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Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype (OMIM 261600), is an inborn error of metabolism causing impaired postnatal cognitive development in the absence of treatment. We used the Pahenu2/enu2 PKU mouse model to study oral enzyme substitution therapy with various chemically modified formulations of phenylalanine ammonia lyase (Av-p.C503S/p.C565S/p.F18A PAL). In vivo studies with the most therapeutically effective formulation (5 kDa PEG-Av-p.C503S/p.C565S/p.F18A PAL) revealed that this conjugate, given orally, yielded statistically significant (p = 0.0029) and therapeutically relevant reduction (~40%) in plasma phenylalanine (Phe) levels. Phe reduction occurred in a dose- and loading-dependent manner; sustained clinically and statistically significant reduction of plasma Phe levels was observed with a treatment ranging between 0.3 IU and 9 IU and with more frequent and smaller dosings. Oral PAL therapy could potentially serve as an adjunct therapy, perhaps with dietary treatment, and will work independently of phenylalanine hydroxylase (PAH), correcting such forms of hyperphenylalaninemias regardless of the PAH mutations carried by the patient.

Phenylalanine ammonia lyase (PAL) has long been recognized as a potential enzyme replacement therapeutic for treatment of phenylketonuria. However, various strategies for the oral delivery of PAL have been complicated by the low intestinal pH, aggressive proteolytic digestion and circulation time in the GI tract. In this work, we report 3 strategies to address these challenges. First, we used site-directed mutagenesis of a chymotrypsin cleavage site to modestly improve protease resistance; second, we used silica sol-gel material as a matrix to demonstrate that a silica matrix can provide protection to entrapped PAL proteins against intestinal proteases, as well as a low pH of 3.5; finally, we demonstrated that PEGylation of AvPAL surface lysines can reduce the inactivation of the enzyme by trypsin.

Phenylketonuria (PKU) is a metabolic disorder, in which loss of phenylalanine hydroxylase activity results in neurotoxic levels of phenylalanine. We used the Pah enu2/enu2 PKU mouse model in short- and long-term studies of enzyme substitution therapy with PEGylated phenylalanine ammonia lyase (PEG-PAL conjugates) from 4 different species. The most therapeutically effective PAL ( Av, Anabaena variabilis ) species was one without the highest specific activity, but with the highest stability; indicating the importance of protein stability in the development of effective protein therapeutics. A PEG- Av -p.C503S/p.C565S-PAL effectively lowered phenylalanine levels in both vascular space and brain tissue over a >90 day trial period, resulting in reduced manifestations associated with PKU, including reversal of PKU-associated hypopigmentation and enhanced animal health. Phenylalanine reduction occurred in a dose- and loading-dependent manner, and PEGylation reduced the neutralizing immune response to the enzyme. Human clinical trials with PEG- Av -p.C503S/p.C565S-PAL as a treatment for PKU are underway.

