Other characteristic medical problems include heart defects or other heart problems, an abnormally small head size microcephaly , and behavioral problems. Women with PKU and uncontrolled phenylalanine levels also have an increased risk of pregnancy loss.
The occurrence of PKU varies among ethnic groups and geographic regions worldwide. Most cases of PKU are detected shortly after birth by newborn screening, and treatment is started promptly. As a result, the severe signs and symptoms of classic PKU are rarely seen. Mutations in the PAH gene cause phenylketonuria. The PAH gene provides instructions for making an enzyme called phenylalanine hydroxylase.
This enzyme converts the amino acid phenylalanine to other important compounds in the body. If gene mutations reduce the activity of phenylalanine hydroxylase, phenylalanine from the diet is not processed effectively. As a result, this amino acid can build up to toxic levels in the blood and other tissues. Because nerve cells in the brain are particularly sensitive to phenylalanine levels, excessive amounts of this substance can cause brain damage.
Classic PKU, the most severe form of the disorder, occurs when phenylalanine hydroxylase activity is severely reduced or absent. People with untreated classic PKU have levels of phenylalanine high enough to cause severe brain damage and other serious health problems. Mutations in the PAH gene that allow the enzyme to retain some activity result in milder versions of this condition, such as variant PKU or non-PKU hyperphenylalaninemia.
Changes in other genes may influence the severity of PKU, but little is known about these additional genetic factors. Those who suffer from PKU must be supplemented with tyrosine to account for PAH deficiency in converting phenylalanine to tyrosine sufficiently.
Dihydrobiopterin reductase activity is to replenish quinonoid-dihydrobiopterin back into its tetrahydrobiopterin form, which is an important cofactor in many metabolic reactions in amino acid metabolism.
Those with this deficiency may produce sufficient levels of PAH, but since tetrahydrobiopterin is a cofactor for PAH activity, deficient dihydrobiopterin reductase renders any PAH enzyme non-functional. Tetrahydrobiopterin is also a cofactor in the production of L-DOPA from tyrosine and 5-hydroxy-l-tryptophan from tryptophan, which must also be supplemented as treatment in addition to the supplements for classical PKU.
Levels of dopamine can be used to distinguish between these two types. Low levels of dopamine lead to high levels of prolactin. By contrast, in classic PKU, prolactin levels would be relatively normal. Tetrahydrobiopterin deficiency can be caused by defects in four different genes. The mean incidence of PKU varies widely in different human populations. Caucasians are affected at a rate of 1 in 10, Turkey has the highest documented rate in the world, with 1 in 2, births, while countries such as Finland and Japan have extremely low rates with fewer than one case of PKU in , births.
Treatment The mainstream treatment for classic PKU patients is a strict PHE-restricted diet supplemented by a medical formula containing amino acids and other nutrients.
These four novel mutations were not detected in control chromosomes. Deletions or duplications were detected in six out of eight patients: five of these patients had exon deletions and one patient had a duplication of exon 4 Figure 2. Among the five exon deletions, four showed the same pattern in MLPA analysis that encompassed exons 5 and 6, while the remaining deletion involved exons 4 through 7.
One patient had a duplication of exon 4. However, gene dosage analysis by MLPA failed to detect any deletions or duplications in two patients. Multiplex ligation-dependent probe amplification MLPA electrophoresis tracings for normal control red-colored and PKU subjects blue-colored with exon deletions or duplication.
The boxes and arrows indicate decreased or increased patient's peaks relative to the control's peaks. C, control; E, exon. Using different intronic primers that covered the region from intron 4 to intron 6, long-range PCR was used to identify the breakpoint of the recurrent large deletion of exons 5 and 6 Figure 3. Sequence analysis of the PCR amplicon revealed an 11,bp deleted region spanning from nucleotide 14 of exon 5 to nucleotide of intron 6.
Long-range PCR encompassing exons 5 and 6 using intronic primers results in an amplicon of Sequence analysis shows a deletion of 11, bp that spans from the nucleotide 14 of exon 5 to nucleotide of intron 6, designated as c. The locations of the two novel missense variants p.
These variations are located in the catalytic domain. Changing the alanine at amino acid position to threonine is expected to weaken the hydrophobic interactions among Ala, Tyr, Tyr, and Leu, and the activity of PAH may be decreased as a result of alterations to the side-chain and polarity that contribute to structural conformation Figure 5A.
