gene. The identified variants were classified according to the ClinGen PAH Expert Panel Specifications to the Association for Molecular Pathology (AMP) and the American College of Medical Genetics and Genomics (ACMG) guidelines (13,14). Carrier testing was carried out for the parents of each family to validate the identified variants.

Ethical consideration

A signed informed consent was obtained from all families. The present study was approved by the institutional review board/ethical committee of the National Center for Diabetes, Endocrinology and Genetics (approval no. IRB-1/2025; Amman, Jordan).

Results

PAH variant spectrum

A total of 23 disease-causing variants in the PAH gene were identified in 77 patients with PKU from 50 families (Tables I and SII). A total of 30 of these families exhibited a single affected child, 15 had 2 affected children, 3 had 3 affected children and 2 had 4 affected children. In total, 154 alleles were found to carry the identified variants. These variants included 9 missense (39.1%) mutations, 6 splice sites (26.1%), 4 frameshift (17.4%), 2 nonsense (8.7%) mutations and 2 in-frame deletions (8.7%) (Fig. 1A).

Figure 1 The categories of the identified variants and their distribution in PAH domains and the PAH gene. (A) Type of variants identified and their frequency. (B) The distribution of PAH variants in PAH domains. (C) The distribution of detected variants in the PAH gene. PAH, phenylalanine hydroxylase.

Table I The variants identified by Sanger sequencing of the PAH gene in patients with PKU. PAH, phenylalanine hydroxylase; PKU, phenylketonuria.

Table I

The variants identified by Sanger sequencing of the PAH gene in patients with PKU. PAH, phenylalanine hydroxylase; PKU, phenylketonuria.

#	Genomic location (GRCh37)	HGVS cDNA (NM_000277.3)	HGVS protein (NP_000268.1)	Variant Type	Classification	Alleles (N=154)	Allele frequency (%)
1	Chr11:103306594	c.143T>C	p.Leu48Ser	Missense	Pathogenic	6	3.9
2	Chr11:103306618	c.116_118del	p.Phe39del	In-frame deletion	Pathogenic	2	1.3
3	Chr11:103306581	c.155del	p.Leu52 Cysfs*9	Frameshift	Pathogenic	2	1.3
4	Chr11:103306568	c.168_168+ 1delinsAA	p.Glu5 Argfs*3	Splice site	Likely pathogenic	21	13.6
5	Chr11:103288693	c.169_171del	p.Glu57del	In-frame deletion	Likely pathogenic	13	8.4
6	Chr11:103271235	c.441+5G>T	p.?	Splice site	Pathogenic	26	16.9
7	Chr11:103260373	c.509+1G>A	p.?	Splice site	Pathogenic	4	2.6
8	Chr11:103249094	c.526C>T	p.Arg176*	Nonsense	Pathogenic	11	7.1
9	Chr11:103249005	c.593_614del	p.Tyr198 Cysfs*136	Frameshift	Pathogenic	8	5.2
10	Chr11:103246708	c.727C>T	p.Arg243*	Nonsense	Pathogenic	3	1.9
11	Chr11:103246653	c.781delC	p.Arg261 Glufs*80	Frameshift	Likely pathogenic	4	2.6
12	Chr11:103246653	c.782G>A	p.Arg261Glu	Missense	Pathogenic	3	1.9
13	Chr11:103246625	c.810A>T	p.Arg270Ser	Missense	Pathogenic	8	5.2
14	Chr11:103246597	c.838G>A	p.Glu280Lys	Missense	Pathogenic	1	0.6
15	Chr11:103246593	c.842C>T	p.Pro281Leu	Missense	Likely pathogenic	2	1.3
16	Chr11:103245479	c.898G>T	p.Ala300Ser	Missense	Pathogenic	1	0.6
17	Chr11:103240668	c.969+5G>A	p.?	Splice site	Pathogenic	1	0.6
18	Chr11:103237568	c.1066-11G>A	p.?	Splice site	Pathogenic	22	14.3
19	Chr11:103237533	c.1089del	p.Lys363 Asnfs*37	Frameshift	Pathogenic	4	2.6
20	Chr11:103237461	c.1162G>A	p.Val388Met	Missense	Pathogenic	8	5.2
21	Chr11:103237423	c.1199+1G>C	p.?	Splice site	Pathogenic	2	1.3
22	Chr11:103234285	c.1208C>T	p.Ala403Val	Missense	Pathogenic	1	0.6
23	Chr11:103234271	c.1222C>T	p.Arg408Trp	Missense	Pathogenic	1	0.6