La population du Saguenay est depuis longtemps reconnue pour l’ampleur des problèmes génétiques auxquels elle fait face. S’agissant en particulier de maladies récessives comme la tyrosinémie, l’ataxie de Friedreich (forme Charlevoix-Saguenay), le rachitisme ou l’agénésie du corps calleux, on a imputé les fortes incidences observées à divers facteurs comme les nombreux mariages consanguins, la stabilité de la population, un effet fondateur particulièrement accentué, un modèle particulier d’immigration, etc. Cependant, les analyses que nous avons commencé à réaliser à l’aide du fichier de population construit par SOREP obligent à réviser ces énoncés. La construction des généalogies par ordinateur et l’étude des comportements démographiques à l’aide de la reconstitution automatique des familles permettent en effet des analyses rétrospectives très approfondies et livrent des aperçus qui étaient hors de portée jusqu’à ce jour. L’enquête que nous rapportons ici revêt un caractère expérimental ; c'était la première fois que nous tentions une utilisation rigoureuse du fichier de population dans cette direction. Les résultats obtenus ont d’ores et déjà des retombées sur le conseil génétique et, en outre, ils modifient substantiellement notre perception de la dynamique démographique au Saguenay., The population of the Saguenay has long been known for the magnitude of the genetic problems it faces. In particular, since the problem is one of recessive illnesses such as tyrosinemia, Friedreich's ataxia (Charlevoix-Saguenay type), rickets or agenesis of corpus callosum, the strong observed effects have been ascribed to several factors such as the numerous mariages between blood relatives, the stability of the population, a particularly strong founder effect, a specific model of immigration, etc. However, the analysis we have begun to carry out with the aid of SOREP's population data base, forces us to revise these statements. The computer construction of genealogies and the study of demographic behaviour using automatic family reconstruction allow, indeed, for very thorough retrospective analyses, and deliver insights that, to this day, were out of reach. The survey on which we report is of an experimental nature; it was the first time we attempted a rigorous use of the population data base, for such purposes. The results obtained have already had an impact on underlying assumptions in the field of genetics, and moreover, they have substantially changed our perception of the demographic dynamics of the Saguenay., Hace tiempo que la población saguenayense es reputada por la envergadura de los problemas genéticos que enfrenta. Se trata particularmente de las enfermedades recesivas como la tirosinemia, la ataxia de Friedreich (tipo Charlevoix-Saguenay), el raquitismo o la agenesia del cuerpo calloso, cuya alta frecuencia se ha atribuido a diversos factores tales como el alto numero de uniones consanguíneas, la estabilidad de la población, un efecto "fundador" especialmente acentuado, un patrón particular de migración, etc. No obstante, los análisis que hemos comenzado a realizar gracias al archivo de población elaborado por SOREP (Sociedad de Investigaciones acerca de las Poblaciones), compelen a examinar de nuevo estos enunciados. La creación de genealogías por medio del computador y el estudio de los coiportamientos demográficos a través de la reconstitución automática de las familias, permiten elaborar análisis retrospectivos refinados que ofrecen una visión que antes estaba fuera de nuestro alcance. La investigación, de la cual damos cuenta en este articulo, es de carácter experimental; por primera ocasión intentamos el uso minucioso del archivo de población en este sentido. Los resultados obtenidos tienen ya su resonancia en la orientación que prodigan los genetistas, además de que modifican substancialmente nuestra percepción acerca de la dinámica demográfica del Saguenay.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

''Inborn errors of metabolism,'' first recognized 100 years ago by Garrod, were seen as transforming evidence for chemical and biological individuality. Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype, was identified in 1934 by Asbjorn Folling. It is a disease with impaired postnatal cognitive development resulting from a neurotoxic effect of hyperphenylalaninemia (HPA). Its metabolic phenotype is accountable to multifactorial origins both in nurture, where the normal nutritional experience introduces L-phenylalanine, and in nature, where mutations (4500 alleles) occur in the phenylalanine hydroxylase gene (PAH) on chromosome 12q23.2 encoding the L-phenylalanine hydroxylase enzyme (EC 1.14.16.1). The PAH enzyme converts phenylalanine to tyrosine in the presence of molecular oxygen and catalytic amounts of tetrahydrobiopterin (BH4), its nonprotein cofactor. PKU is among the first of the human genetic diseases to enter, through newborn screening, the domain of public health, and to show a treatment effect. This effect caused a paradigm shift in attitudes about genetic disease. The PKU story contains many messages, including: a framework on which to appreciate the complexity of PKU in which phenotype reflects both locus-specific and genomic components; what the human PAH gene tells us about human population genetics and evolution of modern humans; and how our interest in PKU is served by a locus-specific mutation database (http://www.pahdb.mcgill.ca; last accessed 20 March 2007). The individual Mendelian PKU phenotype has no ''simple'' or single explanation; every patient has her/his own complex PKU phenotype and will be treated accordingly. Knowledge about PKU reveals genomic components of both disease and health. Hum Mutat 28(9), 831-845, 2007. Published 2007 Wiley-Liss, Inc. y

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6 R )- l - erythro -5,6,7,8-tetrahydrobiopterin (BH 4 )]. Several hypotheses have been put forward to explain BH 4 responsiveness, but the molecular basis for the corrective effect(s) of BH 4 has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH 4 -responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH 4 responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased K m (BH 4 ) and reduced cooperativity of substrate binding, but no decoupling of cofactor (BH 4 ) oxidation. For some, BH 4 seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo . Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH 4 appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

