Significance of utilizing in silico structural analysis and phenotypic data to characterize phenylalanine hydroxylase variants: A PAH landscape.

Phenylketonuria (PKU) is a genetic disorder caused by variations in the phenylalanine hydroxylase (PAH) gene. Among the 3369 reported PAH variants, 33.7% are missense alterations. Unfortunately, 30% of these missense variants are classified as variants of unknown significance (VUS), posing challenges for genetic risk assessment. In our study, we focused on analyzing 836 missense PAH variants following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines specified by ClinGen PAH Variant Curation Expert Panel (VCEP) criteria. We utilized and compared variant annotator tools like Franklin and Varsome, conducted 3D structural analysis of PAH, and examined active and regulatory site hotspots. In addition, we assessed potential splicing effect of apparent missense variants. By evaluating phenotype data from 22962 PKU patients, our aim was to reassess the pathogenicity of missense variants. Our comprehensive approach successfully reclassified 309 VUSs out of 836 missense variants as likely pathogenic or pathogenic (37%), upgraded 370 likely pathogenic variants to pathogenic, and reclassified one previously considered likely benign variant as likely pathogenic. Phenotypic information was available for 636 missense variants, with 441 undergoing 3D structural analysis and active site hotspot identification for 180 variants. After our analysis, only 6% of missense variants were classified as VUSs, and three of them (c.23A>C/p.Asn8Thr, c.59_60delinsCC/p.Gln20Pro, and c.278A >T/p.Asn93Ile) may be influenced by abnormal splicing. Moreover, a pathogenic variant (c.168G>T/p.Glu56Asp) was identified to have a risk exceeding 98% for modifications of the consensus splice site, with high scores indicating a donor loss of 0.94. The integration of ACMG/AMP guidelines with in silico structural analysis and phenotypic data significantly reduced the number of missense VUSs, providing a strong basis for genetic counseling and emphasizing the importance of metabolic phenotype information in variant curation. This study also sheds light on the current landscape of PAH variants.

Comprehensive analyses of phenylalanine hydroxylase variants and phenotypic characteristics of patients in the eastern region of Türkiye.

OBJECTIVES: Phenylalanine hydroxylase (PAH) is predominantly a hepatic enzyme that catalyzes phenylalanine (Phe) into tyrosine, which is the rate-limiting step in Phe catabolism. Biallelic variants in the PAH gene cause PAH enzyme deficiency. Phenylketonuria (PKU) is an autosomal recessive disorder that causes neurologic, behavioral, and dermatological findings. PKU could be divided clinically into three types based on the blood Phe levels: classic phenylketonuria (cPKU), mild-moderate phenylketonuria (mPKU), and mild hyperphenylalaninemia (MHP). This study aimed to determine the phenotypic and genotypic characteristics of Turkish PKU patients in the eastern region of Türkiye. METHODS: Demographic characteristics, serum Phe levels, treatments, and PAH variants of 163 patients with PKU and hyperphenylalaninemia (HPA) were retrospectively evaluated. Blood Phe levels of the patients were analyzed with the high-performance liquid chromatography method. For PAH gene analysis, next-generation sequencing was performed. RESULTS: Of the 163 patients included in the study, 38 (23.3 %) had cPKU, 16 (9.8 %) had mPKU, and 109 (66.9 %) had MHP. Homozygous variants in the PAH gene were detected in 66 (40.5 %) of the patients, while compound heterozygous variants were detected in 97 (59.5 %) patients. Two novel and 35 recurrent variants in the PAH gene were detected. Of the two novel variants, one was missense (p.Phe351Leu) and the other was frameshift (p.Met276Cysfs*65). The most frequently detected variants were p.Thr380Met (18 %), p.Arg261Gln (16.8 %), and p.Ala300Ser (12.8 %). All patients with the homozygous c.1066-11G>A variant exhibited cPKU phenotype. The c.898G>T (p.Ala300Ser), c.1139C>T (p.Thr380Met), and c.1208C>T (p.Ala403Val) variants were statistically related to mild phenotype. On the other hand, c.592_613del (p.Tyr198Serfs*136), c.1028A>G (p.Tyr343Cys), and c.782G>A (p.Arg261Gln) variants were more frequently detected in the cPKU group. CONCLUSIONS: Our study, conducted with patients from the eastern region of Türkiye, demonstrates the genetic heterogeneity in the Turkish population. Simultaneously, our research contributes to genotype-phenotype correlation and expands the genotypic spectrum by identifying novel variants.