Ribbon diagram of a monomer of human PAH depicting the locations of the amino acid replacements newly identified in Korean PAH patients. Substituted amino acid residues are denoted with red-colored balls. Predicted structural changes in human PAH induced by novel mutations. A Changing p. Ala to threonine is expected to weaken the hydrophobic interactions among Ala, Tyr, Tyr and p. B Changing p. As shown in Table 1 , all three patients with the p.
ArgCys mutation showed BH4 responsiveness: patient 30 with p. ArgCys and p. GlyAsp, patient 31 with p. ValMet, and patient 33 with p. An additional three patients with p. GlyArg and p. ValMet, p. ArgGln, and homozygous p.
Ile65Thr mutation also showed BH4 responsiveness Table 1. In the present study, we identified 27 different mutations in 33 unrelated Korean PKU patients, including four novel mutations. ArgGln, p. This finding was consistent with the previous report of Lee et al. In Europe, there are several prevalent founder alleles that represent the expansion, migration, and genetic drift of European population Zschocke, Particularly, the p.
However, this mutation was not found in the present study. Instead, p. In the present study, we also identified four novel mutations in 33 unrelated PKU Korean patients. Three of the 66 alleles 4. It remains to be seen whether uncharacterized alleles correspond to molecular defects that are embedded in the intronic regions and cause splicing aberrations or to impairments of the 5'-end promoter or regulatory regions and 3'-end polyadenylation regions. Large deletions or duplications of the PAH gene may also be sources of the defective alleles that remain to be specified.
Offspring of women who had metabolic control prior to pregnancy had a mean score of Forty-seven percent of offspring whose mothers did not have metabolic control by 20 weeks' gestation had a General Cognitive Index score 2 standard deviations below the norm.
At birth, offspring were examined; they were followed up at 3 months, 6 months, and then annually. Congenital heart defects were found in 31 offspring; of these, 17 also had microcephaly.
Mean phenylalanine levels at 4 to 8 weeks' gestation predicted congenital heart defects P less than 0. An infant with a congenital heart defect had a 3-fold risk of having microcephaly when the mother had higher phenylalanine levels. No direct relationship to the specific PAH mutation was found. None of the women whose offspring had congenital heart defects had blood phenylalanine levels in control during the first 8 weeks of gestation.
One of the children among 50 from mothers with non-PKU mild hyperphenylalaninemia also had congenital heart disease. Coarctation of the aorta and hypoplastic left heart syndrome were overrepresented. Brumm et al. Those with untreated PKU tended to have severe behavioral disturbances, including psychotic disorders, autistic features, hyperactivity, and aggression, as well as self-mutilation.
Among early-treated children and adolescents, discontinuation of treatment was associated with attention-deficit disorder and decreased social competence. Children who continued treatment had fewer behavioral problems. However, most tended to be less happy and confident. Even adults who had early treatment had higher rates of depression, anxiety-related disorders, and social introversion compared to the normal population.
In general, the severity of problems correlated with the timing and degree of exposure to increased blood levels of phenylalanine. Gentile et al. These included difficulties in forming interpersonal relationships, achieving autonomy, attending educational goals, and having healthy emotional development.
The most important way to reduce these problems is strict metabolic control throughout life, with particular importance on the first year of life. Pilotto et al. The patients had a higher prevalence of neurologic symptoms hyperreflexia, kinetic tremor, and slowed horizontal saccades and cognitive and behavioral abnormalities global cognition and depressive and behavioral symptoms compared to controls.
The patients also had increased atrophy of the putamen and right thalamus on MRI compared to controls. CSF metabolites, which were tested in 10 of the patients, had increased beta-amyloid, total tau, and phosphorylated tau compared to controls.
Plasma phenylalanine level in the patients correlated to total number of pathologic cognitive tests, motor-evoked potential latency, and parietal lobe atrophy on brain MRI. In untreated classical PKU, blood levels as high as 2. Bowden and McArthur found that phenylpyruvic acid inhibits pyruvate decarboxylase in brain but not in liver. They suggested that this accounts for the defect in formation of myelin and mental retardation in this disease.
Both the size and the amount of phenylalanine hydroxylase mRNA were normal. The findings confirmed the genetic diagnosis of PKU in the fetus and indicated that the mutations affected translation or stability of the protein.
Tolerance to dietary phenylalanine and therefore the clinical severity of PKU have been presumed to be the consequence of the rate of conversion of phenylalanine into tyrosine.