View Large Image | View Hi-Res Image | Download PowerPoint SlideLet me begin by expressing my thanks to The American Society of Human Genetics (ASHG) for the recognition it is giving with the McKusick Award. Thanks also to David Valle for his very kind words of introduction and also to Neil Risch for the way he managed to astonish me with the news.Previous recipients of the McKusick Award have set the bar very high, and I am surprised to be joining them. This is a good moment for me to thank those in charge of the nominations and selection of candidates. It is also a good moment to suggest that the ASHG is perhaps wrong in honoring only me for the award this year because what is being recognized is in fact the opportunities that by chance came my way. Indeed, the colleagues who translated opportunities into data, discoveries, and services are ultimately honored here.Thanks also to the man from Maine, Victor McKusick, whose style, energy, wisdom, and advice have influenced so many of us. Victor had a memorable way of saying things and of getting things done, and he would certainly have something to say about my speaking to you from Australia under bizarre technical conditions. I can hear Victor saying, “If I had to go to theyre, I would not start from heyah.”Recognition is precious, and it is received accordingly. The ASHG is recognizing me, but in fact, with this award, it is recognizing many, many persons and a team of good people who I will not name individually here so as to avoid errors of omission. My colleagues are identifiable in many other ways, for example, as co-authors of publications and as participants in programs of service and in initiatives in education. Indeed, we cooperated, making the whole more than the sum of its parts and letting cooperation be seen as a successful feature of human evolution. This is my opportunity to express my personal admiration and affection for all those colleagues. In the words of Thomas Merton, they have been “shining like the sun” and are recognized here accordingly.And then there is the family! It has been and remains an inspiration. The words are brief; the feeling is huge.Chance and opportunity, where and when, have played important roles in my life. For example, in 1962, my wife and I were traveling in our Volkswagen Beetle when an impatient driver tried to pass us on the two-lane highway. Unfortunately, there was oncoming traffic in the other lane. The impatient person smashed into us while trying to avoid a head-on collision. Our car veered, and in a fortunate break in the traffic, we rolled (yes, rolled!) through a gap in the oncoming lane of cars. Believing we were about to die, I called out to my wife, “It has been great!” We did survive, and the continuing gift of life has been and continues to be acknowledged in every breath.Where chance and opportunity played their important roles in my life and when they did are relevant facts. For example, I have worked all my academic life at the Montreal Children’s Hospital under the McGill University umbrella in Quebec, Canada. These proved to be enlightened environments. When they played their roles was also important. In the 1950s, Quebec was experiencing the Quiet Revolution, which transformed Quebec society; and while that was happening, the role of the DNA molecule in biology was being recognized. The incidental development of chromatography as a new technology, and its contribution to the discovery of inborn errors of metabolism, for example, greatly influenced me personally. I was participating as a fledgling clinician-scientist in what is now called translational science. I found it could serve scientific inquiry, medical services, and the health of society as a whole. The character of society is revealed in how we treat our weaker members, such as children, the aged, the disabled, and the chronically ill. Perhaps at some other time, I could describe how my own experiences illustrate a relatively humane society in progress. You would discover why it has been possible for me to work with all of those colleagues in this place called the Montreal Children’ s Hospital at McGill University in Montreal, Quebec, Canada.I have always enjoyed literature and poetry. I love this poem by the Russian author Osip Mandelstam; it was composed in 1909. It is a metaphor for the genetics of life itself:What shall I do with the body I have been givenSo much at one with me, so much my own?For the calm happiness of breathing, being ableTo be alive, tell me where I should be grateful.I am gardener, flower too, and un-aloneIn this vast dungeon.My breath, my glow, you can already seeOn the windowpanes of eternity.A pattern is imprinted thereUnknown ’til now.Let this muddle die down, this sediment flow outThe lovely pattern cannot be crossed out.And then there is this wonderfully honest brief comment by W.S. Merwin, the American poet, who at age 84 wrote, “I have with me all that I do not know / I have lost none of it.”Thank you again for the recognition and for this opportunity to share a thought or two.

PAHdb is an online relational locus-specific "mutation database" (http://www.mcgill.ca/pahdb) for the human phenylalanine hydroxylase gene (symbol PAH) and its associated phenotypes (protein, metabolic, clinical). When combined with associated information (population distribution of allele, haplotype association, etc.) PAHdb functions as a knowledgebase. From the outset, and in the absence of raw data (e.g., sequence gels), PAHdb has instead been an annotated repository of information about mutations maintained by a team of curators. It is also disease-oriented, being focused on a variant phenotype (hyperphenylalaninemia (HPA) and its most important form of disease, phenylketonuria (PKU)) resulting from primary dysfunction of the PAH enzyme (EC 1.14.16.1); it is "patient friendly" in that it contains information for those personally involved with HPA/PKU (MIM# 261600). PAHdb also serves its community through direct interaction.