Genetic Landscape and Clinical Features of Hyperphenylalaninemia in North Ossetia-Alania: High Frequency of P281L and P211T Genetic Variants in the PAH Gene.

This study, conducted in the Republic of North Ossetia-Alania (RNOA), aimed to explore the genetic landscape of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) in the Ossetian population using data from newborn screening (NBS). Through comprehensive molecular genetic analysis of 29 patients with HPA from diverse ethnic backgrounds, two major genetic variants in the PAH gene, P281L and P211T, were identified, constituting 50% of all detected pathogenic alleles in Ossetian patients. Remarkably, these variants exhibited an exceptionally high frequency in the Ossetian population, surpassing global prevalence rates. This study unveiled a notable prevalence of mild forms of HPA (78%), underscoring the importance of genetic counseling for carriers of pathogenic variants in the PAH gene. Moreover, the findings emphasized the necessity for ongoing monitoring of patients with mild forms, as they may lack significant symptoms for diagnosis, potentially impacting offspring. Overall, this research offers valuable insights into the genetic landscape of HPA and PKU in the Ossetian population.

[Analysis of PAH gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria].

OBJECTIVE: To explore the characteristics of phenylalanine hydroxylase (PAH) gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria (PKU). METHODS: Forty three PKU pedigrees diagnosed at the First Affiliated Hospital of Zhengzhou University between 2019 and 2021 were selected as the study subjects. Variants of the PAH gene of the probands were screened by high-throughput sequencing, and candidate variants were verified by Sanger sequencing. Negative cases were further analyzed by multiplex ligation-dependent probe amplification (MLPA) to detect large fragment deletions and duplications of the PAH gene. For 43 women undergoing subsequent pregnancy, Sanger sequencing, MLPA, combined with short tandem repeats (STR) sequence-based linkage analysis, were carried out for prenatal diagnosis. RESULTS: Among the 86 alleles carried by the 43 probands, 78 nucleotide variants (90.70%) and 3 large deletions (3.49%) were found based on high-throughput sequencing and MLPA. The 81 mutant alleles had included 21 missense variants, 5 splice site variants, 4 nonsense variants, 2 microdeletions, 1 insertional variant and 2 large fragment deletions. Relatively common variants have included p.Arg243Gln (23.26%), p.Arg111Ter (8.14%), EX6-96A>G (6.98%), p.Val399Val (5.81%) and p.Arg413Pro (4.65%). Most of the variants were located in exons 7, 11, 3, 6 and 12. For the 43 families undergoing prenatal diagnosis, 9 fetuses (20.45%) were diagnosed with PKU, 20 (45.45%) were heterozygous carriers, and 15 (34.09%) did not carry the same pathogenic allele as the proband. All neonates were followed up till 6 months old, and the accuracy of prenatal diagnosis was 100%. CONCLUSION: The combination of high-throughput sequencing, Sanger sequencing, MLPA and linkage analysis can increase the diagnostic rate of PKU and attain accurate prenatal diagnosis.

Unveiling the genetic tapestry: Rare disease genomics of spinal muscular atrophy and phenylketonuria proteins.

