Molecular characterization of phenylketonuria in South Brazil

doi:10.1016/S1096-7192(03)00032-5

Molecular Genetics and Metabolism

Volume 79, Issue 1, May 2003, Pages 17-24

https://doi.org/10.1016/S1096-7192(03)00032-5 Get rights and content

Abstract

Phenylketonuria (PKU) is an autosomal recessive disorder due to phenylalanine hydroxylase (PAH) deficiency. The PAH gene, located at 12q22–q24.1, includes about 90 kb and contains 13 exons. To date, more than 420 different alterations have been identified in the PAH gene. To determine the nature and frequency of PAH mutations in PKU patients from South Brazil, mutation analysis was performed on genomic DNA from 23 unrelated PKU patients. The 13 exons and flanking regions of the PAH gene were amplified by PCR and the amplicons were analyzed by single strand conformation polymorphism (SSCP). Amplicons that showed abnormal migration patterns were analyzed by restriction endonuclease digestion and/or sequencing. Twenty-two previously reported mutations were identified including R261X, R408W, IVS2nt5g → c, R261Q, and V388M. Polymorphisms were observed in 48.8% of the PKU patients, the most frequent being IVS2nt19t → c, V245V, and IVS12nt-35c → t. In addition, two novel sequence variants were identified: 1378g → t in the 3^′-untranslated region in exon 13 which may be disease-causing and an intron 12 polymorphism, IVS12nt-15t → c. The mutation spectrum in the patients from Southern Brazil differed from that observed in patients from other Latin American countries and further defined the molecular heterogeneity of this disease.

Introduction

Phenylketonuria (PKU) is an autosomal recessive disease caused by phenylalanine-4-hydroxylase (PAH) deficiency. PAH is a liver-specific enzyme that catalyzes the hydroxylation of l-phenylalanine (Phe) to l-tyrosine (Tyr) in the presence of the cofactor, tetrahydrobiopterin (BH₄). The deficiency of this enzyme leads to the storage of Phe in the tissues and plasma of patients, interfering in both Tyr and tryptophan (Trp) metabolism. Mental retardation is the main debilitating symptom observed in PKU patients and is prevented by early treatment with a low-phenylalanine diet [1].

The incidence of PKU is variable, being high in Turkey (around 1 in 2600 births), and low in Japan (1 in 120,000 births). The incidence in Caucasians is approximately 1:10,000, giving a heterozygote frequency of 1:50 to 1:70 [1]. Several mechanisms have been proposed to explain the relatively high PKU frequency in humans including founder effect/genetic drift, selective advantage of heterozygotes, reproductive compensation, high mutational rate, and involvement of multiple loci that give rise to similar disease phenotypes [2].

The PAH gene locus is located at chromosome 12q22–24.1, spans about 90 kb and contains 13 exons, that code for a mRNA of about 2.5 kb [3], [4]. To date, over 420 mutations in the PAH gene (at least 385 pathogenic and 27 non-pathogenic) have been identified, which explains the wide clinical variability observed in PKU. In addition to the PAH mutations, modifier genes and other factors presumably are responsible for the variability in disease expression [5], [6]. The frequency and distribution of these mutations have been analyzed in different populations and there are no predominant or common PKU-associated mutations in the general population with the exception of certain ethnic or demographic groups (e.g., [7], [8], [9], [10] and (http://www.pahdb.mcgill.ca/) [11]. The PAH cDNA sequence contains a large number of polymorphisms, which have been used to establish polymorphic haplotypes at the PAH locus [3], [12].

In a previous multicenter study in Brazil, which included some patients from our sample, certain PKU mutations that were present in patients from the Iberian Peninsula (I65T, IVS10nt-11g → a and V388M) and Eastern and Northern European regions (R408W and IVS12nt1g → a) were specifically evaluated [13]. Although analysis of 8 patients did not identify the IVS10nt-11g → a mutation, the I65T, V388M, R408W, and IVS12nt1g → a mutations were found in 20, 6, 6, and 6%, respectively [13].

The population of Southern Brazil is very heterogeneous, with a high proportion of individuals from Portugal, Italy, Germany, Spain, and Poland. In order to investigate the molecular basis of PKU in South Brazil, mutation analysis was performed in 23 unrelated PKU patients, in whom the entire PAH coding region, and adjacent sequences were amplified by PCR and analyzed by restriction endonuclease digestion, SSCP, and/or sequencing. These studies identified common, rare, and novel PAH mutations in patients from South Brazil.

Section snippets

Patients

Twenty-three unrelated PKU patients, born in South Brazil (16 from Rio Grande do Sul state and 7 from Santa Catarina state, were diagnosed in the Medical Genetics Service of Hospital de Clı́nicas de Porto Alegre) and studied with informed consent.

