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Foreword

PAHdb began as a published "database" (John et al, 1990, PMID:1971147); a catalogue of variant alleles at the human PAH locus. Because it did not provide primary data (e.g. scans of sequence gels, etc.), when the original catalogue began to be annotated (e.g. associations between alleles and particular populations or polymorphic haplotypes (Scriver et al. 1993)) PAHdb was then converted to an on-line relational database (Hoang et al. 1996, PMID:8594560).  PAHdb was actually functioning as an "information database" driven by validated submissions from members of the PAH Mutation Analysis Consortium and by the curators culling information from the literature; the information was also being distributed by a Newsletter in hardcopy. When the variety and extent of its modules, (e.g. those for information on in vitro expression analysis, polymorphic haplotypes, mouse models, molecular modelling, a page of clinical information for patients),  links and pointers (e.g. to OMIM, GenBank and HGMD) increased, PAHdb became an on-line "knowledge base" (Nowacki et al. 1997, PMID:9016524 ; ibid 1998, PMID:9399840).

Although the information in PAHdb (for ~ 450 variant alleles, carried on > 8000 independent human chromosomes) is already quite extensive, the curators recognize that significant information is still missing on the web site. For example, it has been difficult to deconstruct articles where data on mutations have been aggregated for a population or region or where relative frequencies are significant information. In 1998, we attempted a solution by commenting on a set of such articles in the Newsletter; however it reached only those receiving the Newsletter. Here, we begin a new PAHdb module in which the curatorial team will, from time to time, deposit comments on published articles that improve our knowledge about the PAH gene, its alleles and their significance for understanding human genetic diversity at this locus.

References

Recent updates
[31.03.09] A Mutation Analysis of the Phenylalanine Hydroxylase (PAH) Gene in the Israeli Population.
Five human phenylalanine hydroxylase proteins identified in mild hyperphenylalaninemia patients are disease-causing variants.
Molecular epidemiology of phenylalanine hydroxylase deficiency in Southern Italy: a 96% detection rate with ten novel mutations.
[25.03.09] Added second reference to those BH4-responsive mutations
[16.04.08] Evolutionary and Biomedical Insights from the Rhesus Macaque Genome
[17.03.08] New development in BH4 responsive PKU/HPA
[04.12.07] The birth prevalence of PKU in populations of European, South Asian and Sub-Saharan African ancestry living in South East England.
[23.11.07] Predicted Effects of Missense Mutations on Native-State Stability Account for Phenotypic Outcome in Phenylketonuria, a Paradigm of Misfolding Diseases
[10.02.06] PAH Mutation Analysis Consortium Database: 1997.  Prototype for relational locus-specific mutation databases.
[09.02.06] The molecular basis of phenylketonuria in Koreans.
[02.02.06] Phenylketonuria mutations in Northern China.
[25.11.05] Hypothesis –  PKU Emerged in Temperate Zone Populations after the “Out of Africa” Diaspora  (Scriver CR, Hurtubise M, Prevost L, Dezateux C, Hardelid P and others).
[17.01.05] Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations.
[28.01.04] Novel PAH Short Tandem Repeat (STR) Markers
[05.03.03] BH4-responsive hyperphenylalaninemia (BH4 Alleles Part IV)
[15.01.03] Tetrahydrobiopterin as an Alternative Treatment for Mild Phenylketonuria.
[16.10.02] PAH Mutations Associated with BH4-Responsive HPA(BH4 Alleles Part III)
[29.08.02] A Phenylalanine Hydroxylase Amino Acid Polymorphism.
[02.07.02] A Silent Mutation Induces Exon Skipping.
[02.07.02] In Vitro Expression Analysis update by Paula J. Waters
[28.03.02] Comments on the Curators' Page (Normal Clinical Outcome...)
[18.03.02] Article and Comments on the Curators' Page (BH4 Alleles Part II)
[12.03.02] Article and Comments on the Curators' Page (BH4 Alleles Part I)
The PAHdb Curators October 7th, 2002
  • Mélanie Hurtubise (MH)
  • Manyphong Phommarinh (MP)
  • Lynne Prevost (LP)
  • Charles R. Scriver (CRS)
  • J. David McDonald (JDM)
  • Christineh Sarkissian (CNS)
  • Raymond Stevens (RS)
  • Paula Waters (PJW)

 

Articles and Curators' Comments
 
[31.03.09]
D. Bercovich, A. Elimelech, T. Yardeni1, S. Korem, J. Zlotogora, N. Gal, N. Goldstein,B. Vilensky, R. Segev, S. Avraham, R. Loewenthal, G. Schwartz and Y. Anikster
Annals of Human Genetics (2008) 72,305-309      doi: 10.1111/j.1469-1809.2007.00425.x
(PMID 18294361)
A Mutation Analysis of the Phenylalanine Hydroxylase (PAH) Gene in the Israeli Population 

[Abstract:]
Hyperphenylalaninemia (HPA) is a group of diseases characterized by a persistent elevation of phenylalanine levels in tissues and biological fluids. The most frequent form is phenylalanine hydroxylase deficiency, causing phenylketonuria (PKU). Among 159 Israeli patients (Jews, Muslim and Christian Arabs and Druze) with HPA, in whom at least one of the mutations was characterized, a total of 43 different mutations were detected, including seven novel ones. PKU was very rare among Ashkenazi Jews and relatively frequent among Jews from Yemen, the Caucasian Mountains, Bukhara and Tunisia. The mutations responsible for the high frequency were: exon3del (Yemenite Jews), L48S (Tunisian Jews) and E178G, P281L and L48S (Jews from the Caucasian Mountains and Bukhara). Among the non-Jewish Israeli citizens, the disease was relatively frequent in the Negev and in the Nazareth vicinity, and in many localities a unique mutation was detected, often in a single family. While marked genetic heterogeneity was observed in the Arab and Jewish populations, only one mutation A300S, was frequent in all of the communities. Several of the other frequent mutations were shared by the non-Ashkenazi Jews and Arabs; none were mutual to Ashkenazi Jews and Arabs.

Daniele A, Cardillo G, Pennino C, Carbone MT, Scognamiglio D, Esposito L, Correra A, Castaldo G, Zagari A, Salvatore F.
Biochimica et Biophysica Acta 1782 (2008) 378-384      doi:10.1016/j.bbadis.2008.01.012
(PMID 18346471)
Five human phenylalanine hydroxylase proteins identified in mild hyperphenylalaninemia patients are disease-causing variants. 

[Abstract:]
Hyperphenylalaninemia is a group of autosomal recessive disorders caused by a wide range of phenylalanine hydroxylase (PAH) gene variants. To study the effects of mutations on PAH activity, we have reproduced five mutations (p.N223Y, p.R297L, p.F382L, p.K398N and p.Q419R) that we recently identified in a population of Southern Italy. Transient expression of mutant full-length cDNAs in human HEK293 cells yielded PAH variants whose l-phenylalanine hydroxylase activity was between 40% and 70% that of the wild-type enzyme. Moreover, Western blot analysis revealed a 50-kD monomer in all mutants thereby indicating normal synthesis of the mutant proteins. Because of the clinical mild nature of the phenotypes we performed an in vivo BH4 loading test. This was positive in all tested patients, which indicates that they are likely to respond to the coenzyme in vivo. We also analysed the environment of each mutation site in the available crystal structures of PAH by using molecular graphics tools. The structural alteration produced by each mutation was elucidated and correlated to the mutated properties of the mutant enzymes. All the data obtained demonstrate the disease-causing nature of the five novel variants.

Daniele A, Cardillo G, Pennino C, Carbone MT, Scognamiglio D, Correra A, Pignero A, Castaldo G, Salvatore F.
Annals of Human Genetics (2006) 71,185-193      doi: 10.1111/j.1469-1809.2006.00328.x
(PMID 17096675)
Molecular epidemiology of phenylalanine hydroxylase deficiency in Southern Italy: a 96% detection rate with ten novel mutations. 