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5 ) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1 ), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe ( i ) an efficient recombinant approach to produce PAL enzyme, ( ii ) testing of PAL in orthologous N -ethyl- N′ -nitrosourea (ENU) mutant mouse strains with HPA, and ( iii ) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

These Guidelines recognize the need for annotated online mutation databases documenting allelic variation (both pathogenic and phenotype modifying, and also neutral polymorphic); the databases will be both generalized (genomic) and specialized (locus specific), and a seamless integration of the two types is intended. Each requires a Document (its “biography”). Different mutation databases will have different content and structure, but a minimum core of content in a shared syntax is a necessity; the core includes: (1) a unique identifier of the allele; (2) the source /report of the data; (3) context of the allele; and (4) the allele itself (the description). The allele description should be validated. There is no single correct way to design a mutation database. The uses to which databases are put dictate the design. Software and deployment together recognize the different needs of specialized and generalized databases, while making them mutually compatible through shared content and the appropriate search facilities. A set of eight Recommendations completes these Guidelines for Content, Design, and Deployment of Mutation Databases. Hum Mutat 13:344–350, 1999. © 1999 Wiley-Liss, Inc.

PAHdb (http://www.mcgill.ca/pahdb ) is a curated relational database (Fig. 1) of nucleotide variation in the human PAH cDNA (GenBank {"type":"entrez-nucleotide","attrs":{"text":"U49897","term_id":"2462721","term_text":"U49897"}}U49897). Among 328 different mutations by state (Fig. 2) the majority are rare mutations causing hyperphenylalaninemia (HPA) (OMIM 261600), the remainder are polymorphic variants without apparent effect on phenotype. PAHdb modules contain mutations, polymorphic haplotypes, genotype-phenotype correlations, expression analysis, sources of information and the reference sequence; the database also contains pages of clinical information and data on three ENU mouse orthologues of human HPA. Only six different mutations account for 60% of human HPA chromosomes worldwide, mutations stratify by population and geographic region, and the Oriental and Caucasian mutation sets are different (Fig. 3). PAHdb provides curated electronic publication and one third of its incoming reports are direct submissions. Each different mutation receives a systematic (nucleotide) name and a unique identifier (UID). Data are accessed both by a Newsletter and a search engine on the website; integrity of the database is ensured by keeping the curated template offline. There have been >6500 online interrogations of the website.

Hyperphenylalaninemia (HPA) resulting from deficient activity of phenylalanine hydroxylase (PAH) is caused by mutations in the human PAH gene (McKusick 261600). Herein, we report a noninvasive method to: 1) estimate whole-body phenylalanine oxidation in patients with HPA and 2) compare effects of mutant genotypes on phenotypes. We used oral L-[1-13C]phenylalanine as a substrate and measured 13CO2 formation in the first hour as an index of phenylalanine oxidation rates in: 1) patients with PKU (n = 6), variant phenylketonuria (PKU) (n = 7) and non-PKU HPA (n = 4); 2) obligate heterozygotes (n = 18); and 3) controls (n = 8). PAH mutations were identified by PCR, denaturing gradient gel electrophoresis, and DNA sequencing. Phenylalanine oxidation rates demonstrated a gene dosage effect; oxidation in heterozygotes was intermediate between probands and controls. The three classes of HPA had different mean oxidation rates (PKU < variant PKU < non-PKU HPA). The in vivo phenotype (HPA class or whole-body oxidation rate) did not always correspond to prediction from in vitro expression analysis of the mutation effect on enzyme activity. The findings indicate that the in vivo metrical trait (phenylalanine oxidation rate) is not a simple equivalent of phenylalanine hydroxylation activity (unit of protein phenotype) and, as expected, is an emergent property under the control of more than the PAH locus.

The PAH Mutation Analysis Consortium (81 investigators, 26 countries) is engaged in mutation detection at the human PAH locus. Ascertainment of probands occurs largely through newborn screening for hyperphenylalaninemia. A relational database records allelic variation (disease-producing and polymorphic) at the locus. Information is distributed by Newsletter, diskette (WINPAHDB software stand-alone executable on IBM compatible hardware), and at a 'real' site on the Worldwide Web (http://www.mcgill.ca/pahdb). The database presently records (Sept. 27, 1995) 248 alleles in 798 different associations (with polymorphic haplotype, geographic region and population) along with additional information. The database, as a record of human genetic diversity, at a particular locus, contributes to the study of human evolution and demic expansion; it also has medical relevance.