Most patients were Caucasians (94.5%), “Brazilian” (20.7%) or descendants of Portuguese (25.5%), Germans (25.5%), Italians (18.4%), or others (9.9%). Blacks represented only 5.4% of the sample. Nine patients were diagnosed late, while 14 were diagnosed

PAH mutations and polymorphisms in 23 classic PKU patients

The combined approach of SSCP, restriction endonuclease digestion and sequencing analysis resulted in the identification of disease-causing mutations in all 23 classic PKU patients as well as detection of a variety of polymorphisms. These included 21 different sequence alterations: 17 disease-associated mutations including one novel sequence variant, 1378g → t, in the 3^′-untranslated region (3^′-UTR) in exon 13, that may be disease-causing (Table 2), and four polymorphisms including one novel

PAH mutations and polymorphisms in classic PKU patients

The Portuguese arrived in Brazil in the 16th century, and the immigration of other Europeans to Brazil began in the 19th century. The South received immigrants mainly from Spain, Italy, Germany, and Poland. These European groups, together with the local Indians, and with Africans that came as slaves until the 19th century, comprise the population of Southern Brazil.

Among the 23 unrelated PKU patients, 17 disease-causing mutations and four polymorphisms were identified, including two novel

Acknowledgments

The authors are grateful to patients and their families, for providing the blood samples, and to physicians of Medical Genetics Service. This work was supported by CAPES, CNPq, FAPERGS, FIPE-HCPA, and PRONEX-MCT. This work was supported in part by grants from the National Institutes of Health including a research grant (R37 DK 34045 Merit Award), a grant (5 MO1 RR00071) for the Mount Sinai General Clinical Research Center Program from the National Center of Research Resources, and a grant (5