[Abstract:]
Hyperphenylalaninemia (HPA) is a group of diseases characterized by a persistent elevation of phenylalanine levels in tissues and biological fluids. The most frequent form is phenylalanine hydroxylase deficiency, causing phenylketonuria (PKU). Among 159 Israeli patients (Jews, Muslim and Christian Arabs and Druze) with HPA, in whom at least one of the mutations was characterized, a total of 43 different mutations were detected, including seven novel ones. PKU was very rare among Ashkenazi Jews and relatively frequent among Jews from Yemen, the Caucasian Mountains, Bukhara and Tunisia. The mutations responsible for the high frequency were: exon3del (Yemenite Jews), L48S (Tunisian Jews) and E178G, P281L and L48S (Jews from the Caucasian Mountains and Bukhara). Among the non-Jewish Israeli citizens, the disease was relatively frequent in the Negev and in the Nazareth vicinity, and in many localities a unique mutation was detected, often in a single family. While marked genetic heterogeneity was observed in the Arab and Jewish populations, only one mutation A300S, was frequent in all of the communities. Several of the other frequent mutations were shared by the non-Ashkenazi Jews and Arabs; none were mutual to Ashkenazi Jews and Arabs.

[16.04.08]
Rhesus Macaque Genome Sequencing and Analysis Consortium
Science 13 April 2007:Vol. 316. no. 5822, pp. 222 - 234 DOI: 10.1126/science.1139247
(PMID 17431167)
Evolutionary and Biomedical Insights from the Rhesus Macaque Genome 

[Abstract:]
The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species.

[Curator's Comment:]
The PAH locus in human subjects, when mutant, can cause hyperphenylalaninemia; but in the Macaque, the orthologous “mutant” phenotype is normal (no increase in blood phenylalanine) when associated with alleles considered to be deleterious, Why? Interesting!


[17.03.08]
New development in BH4 responsive PKU/HPA

Phenylketonuria is the first genetic disease to have an effective therapy, namely dietary restriction of ingested phenylalanine. Recently some patients harbouring missense PAH mutations have shown a therapeutic response to pharmacological doses of tetrahydrobiopterin. A pharmaceutical form of tetrahydrobioipterin, called Sapropterin, as the 6R-epimer, at doses between 5 mg per kg per day and 20 mg per kg per day, brings about a therapeutic response, defined as a fall by 30% or more of the phenylalanine concentration in blood within the first 24 hours post-treatment dose. The findings suggest that there is a new “Orphan Drug” on the horizon. It is called KUVAN and is marketed by BioMarin in North America. Three papers shown below describe: 1. the randomized double blind, placebo-controlled clinical trial demonstrating efficacy and safety of sapropterin (Kuvan); 2. the apparent overall frequency of BH4-responsive PKU/HPA; 3. the guidelines to use Kuvan when discerning whether the patient is or is not BH4 responsive. This is an evolving story. Stay tuned.
Charles Scriver

  1. Harvey Levy, Barbara Burton, Stephen Cederbaum, Charles Scriver
    Molecular Genetics and Metabolism 92 (2007) 287-291
    (PMID: 18036498)

    Recommendations for evaluation of responsiveness to tetrahydrobiopterin (BH(4)) in phenylketonuria and its use in treatment.

    Some individuals with phenylketonuria (PKU) respond to pharmacologic treatment with tetrahydrobiopterin (BH(4)) by a reduction in the blood phenylalanine concentration. This can result in increased dietary tolerance for phenylalanine or, in rare instances, replacement of the phenylalanine-restricted diet. BH(4) is now available as sapropterin dihydrochloride under the name KUVAN, a formulation of natural BH(4). This commentary contains recommendations for determining responsiveness to sapropterin dihydrochloride. The recommendations include challenging with an initial daily dose of 20mg/kg and blood phenylalanine determinations pre-challenge and on days 1, 7, and 14 with the option of an additional continuation to day 28 if required to clarify whether a response has occurred. An algorithm depicting this recommendation for the challenge is included. The most widely accepted standard of response is ≥30% reduction in the blood phenylalanine concentration, but a lower degree of response might also be considered clinically meaningful in some individual circumstances. Issues include the potential treatment of those with mild hyperphenylalaninemia who are not on diet, challenging neonates who have hyperphenylalaninemia identified by newborn screening, and the use of sapropterin dihydrochloride in treatment of maternal PKU pregnancies. These recommendations are intended to provide a basis for the use of sapropterin dihydrochloride in the treatment of PKU but may be altered after close observation of treated patients and carefully performed research.

  2. Harvey L Levy, Andrzej Milanowski, Anupam Chakrapani, Maureen Cleary, Philip Lee, Friedrich K Trefz, Chester B Whitley, François Feillet, Annette S Feigenbaum, Judith D Bebchuk, Heidi Christ-Schmidt, Alex Dorenbaum, for the Sapropterin Research Group.
    Lancet 2007; 370; 504-10
    (PMID: 17693179)

    Efficacy of sapropterin dihydrochloride (tetrahydrobiopterin, 6R-BH4) for reduction of phenylalanine concentration in patients with phenylketonuria: a phase III randomised placebo-controlled study.

    BACKGROUND: Early and strict dietary management of phenylketonuria is the only option to prevent mental retardation. We aimed to test the efficacy of sapropterin, a synthetic form of tetrahydrobiopterin (BH4), for reduction of blood phenylalanine concentration. METHODS: We enrolled 89 patients with phenylketonuria in a Phase III, multicentre, randomised, double-blind, placebo-controlled trial. We randomly assigned 42 patients to receive oral doses of sapropterin (10 mg/kg) and 47 patients to receive placebo, once daily for 6 weeks. The primary endpoint was mean change from baseline in concentration of phenylalanine in blood after 6 weeks. Analysis was on an intention-to-treat basis. The study is registered with ClinicalTrials.gov, number NCT00104247. FINDINGS: 88 of 89 enrolled patients received at least one dose of study drug, and 87 attended the week 6 visit. Mean age was 20 (SD 9.7) years. At baseline, mean concentration of phenylalanine in blood was 843 (300) micromol/L in patients assigned to receive sapropterin, and 888 (323) micromol/L in controls. After 6 weeks of treatment, patients given sapropterin had a decrease in mean blood phenylalanine of 236 (257) micromol/L, compared with a 3 (240) micromol/L increase in the placebo group (p<0.0001). After 6 weeks, 18/41 (44%) patients (95% CI 28-60) in the sapropterin group and 4/47 (9%) controls (95% CI 2-20) had a reduction in blood phenylalanine concentration of 30% or greater from baseline. Blood phenylalanine concentrations fell by about 200 micromol/L after 1 week in the sapropterin group and this reduction persisted for the remaining 5 weeks of the study (p<0.0001). 11/47 (23%) patients in the sapropterin group and 8/41 (20%) in the placebo group experienced adverse events that might have been drug-related (p=0.80). Upper respiratory tract infections were the most common disorder. INTERPRETATION: In some patients with phenylketonuria who are responsive to BH4, sapropterin treatment to reduce blood phenylalanine could be used as an adjunct to a restrictive low-phenylalanine diet, and might even replace the diet in some instances.

  3. B. K. Burton, D. K. Grange, A. Milanowski, G. Vockley, F. Feillet, E. A. Crombez, V. Abadie, C. O. Harding, S. Cederbaum, D. Dobbelaere, A. Smith and A. Dorenbaum
    J Inherit Metab Dis (2007) 30:700-707
    (PMID: 17846916)

    The response of patients with phenylketonuria and elevated serum phenylalanine to treatment with oral sapropterin dihydrochloride (6R-tetrahydrobiopterin): a phase II, multicentre, open-label, screening study

    This study aimed to evaluate the response to and safety of an 8-day course of sapropterin dihydrochloride (6R-tetrahydrobiopterin or 6R-BH4) 10 mg/kg per day in patients with phenylketonuria (PKU), who have elevated blood phenylalanine (Phe) levels, and to identify a suitable cohort of patients who would respond to sapropterin dihydrochloride treatment with a reduction in blood Phe level. Eligible patients were aged ≥8 years, had blood Phe levels ≥450 μmol/L and were not adhering to a Phe-restricted diet. Suitable patients were identified by a ≥30% reduction in blood Phe level from baseline to day 8 following sapropterin dihydrochloride treatment. The proportion of patients who met these criteria was calculated for the overall population and by baseline Phe level (<600, 600 to <900, 900 to <1200 and ≥1200 μmol/L). In total, 485/490 patients completed the study and 20% (96/485) were identified as patients who would respond to sapropterin dihydrochloride. A reduction in Phe level was observed in all subgroups, although response was greater in patients with lower baseline Phe levels. Wide variability in response was seen across all baseline Phe subgroups. The majority of adverse events were mild and all resolved without complications. Sapropterin dihydrochloride was well tolerated and reduced blood Phe levels across all PKU phenotypes tested. Variability in reduction of Phe indicates that the response to sapropterin dihydrochloride cannot be predicted by baseline Phe level.