A total of 11 of the variants (47.8%) were located in the catalytic domain, 4 (17.4%) were present in the regulatory domain and 2 (8.7%) were located in the oligomerization domain (Fig. 1B). The identified variants were distributed across exons (2, 3, 6-8, 11 and 12) and the splicing regions were present within the introns (2, 4, 5, 9-11) of the PAH gene (Fig. 1C). No variants were identified in exons 1, 4, 5, 9, 10 and 13.

The identified variants were classified as either pathogenic (19 variants), or likely pathogenic (4 variants) (Table I). The five most prevalent variants were the following: c.441+5G>T (16.9%), c.1066-11G>A (14.3%), c.168_168+1delGGinsAA (13.6%), c.169_171del (8.4%) and c.526C>T (7.1%). The remaining variants exhibited frequencies ranging from 0.6 to 5.2% (Table I).

A total of 17 homozygous variants were detected in 88.3% of patients (68/77), and seven exhibited a compound heterozygotic state and were found in 19.1% of patients (9/77) (Tables II and SII). Out of 77 patients, 66 (85.7%) were classified as having classic PKU, while the classification was unavailable for the remaining 11 patients (Table SII).

Table II Genotypes of 77 Jordanian patients with PKU.

Table II

Genotypes of 77 Jordanian patients with PKU.

Zygosity	Allele 1	Allele 2	Number of patients (n=77)
Homozygous	c.143T>C	c.143T>C	3
	c.116_118del	c.116_118del	1
	c.155del	c.155del	1
	c.168_168+1delinsAA	c.168_168+1delinsAA	10
	c.169_171del	c.169_171del	6
	c.441+5G>T	c.441+5G>T	11
	c.509+1G>A	c.509+1G>A	2
	c.526C>T	c.526C>T	5
	c.593_614del	c.593_614del	4
	c.781del	c.781del	2
	c.782G>A	c.782G>A	1
	c.810A>T	c.810A>T	4
	c.842C>T	c.842C>T	1
	c.1066-11G>A	c.1066-11G>A	10
	c.1089del	c.1089del	2
	c.1162G>A	c.1162G>A	4
	c.1199+1G>C	c.1199+1G>C	1
Compound heterozygous	c.727C>T	c.441+5G>T	3
	c.838G>A	c.782G>A	1
	c.898 G>T	c.1066-11G>A	1
	c.969+5G>A	c.1208C>T	1
	c.1222C>T	c.1066-11G>A	1
	c.168_168+1delinsAA	c.169_171del	1
	c.526C>T	c.441+5G>T	1

Novel variants

A single novel homozygous variant [c.781del; p.(Arg261Glufs80)] was identified in two affected children from one family. This variant has not been previously reported in the BIOPKU database, in public databases or in the literature. The pretreatment Phe level was performed for one of the children which was high (1,008 µmol/l). The parents were found to be heterozygous for this variant (Fig. 2).

Figure 2 Electropherogram of the novel variant [c.781del; p.(Arg261Glufs80)]. The parents are heterozygous and the affected children are homozygous.

Discussion

PKU is a recessively inherited metabolic disorder caused by alteration in the PAH gene resulting in hyperphenylalaninemia in the brain and blood. PKU screening has been included in the national newborn screening program since 2006. Although the prevalence of PKU in Jordan is not precisely documented, it has been estimated to be ~1 in 5,263 live births (4). The disorder can be effectively managed via dietary restrictions if diagnosed early, emphasizing the critical role of neonatal screening and genetic diagnosis in patient follow-up and improving long-term outcomes. Sanger sequencing of the PAH gene provides an accurate and reliable method to identify disease-causing variants, enabling confirmation of the diagnosis and facilitating future prevention strategies.