However, in a study of 7 classic PKU patients, van Spronsen et al. Kaufman described the derivation of a quantitative model of phenylalanine metabolism in humans. The model was based on the kinetic properties of pure recombinant human PAH and on estimates of the in vivo rates of phenylalanine transamination and protein degradation.
Calculated values for the steady-state concentration of blood phenylalanine, rate of clearance of phenylalanine from the blood after an oral load of the amino acid, and dietary tolerance of phenylalanine all agreed with data from normal as well as from phenylketonuric patients and obligate heterozygotes. Kaufman suggested that these calculated values may help in the decision about the degree of restriction of phenylalanine intake that is necessary to achieve a satisfactory clinical outcome in patients with classic PKU and in those with milder forms of the disease.
It has been postulated that the significant incidence of learning disabilities in treated patients with PKU may be due, in part, to reduced production of neurotransmitters as a result of deficient tyrosine transport across the neuronal cell membrane. In a study of hypotyrosinemia in a PKU population, Hanley et al. This compared to nonfasting plasma tyrosine levels of The data supported previous findings that plasma tyrosine levels are low in PKU.
Leuzzi et al. An abnormal concentration of brain Phe was detected in all patients, but there was wide interindividual variability of concurrent plasma Phe. In late-detected subjects, brain Phe concentration correlated with clinical phenotype better than did plasma Phe. White-matter alterations were found in all patients.
Koch et al. For each individual with PKU, the authors also noted IQ, mutations, whether or not a restricted diet was followed, and age at diagnosis. Patients' brain phenylalanine concentrations were different in spite of similar blood levels.
Interindividual variations of the apparent transport Michaelis constant ranged from 0. Sibs with lower values for the apparent transport constant, higher values for the ratio of the maximal transport velocity over the intracerebral consumption rate, and higher concurrent brain phenylalanine levels showed a lower IQ and a higher degree of cerebral white matter abnormalities.
To determine whether impairments of cerebral metabolism may play a role in acute phenylalanine neurotoxicity, Pietz et al. PKU patients showed slowing of EEG background activity, a sign of impaired brain function, 24 hours after oral phenylalanine challenge. Pietz et al. Most variation in classical PKU is due to heterogeneity in the mutant alleles with many patients being compound heterozygotes rather than homozygotes for one particular mutant allele.
Bartholome et al. Using a cDNA probe for human phenylalanine hydroxylase to analyze human-mouse hybrid cells by Southern hybridization, Lidsky et al. By in situ hybridization, the assignment of the PAH locus was narrowed to chromosome 12qq Eisensmith and Woo reviewed mutations and polymorphisms in the human PAH gene.
About 50 of the mutations were single-base substitutions, including 6 nonsense mutations and 8 splicing mutations, with the remainder being missense mutations.
Of the missense mutations, 12 apparently resulted from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutations had been observed at several sites, producing associations with different haplotypes in different populations. Studies of in vitro expression showed significant correlations between residual PAH activity and severity of the disease phenotype.
Waters et al. Using 3 complementary in vitro protein expression systems and 3D structural localization, Waters et al. Enzyme-specific activity and kinetic properties are not adversely affected, implying that the only way these mutations reduce enzyme activity within cells in vivo is by producing structural changes which provoke the cell to destroy the aberrant protein. The mutations were chosen because of their associations with a spectrum of in vivo hyperphenylalaninemia among patients.
Most PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. Gjetting et al. They used database searches to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase PDH , the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wildtype and 5 mutant forms including G46S, Binding studies showed that the wildtype form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity.
The data suggested that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations. Most missense mutations found in PKU result in misfolding of the phenylalanine hydroxylase protein, increased protein turnover, and loss of enzymatic function. Pey et al. For the 80 mutations for which expression analyses had been performed in eukaryotes, in most cases they found substantial overall correlation between the mutational energetic impact and both in vitro residual activities and patient metabolic phenotype.
This finding confirmed that the decrease in protein stability is the main molecular pathogenic mechanism in PKU and the determinant for phenotypic outcome. Metabolic phenotypes had been shown to be better predicted than in vitro residual activities, probably because of greater stringency in the phenotyping process.
All the remaining PKU missense mutations compiled in the PAH locus knowledgebase PAHdb were analyzed, and their phenotypic outcomes were predicted on the basis of the energetic impact provided by FoldX. Residues in exons and in interdomain regions within the subunit appeared to play an important structural role and constitute hotspots for destabilization.