[04.12.07]
P. Hardelid, M. Cortina-Borja, A. Munro, H. Jones, M. Cleary, M. P. Champion, Y. Foo, C. R. Scriver, C. Dezateux
Annals of Human Genetics 71:1-7, 2007.
(PMID: 18184144)
The birth prevalence of PKU in populations of European, South Asian and Sub-Saharan African ancestry living in South East England.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism (OMIM 261600). Treatment with a low-phenylalanine diet following early ascertainment by newborn screening prevents impaired cognitive development, the major disease phenotype in PKU. The overall birth prevalence of PKU in European, Chinese and Korean populations is approximately 1/10,000. Since the human PAH locus contains PKU-causing alleles and polymorphic core haplotypes that describe and corroborate an out-of-Africa range expansion in modern human populations, it is of interest to know the prevalence of PKU in different ethnic groups with diverse geographical origin. We estimated PKU prevalence in South East England, where a sizeable proportion of the population are of Sub-Saharan African or South Asian ancestry. Over the period 1994 to 2004 167 children were diagnosed with PKU. Using birth registration and census data to derive denominators, PKU birth prevalence per 10,000 live births (95% Bayesian credible intervals) was estimated to be 1.14 (0.96-1.33) among white, 0.11 (0.02-0.37) among black, and 0.29 (0.10-0.63) among Asian ethnic groups. This suggests that PKU is up to an order of magnitude less prevalent in populations with Sub-Saharan African and South Asian ancestry that have migrated to the UK.

The prevalence rates for PKU/HPA in European, Chinese and Korean populations are similar (10-4) (Donlon, Levy, and Scriver C.R. 2004; Gu and Wang 2004; Song et al. 2005; Lee et al. 2004). However the spectra of PAH alleles in the European and Oriental population sets are different (www.pahdb.mcgill.ca/) implying that they may have had origins different in time and source in human evolution. The PAH locus itself is rich in polymorphic haplotypes and they contain evidence to support an “Out-of-Africa” diaspora of the human species some 100,000 years ago (Kidd and Kidd 2005; Kidd et al. 2000; Degioanni and Darlu 1994). Accordingly, it is of interest to know the prevalence rates of PKU for African populations. Because there is no systematic screening for PKU in Africa, it is necessary to rely on indirect indicators of prevalence rates. A search for PKU in mentally retarded Africans found no cases in a small sample (Familusi and Bolodeoku 1976). Estimates in screened mixed populations in the Americas, for example (Graw and Koch 1967; Epps 1968; Knox 1972; Gjetting et al. 2001; Hofman et al. 1991) (also Andersson HC pers. Comm., Nov. 2004), indicate that PKU prevalence rates could be an order of magnitude lower among persons of African descent relative to those of European and Oriental descent. This hypothesis has been tested by examining the relative prevalence of PKU in ethnically defined populations in South East England in a population of 1.5 millions new borns during a 10 years interval. The relative birth prevalence per 10000 live births was 1.14 in white infants, 0.11 among black infants and 0.29 among Asian infants. The relative prevalence of PKU among infants of African heritage is indeed an order of magnitude less prevalent relative to European and Oriental [Hardelid et al. 2007]. Hardelid et al measured PKU prevalence rates in South East England where a sizable proportions of the population have Sub-Saharan and South-Asian ancestry. Prevalence rates were measured over the period 1994-2004 in a population sample of 1.5 million newborns in which 167 children with PKU were diagnosed. With birth registration and census data to derive denominators, PKU birth prevalence for 10,000 live births were estimated to be 0.14 among the white population, 0.11 among the Black population and 0.29 among the Asian ethnic groups indicating that PKU is up to an order of magnitude less prevalent in populations of Sub-Saharan African and South-Asian ancestry that have migrated to the UK. Hardelid et al discuss the possibility that PKU alleles have been associated with heterozygote advantage in the European and Oriental populations.


[23.11.07]
ANGEL L. PEY, FRANÇOIS STRICHER, LUIS SERRANO, AND AURORA MARTINEZ
Am. J. Hum. Genet., 81:1006-1024, 2007.
Predicted Effects of Missense Mutations on Native-State Stability Account for Phenotypic Outcome in Phenylketonuria, a Paradigm of Misfolding Diseases

(PMID 17924342)

The article by Pey et al. is important! PAHdb harbours over 500 variant alleles. Among those associated with hyperphenylalaninemia / PKU, two-thirds are missense mutations. The article by Pey et al. confirms that a “decrease in protein stability associated with a missense mutation is the main molecular pathogenic mechanism in PKU and the determinant for phenotypic outcome”. This new article finesses the hypothesis proposed earlier by Waters et al. (Waters et al. Molec Genet Metab 69 :101-110, 2000 – See commentary in Curators’ page). Pey studied the effect of PAH missense mutations by using the protein design algorithm FoldX to predict the energetic impact on the PAH protein and the native-state stability. PAH enzyme is a homodimeric tetramer. 80 of the mutations studied also had data for in vitro expression analysis in eurkaryotic systems; those data were used for comparison with the corresponding metabolic data allowing the investigators to consolidate the conclusions stated above. An additional 238 PAH mutations were analyzed by Pey et al. with the algorithm to predict the corresponding phenotypes. Residues in exon 7, 8 and 9, and the interdomain regions of the protein, play important structural roles and constitute hotspots for destabilizing the enzyme. Pey et al. offer this interesting opinion: “Our results show a positive prediction for a data set that is representative of a large fraction of mutant phenotypes in vivo, and we consider that this procedure will be useful to predict the phenotype associated with other rare and new mutations detected with PKU”. The authors suggest that the FoldX Web server can be modified to predict the phenotypic effects of single amino acid mutations in this protein (and others ?) associated with Mendelian disorders – an important thought since approximately half of disease causing mutations in the human genome are missense alleles.

The authors also consider the mechanism underlying BH4 responsiveness and suggest that BH4-induced stabilization of PAH protein mutants, with mild stability defects, is responsive to BH4 when it acts as a chemical / pharmacological chaperone (Table 4 and p. 1017 in the article; see also Comment on the Curators’ page on the article by Erlandsen H., Pey AL, Gamez A, et al. PNAS 30:16903-8, 2004). Keep posted …!


[10.02.06]
NOWACKI PM, BYCK S, PREVOST L, SCRIVER CR
Nucleic Acids Research 26:220-225, 1998.
PAH Mutation Analysis Consortium Database: 1997.  Prototype for relational locus-specific mutation databases. 

(PMID 9399840)

This article described PAHdb (www.pahdb.mcgill.ca) at an early stage of its development.  Figure 3 in the paper, and the accompanying text, served the hypothesis that PKU-causing mutations in European and Oriental populations stratified to suggest independent origins of the hyperphenylalaninemia phenotype due to mutations at the PAH locus in the two population sets.   This idea was initially sustained by only 185 Oriental PAH alleles against 3,630 European alleles; at a later time, it was transformed into a diagram-of-descent of PAH alleles in modern human populations to suggest that there was an Out-of-Africa migration of modern human beings followed by demic expansion into two major groups: Caucasians and Orientals.   The idea is now being further developed, with new data about differences in prevalence of hyperphenylalanemia in “African” and “Out of African” populations [see the dated Curators’ comment for 25.11.05 below]. 

Two new Curators’ Comments now appear relating to this theme.   The one dated 02.02.06 describes mutations in (Northern) Chinese populations.   The other dated 09.02.06 describes PAH alleles in Koreans.  Together these two papers embracing 158 alleles in Koreans and 370 alleles in Chinese populations give strong support to the hypothesis that Caucasian and Oriental PAH alleles reflect different origins.  


[09.02.06]
LEE DH, KOO SK, LEE K-S, YEON Y-J, OH H-J, KIM S-W, LEE S-J, KIM S-S, LEE J-E, JO I, JUNG S-C
J Hum Genet. 2004;49:617-21.
The molecular basis of phenylketonuria in Koreans.