In the present study, the PAH variants were analyzed in a cohort of 77 patients with PKU from 50 unrelated families in Jordan. Following Sanger sequencing of the PAH gene, 23 different variants were identified with a detection rate of 100%. All variants were known to cause PKU and were classified as either pathogenic or likely pathogenic (15-23).

Homozygous variants were identified in 88.3% of patients (68/77). This high frequency was expected due to the high degree of consanguinity marriages among families noted in the present study (86%) (first and second cousins). The results are consistent with those reported in previous studies. Dababneh et al (4) reported a similarly high degree of consanguinity among PKU families in Jordan, which was one generation (87.4%) of patients. The first-degree consanguinity was estimated to be 74.2% (4). Two other studies further indicated 57 and 80% of consanguinity (first or second cousins) among PKU families (18,24). In addition, a study performed on Jordanian patients with inborn error of metabolic disorders including PKU reported that 90.7% of families were consanguineous (25). These findings highlight that the high consanguinity rate in Jordan may contribute to the increased prevalence of the disease.

Of the 23 variants, c.441+5G>T, c.1066-11G>A, c.168_168+1delGGinsAA, c.169_171del, and c.526C>T were the most common accounting for ~2/3 of the identified variants (Table I).

The two common variants in the present study are splice site variants (c.441+5G>T and c.1066-11G>A). The c.441+5G>T was the most common mutant allele with a frequency of ~16.9%. This variant has been previously reported in different countries including those in middle East but not in Jordanian patients (14,16,19,26). The (c.1066-11G>A) variant was the second common mutant allele with a frequency of 14.3% (Table II). This variant is known as the ‘Mediterranean mutation’ since it has been reported as the most common variant in the Mediterranean region. This variant has been reported previously in one Jordanian patient (18). However, it was identified in 11 patients in the present study.

The third variant (c.168_168+1delGGinsAA) has been previously identified in multiple affected individuals either as a homozygotic or compound heterozygotic state with other pathogenic variants (17,27). In the present study, this variant was detected in seven families in a homozygotic state (10/77) and as a compound heterozygotic state (1/77), which is consistent with the findings of a previous study that reported it in eight Palestinian families (17).

The fourth variant (c.169_171del) was also detected in five families, with a frequency of 7.1%. It was detected in a homozygotic (5/77) and a compound heterozygotic state (1/77); however, it has been reported previously in a homozygotic state in a single Jordanian patient (18).

The fifth variant (c.526C>T) was also detected in the present study in five families, with a frequency of 8.4%. It was identified as a homozygous (6/77) as well as a compound heterozygous (1/77) state; however, it has been previously reported in a homozygotic state in four Jordanian patients (18).

In the present study, all identified variants were distributed throughout exons (2, 3, 6-8, 11 and 12) and splicing regions in introns (2, 4, 5, 9-11) of the PAH gene (Fig. 1C), which is consistent with the data reported previously in the study conducted on Jordanian patients with PKU in which the reported variants were distributed in the exons (3, 6, 7, 9 and 11) as well as in intron 10(18). This further supports the idea that the distribution of PAH variants varies among race, ethnicity and geographical regions. Moreover, it will be very helpful for the diagnosis of future cases with PKU by focusing on the identified variants (10).

In the present study, one novel variant (c.781del) was identified in two patients from the same family. This variant has never been reported in the BIOPKU database or public databases or in previous literature reports. This variant is predicted to cause a frameshift and premature stop codon (p.Arg261Glufs80), which will likely result in loss-of-function. The pathogenicity of this variant could not be predicted by in silico tools; however, several frameshift variants have been reported as pathogenic or likely pathogenic in the ClinVar database such as c.740del, c.790del and c.722del (Variation ID: 1065372, 371102 and 102806, respectively). Furthermore, the carrier status of this variant was confirmed in both parents. In one of the two probands, the Phe level following pretreatment was 1,008 µmol/l, which correlated with the severity of the disease in the two patients who had an aggressive phenotype and presented with hypopigmentation and increased muscle tone. Based on these findings, this variant can be classified as likely pathogenic according to ACMG/AMP guidelines (PVS1_very strong, PM2_supporting and PP4_supporting). Further experimental studies are needed to clarify the pathogenicity of this variant.