Rare diseases, defined by their low prevalence, present significant challenges, including delayed detection, expensive treatments, and limited research. This study delves into the genetic basis of two noteworthy rare diseases in Saudi Arabia: Phenylketonuria (PKU) and Spinal Muscular Atrophy (SMA). PKU, resulting from mutations in the phenylalanine hydroxylase (PAH) gene, exhibits geographical variability and impacts intellectual abilities. SMA, characterized by motor neuron loss, is linked to mutations in the survival of motor neuron 1 (SMN1) gene. Recognizing the importance of unveiling signature genomics in rare diseases, we conducted a quantitative study on PAH and SMN1 proteins of multiple organisms by employing various quantitative techniques to assess genetic variations. The derived signature-genomics contributes to a deeper understanding of these critical genes, paving the way for enhanced diagnostics for disorders associated with PAH and SMN1.

Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

[In vitro expression and functional analyses of the mutants p.R243Q, p.R241C and p.Y356X of the human phenylalanine hydroxylase]. / äººè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶çªå˜ä½“p.R243Q、p.R241C和p.Y356Xçš„ä½“å¤–è¡¨è¾¾åŠåŠŸèƒ½ç ”ç©¶.

OBJECTIVES: To study the in vitro expression of three phenylalanine hydroxylase (PAH) mutants (p.R243Q, p.R241C, and p.Y356X) and determine their pathogenicity. METHODS: Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein. Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells. Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants, and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay. RESULTS: Bioinformatics analysis predicted that all three mutants were pathogenic. The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control (P>0.05), while the mRNA expression level of the p.Y356X mutant significantly decreased (P<0.05). The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control (P<0.05). The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control (P<0.05), while there was no significant difference between the p.R243Q mutant and the wild type control (P>0.05). CONCLUSIONS: p.R243Q, p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells, with p.R241C and p.Y356X mutants also affecting the function of PAH protein. These three PAH mutants are to be pathogenic.

[Analysis of pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase gene].

OBJECTIVE: To explore the pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase (PAH) gene among patients with PAH deficiency. METHODS: Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed. RESULTS: Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c.158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c.158G>A variants and a heterozygous c.842+2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c.[158G>A,842+2T>A]/c.728G>A and c.[158G>A,842+2T>A]/c.611A>G, respectively, and one with c.[158G>A, c.722G>A]/c.728G>A. The c.158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. CONCLUSION: When the c.158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

Weakened tanning ability is an important mechanism for evolutionary skin lightening in East Asians.

The evolution of light-skin pigmentation among Eurasians is considered as an adaptation to the high-latitude environments. East Asians are ideal populations for studying skin color evolution because of the complex environment of East Asia. Here, we report a strong selection signal for the pigmentation gene phenylalanine hydroxylase (PAH) in light-skinned Han Chinese individuals. The intron mutation rs10778203 in PAH is enriched in East Asians and is significantly associated with skin color of the back of the hand in Han Chinese males (P < 0.05). In vitro luciferase and transcription factor binding assays show that the ancestral allele of rs10778203 could bind to SMAD2 and has a significant enhancer activity for PAH. However, the derived T allele (the major allele in East Asians) of rs10778203 decreases the binding activity of transcription factors and enhancer activity. Meanwhile, the derived T allele of rs10778203 shows a weaker ultraviolet radiation response in A375 cells and zebrafish embryos. Furthermore, rs10778203 decreases melanin production in transgenic zebrafish embryos after ultraviolet B (UVB) treatment. Collectively, PAH is a potential pigmentation gene that regulates skin tanning ability. Natural selection has enriched the adaptive allele, resulting in weakened tanning ability in East Asians, suggesting a unique genetic mechanism for evolutionary skin lightening in East Asians.

PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Systems Biology and Inborn Error of Metabolism: Analytical Strategy in Investigating Different Biochemical/Genetic Parameters.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.

Analysis of gene variation and long-term follow-up in children with phenylalanine hydroxylase deficiency diagnosed by newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥ç¡®è¯Šè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶ç¼ºä¹ç—‡æ‚£è€ åŸºå› å˜å¼‚åŠé•¿æœŸéšè®¿åˆ†æž.