(PMID: 15503242)
This paper describes mutations in the PAH gene in 79 independent Korean individuals ascertained through hyperphenylalaninemia causing clinical phenotypes classified as PKU, moderate PKU, or mild hyperphenylalaninemia (criteria given in article).   The authors succeeded in characterizing 135 alleles in the set of 158 (86% detection efficiency).  They identified 35 different mutations including 10 novel (previously unreported) alleles of which 9 were missense and one was a splice site variant.  The authors show that A259T (a rare allele) has a significant relative frequency (5.7%) in Koreans, whereas it is unobserved or rare in Chinese, Taiwanese and Japanese populations.  The Korean investigators show that Oriental alleles are a set quite different from corresponding alleles in Caucasians; furthermore that there is diversity and stratification within the above-mentioned Oriental populations (Table 3).   The novel missense alleles each affected a conserved amino acid.   Ten alleles of 39 account for 62.1% relative frequency: R243Q, 12.0%; IVS4-1G->A, 10.1%; E6-96A->G, 10.1%; R241C, 5.7%; A259T, 5.7%; Y356X, 5.7%; four other alleles for 12.8%; total 62.1%.  Again, the paradigm of a few alleles accounting for the majority of relative frequency (in this case, almost two thirds) is observed.   Three alleles showed responsiveness to BH4 in pharmacological doses (R53H, R241C, and R408Q).   

[02.02.06]
Song F, Qu U-j, Zhang T, Jin Y-w, Wang H, Zheng X

Molec Genet & Metab 86 (2005) S107-S118.
Phenylketonuria mutations in Northern China
.
(PMID: 16256386)
This paper has two important messages: 
  1. The population prevalence of PKU in this Oriental population equals that in Caucasian populations (see ref 1) where the reference describes (in Chinese) the birth prevalence of PKU and hyperphe in 11 different samples of the mainland Chinese population. 
  2. The profile of allelic stratification in the Chinese population is distinctly different from that in Caucasian populations.   These two findings contribute to our understanding of the evolution of modern human populations over the past 100,000 years.   The authors performed mutation analysis in 185 unrelated PKU patients from Northern China.   Detection efficiency was 94.3% and revealed 70 different alleles, in 109 genotype combinations, on 370 mutant chromosomes.   There were 15 newly discovered mutations (previously unreported) in the population, each occurring at relative frequencies below 1.1%.    The most prevalent mutations (each exceeding 6.5% relative frequency) were:  R243Q, exon 6-96 A->G, R111X, Y356X, and R413P; together these 5 mutations account for 55% of mutant alleles at the PAH locus in this population.  

[25.11.05]
Hypothesis – PKU Emerged in Temperate Zone Populations after the “Out of Africa” Diaspora (Scriver CR, Hurtubise M, Prevost L, Dezateux C, Hardelid P and others).
(PMID : ).
The human gene locus at 12q23.4 (gene symbol PAH) for phenylalanine hydroxylase enzyme (EC 1.14.16.1.4) contains disease-causing alleles that affect enzyme integrity and function. They cause hyperphenylalaninaemia, an autosomal recessive metabolic phenotype (Scriver et al. 2003). The corresponding disease, phenylketonuria (PKU), impairs postnatal cognitive development, a consequence prevented by treatment with low-phenylalanine diet (Donlon, Levy, and Scriver C.R. 2004). PKU was among the first of the human genetic diseases to be recognised as treatable, from which came the rationale for early diagnosis by newborn screening and effective treatment (National Academy of Sciences. 1975). Accordingly, the newborn screening test became one of the most widely applied genetic tests in human populations. As a result, an opportunity arose to observe birth prevalence rates of PKU and related forms of hyperphenylalaninaemia in human populations.

The prevalence rates in European and Chinese populations are similar (q2 ~10-4) (Donlon, Levy, and Scriver C.R. 2004; Gu and Wang 2004). However the spectra of PAH alleles in the two population sets are different (www.pahdb.mcgill.ca/) implying that they may have had origins different in time and source in human evolution. The PAH locus itself is rich in polymorphic haplotypes and they contain evidence to support an “Out-of-Africa” diaspora of the human species some 100,000 years ago (Kidd and Kidd 2005; Kidd et al. 2000; Degioanni and Darlu 1994). Accordingly, it is of interest to know the prevalence rates of PKU for populations in Africa. Because there is no systematic screening for PKU in Africa, it is necessary to rely on indirect indicators of prevalence rates. A search for PKU in mentally retarded Africans found no cases in a small sample (Familusi and Bolodeoku 1976). Estimates in screened mixed populations in the Americas, for example (Graw and Koch 1967; Epps 1968; Knox 1972; Gjetting et al. 2001; Hofman et al. 1991) (also Andersson HC pers. Comm., Nov. 2004), indicate that PKU prevalence rates are an order of magnitude lower among persons of African descent relative to those of European descent. This hypothesis has been tested by examining the relative prevalence of PKU in ethnically defined populations in South East England in a population of 1.5 millions new borns during a 10 years interval. The relative birth prevalence per 10000 live births was 1.14 in white infants, 0.11 among black infants and 0.29 among Asian infants. The relative prevalence of PKU among infants of African heritage is indeed an order of magnitude less prevalent relative to European and Oriental [Hardelid et al. 2007] The possible explanations for these findings are of fundamental interest in human genetics (Donlon, Levy, and Scriver C.R. 2004).
 

References

     1.    Degioanni A and Darlu P (1994) Analysis of the molecular variance at the phenylalanine hydroxylase (PAH) locus. Eur J Hum Genet 2 (3):166-176.

     2.    Donlon J, Levy H, Scriver C.R. (2004) Hyperphenylalaninemia: Phenylalanine Hydroxylase Deficiency.  Chapter 77. Online. http://genetics.accessmedicine.com/. In Scriver CR et al (eds) The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill.

     3.    Epps RP (1968) Phenylketonuria in an American Negro infant. Clin Pediatr 7:607-610.

     4.    Familusi JB and Bolodeoku JO (1976) Blood phenylalanine levels in mentally retarded African children: A study of 138 patients from Ibidan, Nigeria. Trop Geogr Med 28:96-100.

     5.    Gjetting T et al (2001) A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics. Molec Genet & Metab 73:280-284.

     6.    Graw RGJr and Koch R (1967) Phenylketonuria in two American Negroes. Am J Dis Child 114:412-418.

     7.    Gu X and Wang Z (2004) Neonatal screening for phenylketonuria and congenital hypothyroidism in China. Chinese J Preventive Medicine 38:99-102.

     8.    Hardelid P. et al. (2007) The Birth Prevalence of PKU in Populations of European, South Asian and Sub-Saharan African Ancestry Living in South East England. Ann Hum Genet 71:1-7.

     9.    Hofman KJ et al (1991) Phenylketonuria in U.S. blacks: Molecular analysis of the phenylalanine hydroxylase gene. Am J Hum Genet 48:791-798.

   10.    Kidd JR, Kidd KK (2005) The population genetics of PAH.   Online.  Supplement to Chapter 77. The Hyperphenylalaninemias.  Donlon J, Levy H, Scriver CR. In Scriver CR et al (eds) The Metabolic and Molecular Bases of Inherited Disease.  Online.   New York: McGraw-Hill.

   11.    Kidd JR et al (2000) Haplotypes and linkage disequilibrium at the phenylalanine hydroxylase locus, PAH, in a global representation of populations. Am J Hum Genet 66:1882-1899.

   12.    Knox WE (1972) Phenylketonuria. In Stanbury JB, Wyngaarden JB, Fredrickson DS (eds) The Metabolic Basis of Inherited Disease, 3rd ed. New York: McGraw Hill Book Co.

   13.    National Academy of Sciences. 1975. Genetic Screening. Programs, Principles and Research. Washington, D.C.: National Research Council.

   14.    Scriver CR et al (2003) PAHdb 2003: What a locus-specific knowledgebase can do. Hum Mutat 21 (4):333-344.


[17.01.05]
Erlandsen H, Pey AL, Gamez A, Perez B, Desviat LR, Aguado C, Koch R, Surendran S, Tyring S, Matalon R, Scriver CR, Ugarte M, Martinez A, Stevens RC.
Proc Natl Acad Sci U S A. 2004 Nov 30;101(48):16903-8. Epub 2004 Nov 30.

Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations.
(PMID : 15557004).
Tetrahydrobiopterin responsive forms of hyperphenylalaninemia related to mutations at the PAH locus are of great interest.  Estimates of this responsive phenotype are as high as a quarter of patients with persistent hyperphenylalaninemia.  So far all responsive patients are expressing a missense mutation either in homozygous alleles or in a state of haplotype insufficiency.  It has been assumed that the mechanism of BH4 response is either kinetic, overcoming unfavorable cofactor binding or it might be a chaperone-like effect, where BH4 attenuates the disappearance of the misfolding protein from cytoplasm to proteasome.  By means of expression analysis and other procedures, the authors of the paper identified above show that both mechanisms are in evidence and the BH4 response is influenced by the particular allele and its effect on PAH enzyme integrity and function.

[28.01.04]
Novel PAH short tandem repeat (str) markers
Three new STR (dinucleotide markers) named PAH9, PAH26 and PAH32 are described by professor David Croke and his colleague Orna Tighe (Dublin, Ireland). PAH26 and PAH32 lie outside the genomic sequence (AF404777). PAH9 lies within intron 3, about 6.8kbp 3' to the tetranucleotide STR in this intron. These markers are described and shown on the 'Sequence' page in the database; PAH9 is also annotated on the PAH genomic sequence.

[05.03.03]
BH4-responsive PAH alleles (Part IV)
Spaapen LJM and Rubio-Gonzalbo ME
Mol Genet Metab Feb; 78 (2):93-99, 2003.
BH4-responsive hyperphenylalaninemia is the subject of a thoughtful essay (in press) by Spaapen and Rubio-Gonzalbo.
(PMID : 12618080).
They offer an arbitrary definition of the phenotype: a 30% minimum decline in the plasma phenylalanine level following an oral 6R-BH4 challenge (20 mg per kg body weight). Use of the 6R isomer is important. The essay describes how a significant change in the manufacture of BH4 in 1999 led to the subsequent discovery of the BH4-responsive phenotype. The known patients (n=46 in December 2002) and their PAH genotypes are listed in Table 2; 20 missense alleles are categorically called “BH4-responsive” and 10 other missense alleles are candidates (Table 3). The possible mechanisms underlying BH4- responsiveness (hypotheses) include : i.) Kinetic mutations affecting a BH4-binding domain [as described in Erlandsen H and Stevens RC J Inher Metab Dis (2001) 24:213-230]. ii.) Regulation of PAH gene expression [see Blau N and Trefz F Mol Genet Metab (2002) 75:186-187]. iii.) A “chemical chaperone” effect on the misfolded unstable PAH protein [see Scriver CR and Waters PJ Trends in Genetics 15:267-272 1999]. The clinical relevance of these new findings is highlighted in this essay.

[15.01.03]
MUNTAU A, ROSCHINGER W, HABICH M, DEMMELMAIR H, HOFFMANN B SOMMERHOFF C, ROSCHER A. New Engl J Med 347 :2122-2132, 2002.
Tetrahydrobiopterin as an Alternative Treatment for Mild Phenylketonuria.
(PMID : 12501224).
This article analyzes responsiveness to tetrahydrobiopterin (BH4, Schircks Lab) in patients with phenylketonuria or primary non-PKU forms of PAH enzyme deficiency. It describes the most complete investigation of the problem, so far.

There were 10 patients with "mild hyperphenylalanemia", 21 patients with "mild phenylketonuria" and 7 patients with "classic PKU". They were given tetrahydrobiopterin (20 mg/kg by mouth) one hour after ingestion of L-phenylalanine (100 mg/kg body weight). The criteria for mild hyperphenylalaninemia, mild phenylketonuria and classic PKU are given in the paper.

The investigators monitored the BH4 response in two ways:

i) By measuring the time dependent blood phenylalanine values and clearance rate
ii) By measuring the cumulative conversion of L-[1-13C]phenylalanine to 13CO2 (according to the method of Treacy et al. Ped Res 42:430-5, 1997).
A positive response to BH4 was recognized by a more rapid clearance of blood phenylalanine with time (15 hours) and/or by an increased rate of phenylalanine oxidation. The molecular PAH genotypes were identified (74 alleles, 2 alleles not found). As incidental information, 7 previously unreported PAH mutations were identified in this population from Munich, Germany: N61D, I65S, H170Q, P275L, S310Y, P314S, Y417H.

None of the classic PKU patients (n=7) responded to BH4 and 4 patients with "mild PKU" did not respond. Twenty seven patients with variant HPA had a significant response (fall) in blood phenylalanine levels; 23 also had an enhanced rate of phenylalanine oxidation.

Seven alleles (IVS4-5c to g; V177M; V245A; A300S; P314S; E390G; Y417H) were classified as "probably associated" with BH4 responsiveness; 6 other mutations (F39L, I65T, R158Q, S310Y, A403V, D415N) were considered to be "potentially associated" with BH4 responsiveness; four additional mutations (L48S, I65V, R261Q, Y414C) were inconsistent in their response.

The authors discuss the implications in their findings noting that the oxidation rates and the blood phenylalanine clearance rates were independent yet complementary measures of the response to BH4. The homologous allele and its effect of the PAH protein was seen to play a role; the background genotype must also be taken into consideration. Figure 5 in the paper maps the "responsive" (all missense) alleles onto the amino acid sequence and 3D conformation of the PAH subunit. (The mutations listed by Muntau et al. should be compared with the diagram of BH4-responsive PAH mutations shown elsewhere on the Curators’ Page).


 
[16.10.02]
BH4 RESPONSIVE PAH ALLELES (PART III)
BLAU N (personal comm., Sept. 2002) displayed on ERLANDSEN H AND STEVENS RC. Mol Genet Metab 68(2):103-125, Oct. 1999
The Structural Basis of Phenylketonuria.

(PMID : 10527663).
Figure 1: PAH Mutations Associated with BH4-Responsive HPA

[29.08.02]
Gjetting T, Romstad A, Haavik J, Knappskog PM, Acosta AX, Silva WA Jr., Zago MA, Guldberg P, Guttler P.
Mol Genet Metab 73:280-284, 2001.
A Phenylalanine Hydroxylase Amino Acid Polymorphism with Implications for Molecular Diagnostics
.
(PMID: 11461196)
.
Incidence of PKU/HPA is noticeably lower in populations of African derivation; some estimates are ~ 1:100,000. The report by Gjetting et al. describes a Brazilian family in which there is a child with classical PKU; the paternal and maternal origins are African. Complete (rather than partial) PAH gene analysis was performed. The paternally derived mutant allele is c.754C ->T (R252W); a null by expression analysis. The maternal chromosome harboured two alleles in cis; c.820A->G (K274E) and c.953T->C (I318T). In Vitro Expression analysis showed that the I318T allele is pathogenic; it impairs PAH enzyme activity. The K274E allele had no adverse effect on PAH enzyme activity. K274E is an "amino acid polymorphism" and preliminary estimates (unpublished data by the authors) reveal its prevalence is ~ 4% of African-American alleles. Codon K274 has not been rigorously conserved during evolution of the PAH gene; the corresponding lysine is located on the surface of the folded protein where substitutions could be better tolerated; K274E has no apparent effect on folding of the subunit or on oligomerization to form the enzyme.

In summary, this report presents: i. otherwise rare information about "African" PAH mutations; ii. Another example of two PAH alleles in cis; iii. The novel K274E allele which is benign and exists as an "amino acid polymorphism" in African-derived populations.


[02.07.02]
Chao H-K, Hsiao K-J, Su T-S.
  Hum Genet 108:14-19, 2001.
A silent mutation induces exon skipping in the Phenylalanine Hydroxylase gene in Phenylketonuria
.
 
(PMID: 11214902).
Chao and co-authors report an A-T transversion in the PAH gene at cDNA nucleotide 1197 (c.1197A->T). The mutation, which as been reported previously, has been entered into PAHdb as a silent allele: GUA (wild type) and GUU ( mutant) both encode valine in codon 399. The corresponding "trivial" name for the mutation is V399V. Nucleotide c.1197 is in exon 11 at position -3 from the 3' exon-intron junction. Chao et al show that this mutation actually creates a new splice site leading to the deletion of exon 11 in the mRNA. Accordingly mutation c.1197A->T is not "silent"; it creates a null phenotype.