Using recombinant proteins expressed in E. Residual activity was generally high, but allostery was disturbed in almost all variants, suggesting altered protein conformation. This hypothesis was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding, which primarily affected the regulatory domain, in most variants.
Three-dimensional modeling revealed that the misfolding was communicated throughout the protein. Gersting et al. Matalon et al. Woo identified a DNA restriction polymorphism detected by a phenylalanine hydroxylase cDNA probe and tentatively demonstrated the feasibility of carrier detection and prenatal diagnosis, using the haplotypes defined by the DNA polymorphism.
Riess et al. They pointed out that in those cases in which the affected child had died but a phenotypically normal brother or sister is available for investigation, full genetic predictability could be obtained only if this child proved to be homozygously healthy in the phenylalanine-loading heterozygote test. DiLella et al. The results suggested that it is technically feasible to develop a program for carrier detection of the genetic trait in a population of individuals without a family history of PKU.
Ramus et al. Taking advantage of the 'illegitimate' transcription of the PAH gene in circulating lymphocytes, Abadie et al. Furthermore, they identified 3 novel mutations in 2 patients. Kalaydjieva et al. The combined analysis of these markers and 1 or 2 PKU mutations formed a simple panel of diagnostic tests with full informativeness in a large proportion of PKU families.
Forrest et al. Phenylketonuria is treatable by a low phenylalanine diet. In treated patients, severe white matter abnormalities are predominantly associated with blood phenylalanine levels above 15 mg per deciliter Thompson et al.
Ullrich et al. Ten of these patients had a normal cranial MRI whereas 4 showed mild changes of the signal intensity of the white matter on T2-weighted images confined to the parietooccipital region. The affected and unaffected patients could not be distinguished by age, sex, or mean blood phenylalanine concentrations.
From studies in 4 women, Rohr et al. Drogari et al. For minimizing risks of ill effects, preconceptional dietary control was strongly recommended. Brenton and Lilburn reported that by November , 39 pregnancies had been completed in PKU mothers. Dietary control was post-conception in 6; 2 of these offspring died of congenital heart disease and another needed surgery for coarctation. There were no heart defects in the 34 offspring of the 33 pregnancies following preconception diet controlled by Guthrie assays of maternal Phe 3 times weekly.
Excessively high and low values occurred intermittently in many pregnancies, both of which may adversely affect the fetus.
A multicenter follow-up study Holtzman et al. At the first test, examination showed significant blood phenylalanine-correlated neuropsychologic deficits in PKU patients. In spite of raised blood phenylalanine concentrations during the following 3 years, the repeated measurements revealed a significant decrease in patients' deficits compared to controls. Clinical-neurologic status of patients and controls was normal at both test times.
The results indicated decreased vulnerability of PKU patients with respect to their neuropsychologic functioning against elevated phenylalanine levels on aging. Greeves et al. Multiple regression analysis demonstrated a significant reduction in verbal and overall IQ between the ages of 8 and 14 or 18, with a greater reduction in those with a lower predicted residual enzyme activity.
These data suggested that continued dietary control in this latter group, as defined by genotype, may prove beneficial. Recognizing that a low phenylalanine diet is also low in the long-chain polyunsaturated fatty acids LCPUFA necessary for cell membrane formation and normal brain and visual development, Agostoni et al. The children who received supplementation showed a significant increase in docosahexaenoic acid DHA levels of erythrocyte lipids and improved visual function, as measured by a decreased P wave latency.
Huijbregts et al. Patients whose phenylalanine concentrations had decreased by the second assessment showed generally more improvement than controls. Patients whose phenylalanine concentrations had increased showed minimal improvement or deterioration of task performance. The strongest effects were observed when sustained attention and manipulation of working memory content were required. Seventy of the children were located and evaluated in adulthood. The 'on diet' group had only 2 reported episodes of transient depression not requiring psychiatric care.
The neurologic signs related primarily to increased or decreased muscle tone and deep tendon reflex changes. Singh et al. Their paper was accompanied by an American College of Medical Genetics practice guideline authored by Vockley et al.
At least half of patients with phenylketonuria have a mild clinical phenotype. Muntau et al. Tetrahydrobiopterin significantly lowered blood phenylalanine levels in 27 of 31 patients with mild hyperphenylalaninemia 10 patients or mild phenylketonuria 21 patients.