OBJECTIVES: To retrospectively analyze the variation and characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency. METHODS: Clinical data, treatment and follow-up results of 198 patients with PAH deficiency diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis of 55 patients with PAH deficiency diagnosed by newborn screening in Jinan and 213 patients referred from the surrounding areas of Jinan were summarized. Gene variations were checked by a customized Panel gene detection method. Blood phenylalanine-concentration and physical development indicators including height and weight were regularly monitored. Intellectual development was assessed using a neuropsychological development scale for patients aged 0-6 years and academic performance, and brain injury in patients was assessed using brain magnetic resonance imaging. RESULTS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. The genotype of c.158G>A variation is compound heterozygous variation, with mainly a mild hyperpheny-lalaninemia. 168 patients with PAH deficiency who were followed-up regularly had normal physical development without dwarfism or malnutrition. Among the 33 preschool patients who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine patients with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, one case was normal, and 8 cases were abnormal. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. Compared with the other eight patients, the blood phenylalanine concentration of the normal child was better and stably controlled within the ideal range. CONCLUSIONS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. After standardized treatment, most patients with PAH deficiency diagnosed by screening can obtain normal growth and intellectual development in adolescence, but there are different degrees of organic lesions in the cerebral white matter.

Identification of mutations in the pah gene in pku patients in the state of mato grosso / Identificação de mutações no gene da pah em pacientes com fenilcetonúria do estado de mato grosso

ABSTRACT Objective: To identify phenylalanine hydroxylase (PAH) mutations in patients with phenylketonuria (PKU) from the Newborn Screening Service in Mato Grosso, Midwest Brazil. Methods: This is a cross-sectional descriptive study. The sample consisted of 19 PKU patients diagnosed by newborn screening. Molecular analysis: DNA extraction using the "salting-out" method. Detection of IVS10nt-11G>A, V388M, R261Q, R261X, R252W, and R408W mutations by the restriction fragment length polymorphism (RFLP) technique. Results: Two mutant alleles were identified in four patients (21.1%), one allele in five patients (26.2%), and none in the remaining ten patients (52.6%). A total of 13/38 alleles were detected, corresponding to 34.2% of the PAH alleles present. The most prevalent variant was V388M (13.2% of the alleles), followed by R261Q (10.1%) and IVS10nt-11G>A (7.9%). Three variants (R261X, R252W, and R408W) were not found. The most frequent mutation types were: missense mutation in eight alleles (18.4%) and splicing in four alleles (10.5%). The model proposed by Guldberg to determine a genotype/phenotype correlation was applied to four classical PKU patients with two identified mutations. In three of them, the predicted moderate/moderate or moderate PKU phenotype did not coincide with the actual diagnosis. The prediction coincided with the diagnosis of one classic PKU patient. The estimated incidence of PKU for Mato Grosso, Brazil, was 1:33,342 live births from 2003 to 2015. Conclusion: The only mutations found in the analyzed samples were the IVS10nt-11G>A, V388M, and R261Q. The genotype/phenotype correlation only occurred in four (5.3%) patients.