Two additional mutations, T323T (c.969A->G) and K398K (c.1194A->G), are proposed to generate splice sites and are not silent (Zschocke J, and Hoffman GF Hum Genet 104:390-398, 1999).

The discovery raises the question about classification of alleles and their phenotypic effect by assuming their presumed effect. Elsewhere (Cartegni L, Chew SL, Krainer AR Nature Reviews 3:285-298, 2002 PMID 11967553) review the "increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing". Chao et al have given us a cautionary tale that fits into the larger perspective provided by Cartegni et al. 2002. The theme is echoed by the Curator (P. Waters) of PAHdb in vitro expression analysis data; see her Commentary (June 17, 2002. final paragraph, under "Expression Systems").


[17.06.02]
In vitro expression analysis update BY PAULA J. WATERS.
Data concerning In Vitro Expression analysis of naturally-occurring mutations in the human PAH gene have recently been extensively updated. The searchable table In Vitro Expression (human) now contains information on the analysis of 81 PAH mutations (in a total of 225 records).

A commentary, designed both to assist in interpretation of the data from this table and to draw together some key messages, has also been deposited.


 
[28.03.02] Correction!
Weglage J, Pietsch M, Feldmann R, Koch H-G, Zschocke J et al.
 
Pediatric Research 49:532-536, 2001
Normal Clinical Outcome in Untreated Subjects with mild Hyperphenylalaninemia.
  
(PMID: 11264437)
See also LEVY ET AL. New Engl J Med 285:424-429, 1971.
This paper is significant. It addresses the question whether mild hyperphenylalaninemia (360-600 µM) should be treated with a low phenylalanine diet. Existing reports on treated "mild" phenotypes indicate outcome deficits in cognitive performance. (The question then arises whether dietary treatment was insufficient in process or goal, or whether the treatment itself contributed to the deficient outcome). This new paper studies 31 never-treated adolescent and adult patients who had persistent mild hyperphenylalaninemia in the range described. The assessment program included comprehensive psychological testing, MRI of the head, magnetic resonance spectroscopy and genotyping. All patients had mutant genotypes with compound heterozygosity at the PAH locus for a "mild" HPA mutation and a PKU-causing allele (6 alleles were not identified in the set of 62). The outcome measurements revealed no deficits. The authors concluded that "dietary treatment is unlikely to be of value in patients with mild hyperphenylalaninemia (< 600 mM)". Their findings imply some cost to outcome in the treatment modality itself. The authors were prudent and advised that every patient potentially in this class should be followed carefully in the first year of life to detect change of status (e.g. to PKU class). They also reminded us that the situation of maternal of hyperphenylalaninemia must also be kept in mind. 

NB This finding supports an observation long present in the literature, and largely forgotten. Levy and colleagues said, in 1971, that "dietary therapy appears not to be needed for persistent mild hyperphenylalaninemia". [Levy HL, Shih VE, Karolkewicz BA et al. Persistent mild hyperphenylalaninemia in the untreated state. A prospective study. New Engl J Med 285:424-429, 1971] (PMID 5557279)]

[26.03.02]
CpG SITES
IN THE PAH GENOMIC SEQUENCE.
PAHdb houses the genomic sequence for the PAH locus (~ 170 kb) submitted by Dr. David Konecki. Undoubtedly this sequence contains many polymorphic sites; one likely set of sites would be CpG dinucleotide sequences. There are 1198 of these in the PAH sequence. Their positions are highlighted in a table; comments are welcome.

 
[18.03.02]
BH4-responsive PAH alleles (PART II)
Blau N and Trefz FK. Letter to the Editor
.
Mol Genet Metab 75:186-187, 2002.
Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency: Possible regulation of gene expression in a patient with the homozygous L48S mutation.

(PMID: 11855940).
The story of BH4-responsive PAH alleles continues to unfold. The present paper indicates a forthcoming publication (Lassker U et al. J Inherit Metab Dis 2002) and lists an extended number of PAH alleles that may be BH4 responsive. These new alleles include L48S, I65T, R261Q, A395P and Y414C; they are listed in earlier papers (Kayaalp et al. Am J Hum Genet 61:1309-1317, 1997; and Guldberg P. et al. Am J Hum Genet 63:71-79, 1998) as ones with "inconsistent phenotype expression". Blau and Trefz suggest they may be BH4-responsive. In their present paper they observed BH4-responsiveness at therapeutic doses (20 mg/kg/day) in a patient homozygous for L48S. This amino acid maps to the regulatory domain and is not known to be a residue involved in cofactor binding or affinity. The authors suggest that the cofactor, at pharmacological doses, in this case, is regulating PAH gene expression. The plot thickens.

[12.03.02]
BH4 responsive PAH alleles (Part i).
[Kure S, Hou D-C, Obura T et al.
J Pediat 135:375-8, (1999)
Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

(PMID: 10484807);
Trefz T, Blau N, Aulehz-Scholz C et al
. Treatment of mild phenylketonuria (PKU) by tetrahydrobiopterin BH4. J Inherit Metab Dis 23:Suppl. 1, 47 (2000) (abstract);
Spaapen LJM, Bakker JA, Velter C et al
. Inherit Metab Dis 24:352-58, (2001).
Tetrahydrobiopterin response phenylalanine hydroxylase deficiency in Dutch neonates.

(PMID: 11486900).
Three separate reports record patients with hyperphenylalaninemia and loss-of-function missense alleles at the PAH locus. These patients responded to tetrahydrobiopterin in loading (20 mg/kg) or maintenance (5-10 mg/kg/d) doses. There was no abnormality in synthesis or recycling of tetrahydrobiopterin.

The PAH alleles that apparently confer responsiveness to BH4 at pharmacologic doses, at present, include V190A, R241C, A300S, A313T, A373T, E390G, A403V and P407S (under certain conditions).

Note: Four of the 8 alleles expressed themselves as a homopolymeric tetramer; the others as heteropolymeric enzymes, implying that subunit interactions contribute to BH4 responsiveness.

This important and newly recognized PAH enzyme phenotype has been analyzed in an elegant paper (Erlandsen H and Stevens RC. A structural hypothesis for BH4 responsiveness in patients with mild forms of hyperphenylalaninemia and phenylketonuria. J Inherit Metab Dis 24:213-30, 2001. PubMed 11405341). The authors are structural biologists who have made major contributions to our understanding of the PAH monomer and the functional homotetrameric enzyme. They present a hypothesis for the in vivo BH4 responsiveness. They propose that some mutations result in mutant enzymes that are KM variants with lower affinity for BH4 binding. Pharmacological doses of BH4 overcome the low binding affinity and saturate the enzyme surface to allow the hydroxylation reaction to take place. The majority (but not all) of the BH4 responsive PAH alleles map to the catalytic domain of the PAH monomer in one of two regions; either in cofactor binding regions or in regions that interact with the secondary elements involved in binding. The findings imply that mutation analysis of every proband could have practical significance for therapy. The possibility of BH4 responsiveness should be considered, certainly in patients with variant or non-PKU forms of hyperphenylalaninemia.


Giannattasio S, Jurgelevicius V, Lattanzio P, Cimbalistiene L, Marra E, Kučinskas V. Hum Hered 47:155-160, 1997.
Phenylketonuria Mutations and Linked Haplotypes in the Lithuanian Population: Origin of the Most Common R408W Mutation.   
(PMId: 9156326
)
The authors describe PAH mutations associated with phenylketonuria on 130 (presumably independent) Lithuanian chromosomes; subdivided by putative origins into 95 "Baltic", 28 "Slavonic", and 7 "unknown" origins. The principal mutation was R408W on haplotype 2 bearing VNTR3, STR 240 alleles. The relative frequency of R408W was ~ 70% overall in the 3 populations, separate and together. This finding is compatible with earlier observations that the center of diffusion for R408W on this haplotype is in Northeastern Europe in the Lithuanian region (Kučinskas V. et al. Hum Hered 44:110-113 (1994)). Giannattasio and colleagues speculate that the mutant R408W chromosome arose in ancient times among pre-IndoEuropean people; a "founder effect" is postulated to explain its present-day appearance in Europeans (and its relative absence in Finnish populations).