Phenylalanine oxidation was significantly enhanced in 23 of these 31 patients. Conversely, none of the 7 patients with classic phenylketonuria had a response to tetrahydrobiopterin.
Long-term treatment with tetrahydrobiopterin in 5 children increased daily phenylalanine tolerance, allowing them to discontinue their restricted diets. Mutations connected to tetrahydrobiopterin responsiveness were predominantly in the catalytic domain of the PAH protein and were not directly involved in cofactor binding. Lassker et al. Both patients showed no effect of tetrahydrobiopterin at 7. Patients who responded were found to have mutations in the PAH gene within the catalytic, regulatory, oligomerization, and BH4-binding domains.
Steinfeld et al. They no longer needed dietary restriction and showed normal development after 2 years. No side effects were observed. Keil et al. Median phenylalanine Phe tolerance increased 3. Compared with diet alone, improvement in quality of life was reported in No severe adverse events were reported.
Intellectual functioning as measured by the full-scale intelligent quotient FSIQ and growth rates were maintained over a follow-up period of 7 years and stayed in the normal range. All of the patients had at least one adverse event during the study period; the most common adverse events were upper respiratory tract infections, abdominal pain and vomiting, and diarrhea.
Hoskins et al. Preliminary studies suggested that it may have a place in the treatment of PKU. Sarkissian et al. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. They concluded that the appropriate dosage of orally administered PAL, perhaps in combination with a controlled and modestly low protein diet, should effectively control the phenylalanine pool size through its effect on the gastrointestinal tract.
These findings opened a new avenue to the treatment of this classic genetic disorder. Zori et al. Pegvaliase-treated individuals also had diets with significantly higher protein intake. Stegink et al. They found moderate elevations in phenylalanine levels above baseline for heterozygotes for PKU 2.
Liver transplantation is not a usual therapy for PKU because of the usually good results achieved with early dietary restriction and because liver disease is not part of the clinical picture of PKU. Vajro et al. Eisensmith and Woo reviewed the current state of gene therapy for phenylketonuria. Of the 3 basic steps required, 2 have been accomplished: a cDNA clone expressing human phenylalanine hydroxylase and a phenylalanine hydroxylase-deficient animal model have been developed, while vectors for efficient gene transfer in vivo have yet to be developed.
Retroviral vectors, while effective in vitro, have a low transduction efficiency in vivo. Recombinant adenoviral vectors, although completely successful in the short term, did not persist beyond a few weeks due to an immune response against the adenoviral vector. PKU occurs in about 1 in 10, births Steinfeld et al. Peculiarities in the distribution of phenylketonuria have been noted. The disorder is rare in Ashkenazi Jews Cohen et al.
Carter and Woolf noted that of the cases seen in London and presently living in southeast England, a disproportionately large number had parents and grandparents born in Ireland or West Scotland. The frequency at birth in northern Europeans may be about 1 per 10, Guthrie and Susi, In Kuwait, Teebi et al.
Rh, Kell, and PGM-1 types support the suggestion. PKU was first discovered in Norway by Folling From the increase in frequency of parental consanguinity, Romeo et al. Flatz et al. The PKU gene has been considered to be Celtic in origin.
Perhaps surprisingly, DiLella et al. Furthermore, the association with RFLP haplotype 3 was preserved in these populations. This is a difficult finding to explain in population genetics terms that are compatible with demographic history. Guttler and Woo identified 12 different haplotypes in Danish PKU families; however, of chromosomes analyzed from 66 obligate heterozygotes, 59 of 66 PKU genes were associated with only 4 haplotypes. In Denmark, Guttler et al.
Patients who were either homozygous or heterozygous for the mutant alleles of haplotypes 2 or 3 had a severe clinical course, whereas patients who had a mutant allele of haplotypes 1 or 4 usually had a less severe clinical phenotype.
Ninety percent of all mutant alleles in Danes are associated with only 4 haplotypes, of which 2 had been fully characterized at the molecular level. The GT-to-AT transition at the canonical splice donor site of intron 12, causing skipping of the preceding exon during RNA splicing, is associated with a mutant haplotype 3. The missense mutation involving an arginine-to-tryptophan substitution at residue Both mutant alleles are in linkage disequilibrium with the corresponding RFLP haplotypes throughout Europe, suggesting that 2 mutational events occurred on background chromosomes of the 2 haplotypes, followed by spread and expansion in the Caucasian population.
In 37 French kindreds, Rey et al.
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