American Pediatric Society Presidential Address 1995: Disease, War, and Biology: Languages for Medicine—and Pediatrics

Phenylalanine hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperphenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity and without dietary restriction of phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperphenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperphenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperphenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperphenylalaninemia involves a low-protein diet and use of a phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma phenylalanine and tyrosine concentrations is necessary. Targets of plasma phenylalanine of 120-360 μmol/L (2-6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be given to adult women with hyperphenylalaninemia because of the teratogenic effects of phenylalanine. Women with phenylalanine hydroxylase deficiency considering pregnancy should follow special guidelines and assure adequate energy intake with the proper proportion of protein, fat, and carbohydrates to minimize risks to the developing fetus. Molecular genetic testing of the phenylalanine hydroxylase gene is available for genetic counseling purposes to determine carrier status of at-risk relatives and for prenatal testing.

Prospectively enrolled phenylketonuria patients (n=485) participated in an international Phase II clinical trial to identify the prevalence of a therapeutic response to daily doses of sapropterin dihydrochloride (sapropterin, KUVAN(®)). Responsive patients were then enrolled in two subsequent Phase III clinical trials to examine safety, ability to reduce blood Phenylalanine levels, dosage (5-20 mg/kg/day) and response, and bioavailability of sapropterin. We combined phenotypic findings in the Phase II and III clinical trials to classify study-related responsiveness associated with specific alleles and genotypes identified in the patients. We found that 17% of patients showed a response to sapropterin. The patients harbored 245 different genotypes derived from 122 different alleles, among which ten alleles were newly discovered. Only 16.3% of the genotypes clearly conferred a sapropterin-responsive phenotype. Among the different PAH alleles, only 5% conferred a responsive phenotype. The responsive alleles were largely but not solely missense mutations known to or likely to cause misfolding of the PAH subunit. However, the metabolic response was not robustly predictable from the PAH genotypes, based on the study design adopted for these clinical trials, and accordingly it seems prudent to test each person for this phenotype with a standardized protocol.

SummaryThe expectation for a gene dose effect in an X-linked phenotype is that the corresponding metrical trait in heterozygous females will lie between values for affected hemizygous males and unaffected males and females. We made sequential measurements (at 30, 60, 90, 120 and 150 days) of serum phosphate concentration and tail length in mice with X-linked hypophosphatemia (genotypes: Hyp/Y, Hyp/+ and Hyp/Hyp) and in their normal litter-mates (genotypes: +/Y, +/+). We also measured renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity in 5 to 7-month-old mice fed control and low phosphate diets and representing all five genotypes. The animals were obtained by controlled breeding under uniform environmental conditions. The mutant animals all had uniformly and significantly lower serum phosphate levels, shorter tail length and higher 24-hydroxylase activity relative to unaffected litter-mates. There was no evidence of a gene dose effect because values were not significantly different among the three mutant genotypes. We also studied the influence of gamete of origin on serum phosphate, tail length and renal mitochondrial 24-hydroxylase activity in the Hyp/+ offspring of affected males (Hyp/Y) or affected females (Hyp/+ or Hyp/Hyp). We found no effect on the distribution of trait values. We conclude that parental origin of mutant allele does not explain the absence of a gene dose effect in Hyp mice.

We analyzed mutations, RFLP haplotypes (H), and a VNTR polymorphism at the phenylalanine hydroxylase locus (PAH) in 12 French-Canadian patients with phenylketonuria (PKU) from the eastern region of Quebec province and 13 non-French-Canadian PKU patients from the Montreal region. There were 10 different mutation/H/VNTR haplotype combinations on the 50 PKU chromosomes: one set of 5 and another of 8 accounted for 88 and 77% of these chromosomes in the French-Canadian and non-French-Canadian patients, respectively. The differences in PKU haplotypes between the two groups of probands reflect the different histories of the two populations. Three PKU haplotype combinations were shared by the two groups: IVS12nt1:H-3:VNTR-8, I65T:H-9:VNTR-8, and R408W:H-1:VNTR-8. The IVS12nt1 mutation (18% of the total sample) is prevalent in northern Europeans. The I65T-H-9 and R408W:H-1 haplotypes have seldom been reported in Europeans but when encountered tend to be found in northwestern regions. The R408W mutation is usually on H-2 in Europeans. In Quebec the R408W:H-1 and I65T:H-9 haplotypes accounted for 20% of PKU chromosomes, clustered in two geographic regions, and in every family where they occurred an Irish or Scottish ('Celtic') ancestor could be inferred. We propose that I65T:H-9:VNTR-8 and R408W:H-1:VNTR-8 chromosomes are markers for a diaspora of 'Celtic' descendants. Our findings predict the distributions of these unusual PKU haplotypes in contemporary Europeans.