Carducci et al (18) previously reported 10 known pathogenic variants in 20 Jordanian patients with PKU, six of whom were detected in the cohort. Compared with that study, the cohort of the present study revealed a higher number of variants, suggesting a genetic diversity of PKU within the Jordanian population. Moreover, the larger size of the cohort provides a more comprehensive analysis. These findings reinforce the need for comprehensive genetic screening programs to fully characterize the spectrum of PKU variants and their distribution in the population examined. Following comparison with the study reported by Carducci et al (18), a total of 27 variants have been identified among Jordanian patients with PKU, which indicated the high prevalence of the disease in the population examined.

While the findings of the present study provide valuable insights into the spectrum of the PAH gene variants in the studied cohort, certain limitations need to be highlighted. Firstly, although the sample size of 77 patients was considerable for a monogenic disorder such as PKU, and causative variants were successfully identified in all cases, the exclusive use of Sanger sequencing represents a limitation, as it is not suitable for detecting large deletions or duplications, which account for approximately 12 and 2.1% of the reported PAH mutations (8). Secondly, since most cases have received treatment prior to performing the genetic test, detailed clinical descriptions and Phe levels before and after treatment were unavailable for the majority of cases, which precluded genotype/phenotype correlation. Since disease severity is influenced by residual enzyme activity and variant-based dietary response, future work integrating comprehensive genetic and clinical analysis is required to fully elucidate the relationship between specific PAH variants and phenotypic outcomes (28,29).

The results of the present study were helpful for at least 8 families who had planned a subsequent pregnancy. Prenatal diagnosis was offered for these families, resulting in six healthy children.

In the present study, a PAH gene variant spectrum among Jordanian patients with PKU were described. A total of 22 previously published disease-causing variants and one novel variant in 77 patients were successfully identified. The findings of the present study underscore the effectiveness of full gene sequencing in the diagnosis of PKU, which can be implemented in medical practice as a reliable and effective molecular diagnostic tool for clinical practice. Furthermore, the results of the present study will contribute to the development of genetic screening for common PAH gene variants in the region of the examined population. Genetic diagnosis will improve counseling, enabling early treatment and management of patients and risk assessment and prenatal diagnosis of patients with PKU.

Furthermore, the results of the present study will contribute to the establishment of screening for common variants in the examined region. Genetic diagnosis will enhance counseling of the families, enabling timely treatment and management of patients and support informed reproductive decision-making for families affected by PKU.

Supplementary Material

Oligonucleotide primers used for PAH gene sequencing.

Summary of demographic and clinical characteristics of patients with PKU.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author. The data generated in the present study may be found in the BIOPKU database under accession number PAH3467 or at the following URL: https://www.biopku.org/home/home.asp; and in the ClinVar database under accession number 508849 or at the following URL: https://www.ncbi.nlm.nih.gov/clinvar/submitters/508849.

Authors' contributions

RF, KA, AAR and HA performed the experiments and collected the samples. RF and KAR prepared the original draft. LM performed clinical assessment. MEK performed supervision, project administration, prepared the original draft, reviewed and edited the final draft. MS aided in the conceptualization, methodology, validation, raw data verification and analysis, supervision, preparation of the original draft, review and editing of the final draft. MS and MEK confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Ethical approval was obtained from the Institutional Review Board (IRB) committee (Protocol number IRB-1/2025) of the National Center for Diabetes, Endocrinology and Genetics (Amman, Jordan). An informed consent was obtained from all participants and parents or legal guardians prior to their enrollment in the study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References