Giannattasio S, Dianzani I, Lattanzio P, Spada M, Romano V, Cali F, Andria G, Ponzone A, Marra E, Piazza A. Hum Hered 52:154-159, 2001. 
Genetic Heterogeneity in Five Italian Regions: Analysis of PAH Mutations and Minihaplotypes.
 
(PMID: 11588399
)
The authors analyzed PAH mutations on 289 chromosomes in Italian populations from 5 widely separated geographical regions in Italy. They identified 24 different PAH mutations through patients with hyperphenylalaninemia/PKU. The mutations were associated with 21 different "minihaplotypes" (comprising only the STR and VNTR alleles). The authors conclude that there is genetic stratification in Italy more complex than described in an earlier study (Dianzani I et al. Am J Hum Genet 55:851-853, 1994). The present findings at the PAH locus complement those described at the CFTR locus (Rendine S. et al. Ann Hum Genet 61:411-424 (1997)).

Kidd JR, Pakstis AJ, Zhao H, Lu R-B, Okonofua FE, Odunsi A, Grigorenko E, Bonne-Tamir B, Friedlaender J, Schulz LO, Parnas J, Kidd KK . Am J Hum Genet 66:1882-1899, 2000.
Haplotypes and Linkage Disequilibrium at the Phenylalanine Hydroxylase Locus, PAH, in a Global Representation of Populations
. 
(PMID: 10788337)
Kidd and her colleagues used four single nucleotide polymorphisms (SNPs) in the PAH gene spanning 75 kb of its dimension; two of the polymorphisms were located near each end of the gene. The particular set was selected to study linkage disequilibrium in 50 individuals (on average) in each of 29 different populations from Europe, Asia, Africa and the Americas. All four sites are polymorphic in all 29 populations; all but five of the 16 possible haplotypes were present at more than 5% frequency in at least one population. Not one haplotype was found in every population. In European populations linkage disequilibrium was found generally not to exist between RFLPs at opposite ends of the gene but existed between the markers clustered at each end. The linkage disequilibrium data were compatible with an out-of-Africa hypothesis with a low overall linkage disequilibrium coefficient across the gene in African populations (yet showing variation within Africa). North American Indians have a pattern of haplotype frequencies markedly different from that of eastern Asians, and linkage disequilibrium between opposite ends of the PAH gene is significant only in native American populations (and in one African population).

Scriver CR, Kaufman S (2001) Hyperphenylalanemia: phenylalanine hydroxylase deficiency. in Scriver CR, Baudet AL, Sly WS, Valle D (eds); Childs B, Kinzler K, Vogelstein B (assoc. eds.).
The Metabolics and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill (Med Pub. Div). New York. pp. 1667-1724
.
The article is actually chapter 77 in the book.  It is a comprehensive review of clinical, metabolic, enzymatic and genetic aspects of hyperphenylalanemia.  The chapter was printed in January 2001, updates were made in the text up to the time of page proofs (mid 2000).  7 updates await submission to the web online version of MMBID due for release mid 2002.  Access to this material is available directly from the author (Scriver).

CARTER KC, BYCK S, WATERS PJ, RICHARD B, NOWACKI PM, LAFRAMBOISE R, LAMBERT M, TREACY E, SCRIVER CR. Eur J Hum Genet 6:61-70, 1998.
Mutation at the phenylalanine hydroxylase gene (PAH) and its use to document population genetic variation: the Quebec experience
. 
(PMID: 9781015)
The article describes allelic variation at the PAH locus in contemporary populations of Quebec province, Canada (7 million persons). The Quebec population reflects range expansion of Europeans: French until 1759, English, Scots, Irish and Loyalists (by migration) after 1759; and other Europeans and Asians after 1945. The province has had universal newborn screening for hyperphenylalaninemia since 1971. All patients with PKU identified during a 20 year interval and available for analysis (141 independent chromosomes), and 4 additional probands with non-PKU HPA (8 chromosomes), were studied; 96% of the PKU sample and all non-PKU HPA chromosomes were successfully analyzed. There were 45 different PAH alleles classified as: i) 7 polymorphisms (IVS2nt19, IV3nt-22, IVS6nt-55, Q232Q, V245V, L385L, Y414Y). ii) 4 mutations causing non-PKU HPA (T92I, E390G, R408Q, D415N); iii). 34 mutations causing PKU; 74 % of the alleles are missense. Only 6 mutations (M1V, R261Q, F299C, S349P, R408W, and IVS12nt1) occurred at relative frequencies above 5%; they accounted for half of the total. Alleles stratified in the population by geographic region, demography and population history. Two PKU mutations (E280K on haplotype 2 and R408W on haplotype 1) occurred on non-conventional polymorphic haplotypes in Quebec and gave evidence of being recurrent mutations. Four alleles (S67P, G218V, V245A and IVS12nt1) also occurred on non-conventional haplotypes that could be best explained by intragenic recombination. Ten alleles were first described in Quebec and 5 remain unique to this region; three novel alleles are reported here for the first time (c.125A->T (K42I); [c.470G->A, c.471A->C] (R157N); c.707nt-55 [IVS6nt-55]). The homozygosity value (j) is 0.06 for the whole Quebec region with similar values for Eastern and Western Quebec and Montreal, each with a different demographic history (the abstract errs in stating a range value of 0.08 to 0.5); homoallelic HPA genotypes were 24% of the total (CRS 29, June 1999).

Tyfield, LA. Mol Pathol 50(4):169-74, Aug 1997.
Phenylketonuria in Britain: genetic analysis gives a historical perspective of the disorder but will it predict the future for affected individuals?

(PMID: 9350299)
This article is a review of the core general information about the PAH gene and its mutations; it also provides information about hypermutable codons in PAH (from Table 1 in Byck et al. Hum Mut 9:316-21, 1997 (PMID:9101291)) and the distributions of alleles within the physical structure of the gene (from Figure 1, in PAHdb Newsletter). Figure 2 by Tyfield is a map of England, Wales, Scotland and Ireland showing relative regional frequencies of 5 major European disease-causing alleles (R408W, IVS10nt-11, IVS12nt7, L48S, I65T). Figure 3 relates the diagnostic blood phe level to the mutant genotype in over 50 patients with HPA in the UK; genotype-phenotype correlations (plus non correlations) and predictability of phenotype from genotype are discussed (CRS, 30 June 1999).

Leandro P., Rivera I., Lechner M.C., Tavares D.E., Almeida I. and Konecki D. Molec Genet Metab 69:204-212, 2000.
The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase.
 
(PMID: 10767175)
The majority of mutations in the human genome according to the Human Mutation Genome Database (Cardiff) are missense mutations. The PAH gene harbours many missense mutations, representing ~ 60% of the total so far recorded; most do not map to critical residues framing the catalytic site of PAH enzyme (Erlandsen and Stevens 1999). Therefore mutations affecting kinetics of PAH function are of special interest. The V388M mutation affects the kinetic properties of human PAH. In Vitro Expression analysis of this allele resulted in reduced specific activity (30% wild type) in the presence of synthetic cofactor, and even lower values with the natural cofactor. The allele conferred reduced affinity for both substrate and natural cofactor.

RIVERA I, CABRAL A, ALMEIDA M, LEANDRO P, CARMONA C, EUSEBIO F, TASSO T, VILARINHO L, MARTINS E, LECHNER MC, TAVARES DE ALMEIDA I, KONECKI DS, LICHTER-KONECKI U. Molec Genet Metab 69:195-203, 2000.
The correlation of genotype and phenotype in Portuguese hyperphenylalaninemic patients.

(PMID: 10767174)
This article describes findings in 61 patients with phenylalanine hydroxylase deficiency and hyperphenylalaninemia: 20 different mutant alleles in 36 different genotype combinations are described. There was a "strong correlation" between predicted enzyme activity (in vitro) and the observed metabolic phenotype (and dietary tolerance for phenylalanine). The paper describes a Portuguese population; description of these alleles (Table 1 in paper) is relevant for the population genetics of PAH alleles. The genotype-phenotype correlations fit broadly into the findings of earlier reports (e,g. Kayaalp et al. 1997 (PMID 9399896); Guldberg et al. 1998 (PMID 98299800)).