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Frequency of INherited Disorders database (FINDbase) (http://www.findbase.org) is a relational database, derived from the ETHNOS software, recording frequencies of causative mutations leading to inherited disorders worldwide. Database records include the population and ethnic group, the disorder name and the related gene, accompanied by links to any corresponding locus-specific mutation database, to the respective Online Mendelian Inheritance in Man entries and the mutation together with its frequency in that population. The initial information is derived from the published literature, locus-specific databases and genetic disease consortia. FINDbase offers a user-friendly query interface, providing instant access to the list and frequencies of the different mutations. Query outputs can be either in a table or graphical format, accompanied by reference(s) on the data source. Registered users from three different groups, namely administrator, national coordinator and curator, are responsible for database curation and/or data entry/correction online via a password-protected interface. Databaseaccess is free of charge and there are no registration requirements for data querying. FINDbase provides a simple, web-based system for population-based mutation data collection and retrieval and can serve not only as a valuable online tool for molecular genetic testing of inherited disorders but also as a non-profit model for sustainable database funding, in the form of a 'database-journal'.

The human PAH gene (GenBank: U49897.1 (cDNA), AF404777 (gDNA)) harbors alleles that either cause or are associated with hyperphenylalaninemia and phenylketonuria (http://www.pahdb.mcgill.ca). Mutation analysis has identified approximately 500 alleles of which approximately 30 produce polymorphic core haplotypes. The c.1222C>T allele (p.R408W) is the most prevalent and widely encountered PKU-causing allele. Because it occurs on multiple locus-specific polymorphic haplotypes, it is probably not identical by descent in different populations. This mutation involves a CpG dinucleotide in a so-called "hypermutable" codon suggesting that c.1222C>T could be a recurrent allele following spontaneous methylation-mediated deamination of 5 mC. This concept is widely assumed and accepted but the 5mC status of hypermutable codons has seldom been confirmed. We show that the PAH c.1222C nucleotide is indeed methylated (c.1222 mC) in somatic genomes (leukocyte and brain) of H. sapiens. Examination of a representative region in exon 12 (and also in exon 7) in the PAH gene shows that 5 mC is restricted to cytosines in CpG dinucleotides in the hypermutable codons. The methylation pattern seen in human PAH exon 12 was also observed in the corresponding codon in three nonhuman primates. The finding offers at least one explanation for the high relative frequency of the c.1222C>T (p.R408W) allele in the human population.

Phenylketonuria (PKU) is a metabolic disorder due primarily to mutations in the PAH gene that impair both phenylalanine hydroxylase activity and disposal of l-phenylalanine from the normal diet. Excess phenylalanine is toxic to cognitive development and a low-phenylalanine diet prevents mental retardation, but it is a difficult therapeutic option. Previous studies with recombinant phenylalanine ammonia-lyase, PAL, demonstrated pharmacologic and physiologic proofs of principle for PAL as an alternative therapy for PKU but its immunogenicity was problematic. From a series of formulations of linear and branched polyethylene glycols chemically conjugated to PAL, we have created a parenteral therapeutic agent for PKU treatment. All the pegylated molecules were fully characterized in vitro and the most promising formulations were then tested in vivo in the PKU mouse model. The linear 20-kDa PEG-PAL combination abolished in vivo immunogenicity after repeated challenge while retaining full catabolic activity against phenylalanine, suggesting potential as a novel PKU therapeutic.