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Phenylalanine hydroxylase deficiency in south Italy: Genotype-phenotype correlations, identification of a novel mutant PAH allele and prediction of BH<inf>4</inf> responsiveness
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We identify the same mutation associated with p.A403V, which has a low degree of severity, in a patient belonging to another group of our patients showing the MHP phenotype. So far, the variant c.1378G>T was previously reported in literature in only one other patient who, though, inherited in trans the severe mutation p.R408Q [23]. Although the effect of this lesion was not determined, it is likely that this 3′-UTR substitution could alter mRNA polyadenylation and/or stability with a great impact upon characteristics of the PAH enzyme.
We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 33 Italian PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed by direct sequencing of the patients' genomic DNA.
Thirty-three different disease causing mutations were identified in our patient group, including 19 missense, 6 splicing, 3 nonsense, 5 deletions, with a detection rate of 100%. The most prevalent mutation was the IVS10−11G>A, accounting for 12.1% of PKU alleles studied. Other frequent mutations were: p.R261Q (9.1%), p.P281L (7.6%), and p.R408W (6.1%). We also identified one novel missense mutation, p.H290Q. A spectrum of 31 different genotypes was observed and a genotype based predictions of BH₄-responsiveness were assessed. Among all genotypes, 13 were predicted to be BH₄-responsive represented by thirteen PKU families. In addition, genotype–phenotype correlations were performed. This study reveals the importance of a full genotyping of PKU patients and the prediction of BH₄-responsiveness, not only because of the definitive diagnosis and prediction of the optimal diet, but also to point out those patients that could benefit from new therapeutic approach. They may potentially benefit from BH₄ therapy which, combined with a less strict diet, or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions.
Mutation spectrum of phenylketonuria in Syrian population: Genotype-phenotype correlation
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Some associations between mutations and polymorphisms were observed in this study. The IVS2+5G>C mutation occurred on the same allele with the IVS2+19T>C polymorphisms in two patients; similar results were previously reported in the Egyptian and Brazilian patients (Hashem et al., 1996; Santana da Silva et al., 2003). As well as the p.F55>Lfs mutation occurred on the same allele with the IVS9+43G>T polymorphisms in two patients, and the p.S310F mutation occurred on the same allele with the Q232Q polymorphisms in two patients.
Characterization of the molecular basis of phenylketonuria (PKU) in Syria has been accomplished through the analysis of 78 unrelated chromosomes from 39 Syrian patients with PKU. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by using molecular detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR and direct DNA sequencing. 56.4% of the patients had cPKU. A mutation detection rate of 79.49% was achieved and sixteen different mutations were found: missense 56.25%, splice site 37.5%, and frameshift 6.25%. The predominant mutation in this population sample was p.R261Q G>A, p.F55>Lfs and p.R243Q G>A. No mutation in six PKU patients was observed. In 57.9% of patient genotypes, the metabolic phenotype could be predicted. The identification of the mutations in the PAH gene and the genotype–phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counseling for the patient's relatives.
Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran
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Also S231P and R176X mutations with Q232Q polymorphism were on the same allele in cis form. Similar associations have been reported in other population such as Brazilian patients (Santana da Silava et al., 2003). Exon 6, 7 and 11 and their adjacent intronic regions carry 74% of the mutant alleles.
Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.
Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.
A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).
With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.
Mutation spectrum of phenylketonuria in Iranian population
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Also, the IVS2+5G>C mutation and IVS2+19T>C and IVS12-35C>T polymorphisms associated in cis form. Similar associations have been reported in other population, pointing to a common ancestral origin, but in this study there is no association between p.R261X mutation and IVS2+19T>C polymorphism [20]. In the present study, we chose SSCP over other techniques (e.g. CSGE, DGGE) for scanning mutations because it is easy to perform, cost-effective and sensitive [21].
Identification of molecular basis of phenylketonuria (PKU) in Iran has been accomplished through the analysis of 248 unrelated chromosomes from 124 Iranian classic PKU subjects. Phenylalanine hydroxylase (PAH) gene mutations were analyzed through a combined approach in which p.S67P, p.R252W, p.R261Q, p.R261X, p.L333F, IVS10-11G>A, IVS11+1G>C, p.L364del, p.R408Q and p.R408W mutations were first screened by PCR of PAH gene exons 3, 7, 10, 11 and 12, followed by digestion with the appropriate digestion enzymes. Subsequently SSCP analysis for exons 2, 6, 7 and 11 of the PAH gene and finally, sequencing of 13 PAH gene exons have been used to study uncharacterized PKU chromosomes. 26 different mutations were found. The predominant mutation in this population sample was IVS10-11G>A, with a frequency of 24.6%. Nine mutations (IVS10-11G>A, p.R261Q, p.P281L, IVS11+1G>C, p.K363>NFS, p.R243X, IVS2+5G>C, p.R261X and p.R252W) represent almost 84% of all PKU chromosomes studied. IVS10-11G>A mutation is the major PKU-causing mutation throughout the Mediterranean region. The finding of the high prevalence of this mutation in Iranian population is consistent with the historical and geographical links between Iranian and Mediterranean populations.
Phenylalanine hydroxylase gene mutations in phenylketonuria patients from India: Identification of novel mutations that affect PAH RNA
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Analysis of seven Indian phenylketonuria families has revealed four novel mutations in the phenylalanine hydroxylase gene; two affected consensus splice sequence and the 3′ UTR, respectively, while the other two were single base insertion and deletion mutations, respectively. A novel 3′ splice site mutation c.168-2A>G resulted in the activation of a cryptic 3′ splice site that generated a premature termination codon leading to very low levels of the mutant transcript, probably due to activation of the nonsense-mediated decay (NMD) pathway. This is probably the first report of PKU caused by the activation of NMD.
Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles
2007, Molecular Genetics and Metabolism
Phenylketonuria (PKU, MIM 261600; EC 1.14.16.1) results from mutations in the phenylalanine hydroxylase (PAH) gene. Newborn metabolic disease screening uses blood dried on filter paper (DBS) to prospectively identify candidate newborns affected with PKU via an elevated concentration of phenylalanine. However, it is then important to confirm the specific category of PKU since classical PKU requires a stringent diet while milder categories may not require diet and a very important BH4-responsive category may be treated with the PAH cofactor 6R-tetrahydrobiopterin (BH4). Since there is a close genotype–phenotype correlation in PKU, determining the PAH genotype can be extremely important for therapy as well as prognosis. A simple and rapid method of accurately determining the PAH genotype would be a valuable addition to the diagnosis of PKU. Described herein is a means to identify variants in the PAH gene using high-resolution melt profiling, which compares the thermal denaturation profile of a patient sample to that of a control. Regions where the patient and control samples produce a common profile were not further evaluated, while those regions where the patient profile deviates from the control were assessed by DNA sequencing. Additionally described is a scheme utilizing redundant analysis with melt profile controls and a novel multiplex genotyping assay to triage deviation owing to known polymorphisms. Two mutations were identified in 93 of the 95 patients assessed and in the remaining two patients a single mutation was identified. Melt profiling provided 99% sensitivity to identify sequence variants in the PAH gene.

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Molecular characterization of phenylketonuria in South Brazil

Abstract

Introduction

Section snippets

Patients

PAH mutations and polymorphisms in 23 classic PKU patients

PAH mutations and polymorphisms in classic PKU patients

Acknowledgments

Adv. Genet.

Am. J. Hum. Genet.

Am. J. Hum. Genet.

Clin. Chim. Acta

Hyperphenylalaninaemia: Phenylalanine hydroxylase deficiency

Cloned human phenylalanine hydroxylase gene allows prenatal diagnosis and carrier detection of classical phenylketonuria

Nature

Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene

Biochemistry

Molecular basis of phenotypic heterogeneity in phenylketonuria

N. Engl. J. Med.

Molecular basis of phenylketonuria and a correlation between genotype and phenotype in a heterogeneous southeastern US population

Pediatrics

Mutation spectrum and phenylalanine hydroxylase RFLP/VNTR background in 44 Romanian phenylketonuric alleles

Hum. Mutat.

PAHdb: a locus-specific knowledgebase

Hum. Mutat.

Extensive restriction site polymorphism at the human phenylalanine hydroxylase locus and application in prenatal diagnosis of phenylketonuria

Am. J. Hum. Genet.

Mutation analysis of phenylketonuria in South Brazil

Hum. Mutat.

A simple salting out procedure for extracting DNA from human nucleated cells

Nucleic Acids Res.

The Design of Experiments