[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.

[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.

[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.

[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!

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.

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.

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 …!

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.  

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.  

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.

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).

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).

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.

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").

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.

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.