Every physician interprets manifestations of illness. Osler, more than anyone, was the first to move medical practice beyond the level of a trade and give it an intellectual foundation and language. Osler interpreted the manifestations of disease with a vocabulary of signs and symptoms that gave

The R408W phenylketonuria mutation in Europe has arisen by recurrent mutation in the human phenylalanine hydroxylase (PAH) locus and is associated with two major PAH haplotypes. R408W-2.3 exhibits a west-to-east cline of relative frequency reaching its maximum in the Balto–Slavic region, while R408W-1.8 exhibits an east-to-west cline peaking in Connacht, the most westerly province of Ireland. Spatial autocorrelation analysis has demonstrated that the R408W-2.3 cline, like that of R408W-1.8, is consistent with a pattern likely to have been established by human dispersal. Genetic diversity within wild-type and R408W chromosomes in Europe was assessed through variable number tandem repeat (VNTR) nucleotide sequence variation and tetranucleotide short tandem repeat (STR) allelic associations. Wild-type VNTR-8 chromosomes exhibited two major cassette sequence organizations: (a1)5-b3-b2-c1 and (a1)5-b5-b2-c1. R408W-1.8 was predominantly associated with (a1)5-b5-b2-c1. Both wild-type VNTR-3 and R408W-2.3 chromosomes exhibited a single invariant cassette sequence organization, a2-b2-c1. STR allele distributions associated with the cassette variants were consistent with greater diversity in the wild-type VNTR-8 lineage and were suggestive of different levels of diversity between R408W-1.8 and R408W-2.3. The finding of greater genetic diversity within the wild-type VNTR-8 lineage compared to VNTR-3 suggests that VNTR-8 may be older within the European population. However, in the absence of a more extensive STR data-set, no such conclusions are possible for the respective R408W mutant lineages. Hum Mutat 21:387–393, 2003. r 2003 Wiley-Liss, Inc.

American Pediatric Society's 2010 John Howland Award Acceptance Lecture: Some Things Considered

We describe variation at the PAH locus in the population of Quebec. We successfully analyzed 135 of 141 chromosomes from phenylketonuria (PKU) probands (95.7% of the sample), and eight additional chromosomes from a small number of probands with non-PKU hyperphenylalaninemia (HPA). The full set of chromosomes harboured 45 different PAH mutations: i) seven polymorphisms (IVS2nt19, IVS3nt-22, IVS6nt-55, Q232Q, V245V, L385L, Y414Y); ii) four mutations causing non-PKU HPA (T92I, E390G, R408Q, D415N); iii) 34 mutations causing PKU. Only six mutations (M1V, R261Q, F299C, S349P, R408W and IVS12nt1) occurred in the whole province at relative frequencies > 5%: most are rare and probably identical by descent. By studying associations of mutations with polymorphic haplotype alleles, we found examples of mutations on different haplotypes that were identical by state, but not by descent because they were recurrent mutations (E280K and R408W); and examples of mutations identical both by state and by descent because of intragenic recombination (S67P, G218V, V245A and IVS12nt1). Ten mutations were first described in Quebec and five are still unique there; three of these 'Quebec' mutations are reported here for the first time (c.125A-->T (K42I); [c.470G-->A; c.471A--C] (R157N); c.707nt-55 (IVS6nt-55). The PAH mutations stratify by geographic region and population, their distributions validating hypotheses about European range expansion to North America during three separate phases of immigration and demographic expansion in the Quebec region over the past four centuries. The PAH homozygosity value (j) is 0.06 for the total Quebec sample (0.5-0.08 by regions), and the corresponding homoallelic fraction of mutant PAH genotypes is 24%. These findings are a documentation of genetic diversity in the Quebec population.

The rapid accumulation of mutation data has led to the creation of nearly 300 locus-specific mutation databases. These sites may contain a few dozen to almost 20,000 mutations for a given gene. Many of the mutations are uncharacterised and have no known effects on the gene product, the 'variant of uncertain significance'. Here, the statistics of mutation distribution are examined for six different gene databases: BRCA1 and BRCA2, haemoglobin-beta (HBB), HPRT1, CFTR and TP53. The percentage of all possible point mutations for a protein (the mutation space) is calculated for each gene and the question 'How much mutation data is enough?' is raised.