RESUMO Objetivo: Identificar mutações da fenilalanina hidroxilase (PAH) em pacientes com PKU (fenilcetonúria) do Serviço de Triagem Neonatal em Mato Grosso. Métodos: Estudo de corte transversal. Amostra composta de 19 pacientes com PKU através do exame de triagem neonatal biológica. Análise molecular: a) extração de DNA pela metodologia "salting out". B) detecção de mutações IVS10nt-11G>A, V388M, R261Q, R261X, R252W e R408W pela técnica de polimorfismo de comprimento de fragmento de restrição (RFLP). Resultados: Dois alelos foram identificados em quatro pacientes (21,1%), um alelo em cinco pacientes (26,2%) e nenhum nos dez pacientes restantes (52,6%). Um total de 13/38 alelos foram identificados, correspondendo a 34,2% dos alelos PAH presentes. A variante mais prevalente foi a V388M (13,2% dos alelos), seguida de R261Q (10,1%) e IVS10nt-11G>A (7,9%). Três variantes (R261X, R252W e R408W) não foram encontradas. Os tipos de mutações mais frequentes foram: troca de sentido em oito alelos (18,4%) e emenda em quatro alelos (10,5%). O modelo proposto por Guldberg para determinar uma correlação genótipo/fenótipo foi aplicado para quatro pacientes clássicos de PKU, com duas mutações identificadas. Em três, o fenótipo previsto de PKU moderada/moderada ou moderada não coincidiu com o diagnóstico real. A predição coincidiu com o diagnóstico de um paciente PKU clássico. A incidência de PKU estimada para Mato Grosso, Brasil foi de 1:33.342 nascidos vivos para o período de 2003 a 2015. Conclusões: Foram encontradas apenas as mutações IVS10nt-11G>A, V388M, R261Q nas amostras analisadas. A correlação genótipo/fenótipo ocorreu em quatro (5,3%) pacientes.

¿Qué debe saber el pediatra de las hiperfenilalaninemias? / What should the paediatrician know about hyperphenylalaninaemia?

Las hiperfenilalaninemias se definen por un nivel sanguíneo de fenilalanina sobre 2 mg/dl. La principal causa es una mutación en el gen que codifica la fenilalanina hidroxilasa que cataliza la reacción que transforma la fenilalanina en tirosina. Las hiperfenilalaninemias se clasifican en benignas o leves, y las fenilcetonurias en leves, moderadas y clásicas. Debido a que su detección más allá del periodo neonatal causa retardo mental severo, desde 1992 en Chile su detección, junto con la del hipotirodismo congénito, es parte del Programa Nacional de Pesquisa Neonatal. Este artículo pretende responder las preguntas más comunes que se puede hacer el pediatra cuando enfrenta a un paciente con hiperfenilalaninemias.

Hyperphenylalaninaemias are defined by a blood phenylalanine over 2 mg/dl. The main cause is due to a mutation in the gene that codes the phenylalanine hydroxylase that catalyses the reaction that converts phenylalanine into tyrosine. The hyperphenylalaninaemias are classified into benign or mild hyperphenylalaninaemias, or mild, moderate or classic phenylketonurias. Due to its delayed detection outside the neonatal period it causes severe mental retardation. Its detection along with congenital hypothyroidism has been part of the National Neonatal Screening Program since 1992 in Chile. This article aims to answer the most common questions asked by the paediatrician when faced with a patient with hyperphenylalaninaemias.

Frequencies of phenylalanine hydroxylase mutations I65T, R252W, R261Q, R261X, IVS10nt11, V388M, R408W, Y414C, and IVS12nt1 in Minas Gerais, Brazil

In order to determine the phenylketonuria (PKU) mutation spectrum in the population of Minas Gerais State, Brazil, 78 unrelated PKU patients found by the neonatal screening program from 1993 to 2003 were tested for nine phenylalanine hydroxylase mutations. These mutations were selected due to their high frequencies in other Brazilian populations and in Portugal, where the largest contingent of the Caucasian component of the Brazilian population originated from. The most frequent mutations were V388M (21%), R261Q (16%), IVS10nt11 (13.4%), I65T (5.7%), and R252W (5%). The frequencies of the other four mutations (R261X, R408W, Y414C, and IVS12nt1) did not reach 2%. By testing these nine mutations, we were able to identify 64% of the PKU alleles in our sample. V388M frequency was higher than in any other known population and almost three times larger than that observed in Portugal, probably reflecting genetic drift. The mutation profile, as well as the relative frequency of the different mutations, suggest that the Minas Gerais population more closely resembles that of Portugal than do the other Brazilian populations that have already been tested.