WATERS, P,J., PARNIAK, M.A., AKERMAN, B.R., and SCRIVER, C.R. Molec Genet and Metab 69:101-110, 2000.
Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype.
(PMID: 10720436)

This paper is one in a series by Waters and colleagues describing the effect of PAH mutations in a variety of expression systems. Mutations that do not map to the catalytic domain or residues critic for catalytic function are described in the present report. These missense mutations have their effect by mechanisms remote from a null effect on protein synthesis and formation of the enzyme or a primary kinetic effect on the hydroxylation reaction. The data show that the missense mutations studied here effect the stability of the protein of the polypeptide, presumably by misfolding. The mutant polypeptide is likely to aggregate and enter a proteolytic pathway. Since 60% of the disease causing mutations in the PAH gene are missense, the majority of these may have their effect through impaired protein stability. Thus PAH mutations indicate a new paradigm for understanding the effect of mutations on enzymic function. 

The paradigm of defective folding and rapid degradation of mutant proteins is gaining rapid acceptance and has been discussed elegantly by Gregersen and colleagues in a recent review (J. Inher Metab Dis 23:441-447, 2000, PMID: 10947197)


SARKISSIAN, C.N., BOULAIS, D.M., MACDONALD, J.D. and SCRIVER, C.R. Molec Genet and Metab 69:188-194, 2000.
A heteroallelic mutation mouse model: A new orthologue for human hyperphenylalaninemia.

(PMID: 10767173)
This paper describes the ethyl nitrosourea-generated mutant mouse model developed initially by William Dove and colleagues (Wisconsin University) and further developed as described in this paper. The animals have mutations in exon 3 or exon 7 conferring non-PKU hyperphenylalaninemia or PKU-like phenotypes respectively in the autosomal recessive state. A hybrid animal with homozygous phenotype but heteroallelic for the exon 3 and exon 7 mutations provides advantages for a variety of studies and is analogous to the heteroallelic state that characterizes about 75% of human probands. Detailed descriptions are available also on this web site (mouse module).

ERLANDSEN H. and STEVENS R.C. Molec Genet and Metab 68: 103-125, 1999.
The structure basis of phenylketonuria.

(PMID: 10527663)
This important paper is part of a series in which collaborators in Norway and California worked out the molecular structure of the PAH enzyme at 2 AXXX resolution. The authors used PAHdb as a source of mutations which they then mapped onto the crystal structure as a structural scaffold for explaining the effects of some of the mutations. The paper provides a table listing the nucleotide change of missense mutations, the change in residue, the structural contacts involved, the predicted and calculated frequency of mutation, and the primary reference for the source of the mutation report. A series of figures illustrate the regulatory, catalytic and tetramerization domains, the tetrameric structure, the active site surrounding the catalytic iron in PAH and other views of the protein structure and critical residues that are affected by specific mutations.

SCRIVER CR, WATERS PJ. Trends in Genetics 15(7): 267-272, 1999.
Monogenic traits are not simple: lessons from phenylketonuria.
(PMID: 10384369)

The classification of genetic disease into chromosomal, monogenic and multifactorial categories is an oversimplification. Phenylketonuria (PKU) and PAH-locus related forms of hyperphenylalaninemia are classic "monogenic" phenotypes inherited as autosomal recessives. It has been assumed that mutation in the major gene is sufficient to explain the impaired function of the enzyme (phenylalanine hydroxylase (monooxygenase), E.C. 1.14.16.1), and the attendant hyperphenylalaninemia (the metabolic phenotype). Impaired cognitive development (the clinical phenotype) is presumed to be a consequence of hyperphenylalaninemia. Kayaalp E et al. (1997) Am J Hum Genet 61:1309-1317 (PMID 9399896) and Guldberg P et al. (1998) Am J Hum Genet 63:71-79 (PMID 9634518), by metanalyses of PAH genotype-metabolic/clinical phenotype correlations observed examples where genotype was not associated with the predicted phenotype. The paper by Scriver and Waters examines events that might explain such genotype/phenotype discordances in a classic "monogenic" disease, for example: extensive allelic variation, locus heterogeneity concerning tetrahydrobiopterin homeostasis and the multifactorial nature of the phenotype (since both mutation and dietary phenylalanine intake are necessary conditions for hyperphenylalaninemia). Features of a complex trait begin to emerge at three phenotypic levels:

  1. enzymic, where many of the missense mutations primarily alter protein folding and stability, and secondarily affect enzymic activity; anything that would modify flux of the misfolded protein through the non productive proteolysis pathway (e.g. chaperone "allelism") could modulate enzyme activity.

  2. The rate of catabolic outflow of phenylalanine by the transaminase pathway in PKU, which modulates phenylalanine pool size and tolerance for diet phenylalanine, can vary between siblings with identical mutant PAH genotypes (Treacy EP et al. (1996) J Inher Metab Dis 19:595-602 (PMID 8892014)).

  3. Impaired cognitive development is related to phenylalanine concentration in the brain compartment; influx of phenylalanine acts as a transporter across the blood brain barrier and is a variable parameter suggesting that physiological (or allelic) variation is a function that will modify clinical (cognitive) phenotype (see Weglage J et al (1998) J Inher Metab Dis 21:81-83 (PMID 9584274); Moller HE et al (1998) J Cereb Blood Flow Metab 18:1184-1191 (PMID 9809507).

Scriver and Waters point out that the genotype-phenotype discordance theme can be traced back to Penrose in his inaugural address as Galton professor at the University of London in 1946 (see Penrose LS (1998) Ann Hum Genet 62:193-202, a reprint of original paper from 1946 (PMID 9803263).

That monogenic diseases do not show consistent relationships between their genotypes and phenotypes is an old awareness reappearing and addressed in other articles (Summers KM (1996) Hum Mutat 7:283-293 (PMID 8723677); Dipple KM and McCabe ERB (2000) Am J Hum Genet 66:1729-1735 (PMID 10793008); Dipple KM, McCabe ERB (2000) Mol Genet Metab 71:43-50 (PMID 11001794)). Should we be surprised by discordance? Probably not. "Genomes speak biochemistry not phenotype" (Plasterk RH (1999) Nat Genet 21:63-64 (PMID 9916788)). What goes on in complex metabolic networks, cycles and pathways, and how the effect of a mutant allele is buffered, has been discussed since the days of RA Fisher, Sewall Wright, and Henrik Kacser, for example, and it continues (Hartman JH et al (2001) Science 291:1001-1004 (PMID 11232561) ). Barkai N and Leibler S (1997) Nature 387:913-917 (PMID 10659837); Hartwell LH et al (1999) Nature 402:C47-C52 (PMID 10591225); Jeong H et al (2000) Nature 407:651-654 (PMID 11034217). Whereas there is robustness in "standardized" evolutionary biochemical networks, at levels of molecular or modular cell biology, it is redundancy (in alleles, genes and feedback in regulatory pathways, that both randomize and co-opts the "noisy business" of regulatory control and homeostasis. organization of metabolic networks. (McAdams HH and Arkin A (1999) Trends Genet 15:65-69. 1999 (PMID 10098409)).

CONSENSUS DEVELOPMENT CONFERENCE ON PHENYLALANINE: SCREENING AND MANAGEMENT (OCT. 16-18, 2000). Bethesda, Maryland.
Report Published: Jan. 2001. US Department of Health and Human Services; Public Health Service; National Institutes of Health; National Institute of Child Health and Human Development.

The NIH Consensus Conference was held to develop a response in the North American context, to guidelines previously published from the United Kingdom (Medical Research Council Working Party on Phenylketonuria (UK) (1993) BMJ 306:115-119 (PMID 8435608). Burgard P et al. Eur J Pediatr (1999) 158:46-54 (PMID 9950308). The present document offers balanced opinions about screening, treatment; in particular, treatment of hyperphenylalaninemia/PKU beyond adolescence and childhood.

Appendix A of the Consensus Report contains tables of PAH mutations (with systematic and trivial names) classified by State in the USA, in the other Americas (not USA), in the British Isles, in Europe - Western, Central and Eastern regions, and in Africa, Asia and Australia.

Copyright © 2003 DeBelle Laboratory - Created [2002.10.17.208076] - Updated [2009.08.31]