SummaryWe analyzed correlations between mutant genotypes at the human phenylalanine hydroxylase locus (gene symbol PAH) and the corresponding hyperphenylalaninemia (HPA) phenotypes (notably, phenylketonuria [OMIM 261600]). We used reports, both published and in the PAH Mutation Analysis Consortium Database, on 365 patients harboring 73 different PAH mutations in 161 different genotypes. HPA phenotypes were classified as phenylketonuria (PKU), variant PKU, and non-PKU HPA. By analysis both of homoallelic mutant genotypes and of “functionally hemizygous” heteroallelic genotypes, we characterized the phenotypic effect of 48 of the 73 different, largely missense mutations. Among those with consistent in vivo expression, 24 caused PKU, 3 caused variant PKU, and 10 caused non-PKU HPA. However, 11 mutations were inconsistent in their effect: 9 appeared in two different phenotype classes, and 2 (I65T and Y414C) appeared in all three classes. Seven mutations were inconsistent in phenotypic effect when in vitro (unit-protein) expression was compared with the corresponding in vivo phenotype (an emergent property). We conclude that the majority of PAH mutations confer a consistent phenotype and that this is concordant with their effects, when known, predicted from in vitro expression analysis. However, significant inconsistencies, both between in vitro and in vivo phenotypes and between different individuals with similar PAH genotypes, reveal that the HPA-phenotype is more complex than that predicted by Mendelian inheritance of alleles at the PAH locus.

A website (http://www.mcgill.ca/pahdb ) is maintained by the curators for a Consortium (88 investigators, 28 countries) and all other users; it serves a relational database for human locus-specific genetic variation in a defined DNA sequence (GenBank U49897); (100 kb on human chromosome 12q24.1, gene symbol PAH). The intragenic nucleotide variation is both rare (Q< 0.01), extensive (>320 different mutations) and phenotype modifying, causing hyperphenylalaninemia by impairing phenylalanine hydroxylase function (see OMIM 261600), as well as polymorphic and neutral, the latter providing informative locus-specific haplotypes (>1200 different mutation/haplotype associations). The PAH database contains both offline core components (mutations, population associations and data source information) and several accessory online components: (i) relative frequencies of mutations by populations/regions (expanding file); (ii) data on genotype- phenotype correlations both in vitro and in vivo (new file); (iii) polymorphic haplotype structures (new file); (iv) intron sequence data (new file for design of primers); (v) description of mouse homologues (new file for mutations and phenotypes); (vi) the predicted PAH gene mutability profile (improved graphic); (vii) a clinical field for patient use (new interface with database). The website home page has been revised and a counter is recording >15 visits per day. Linkages to other mutation databases and an alliance of mutation database curators (new) are expanding. The primary 'electronic publication' reports now vastly exceed print reports. PAHdb serves as a prototype for obtaining, storing and distributing records of human genetic variation.

Exactly 50 years after Bickel et al. [1] initiated dietary treatment by phenylalanine restriction clinicians and researchers worldwide are still searching for an alternative approach to the treatment of phenylketonuria (PKU). Today we know that maintaining the restricted diet is beneficial if not essential to prevent brain damage, but there are still disagreements as to how long this diet should be continued. A number of nutritional products with improved quality are available in most countries, but many adolescents and young adults generally do not comply with the recommendations for monitoring and control of phenylalanine concentrations [2], and two thirds of pregnant women in the United States did not follow the diet before becoming pregnant [3]. There is a need for an alternative approach to the treatment of PKU. As for many other inherited metabolic disorders, somatic gene therapy for PKU offers hope for the future. The paper by Ding et al. published in this issue of Molecular Genetics and Metabolism summarizes the present knowledge of different strategies in PKU gene therapy. With the help of the recently available animal models for PKU and hyperphenylalaninemia [4] it became possible to test different gene transfer vehicles, however, the reality is quite different from what had been expected. Both, liver phenylalanine hydroxylase (PAH) gene transfer in vitro and in vivo, as well as autologous non-hepatic gene targeting attempts failed due to poor efficiency of gene delivery and/or lack of essential cofactor tetrahydrobiopterin (BH4). The final statement by Ding et al. is rather sobering: ‘‘. . . despite different non-viral and viral gene transfer approaches that have been examined, none of them seems to hold

We report findings in phase II of a pilot study of cystic fibrosis (CF) carrier screening/testing by mutation analysis. Phase I has been reported elsewhere. Eligible participants in phase II (n = 815) were students (15 to 17 years of age) in public high schools. An educational component (exchange of information and discussion about common genetic disorders including CF) preceded, by one week or more, voluntary participation in the screening component which required a blood sample. The uptake rate for screening was 42%. Nine carriers (2pq = 0.0260) were identified, all with the delta F508 mutation; students were also tested for G551D, G542X, W1282X, and -549-mutations, but no carriers of these alleles were found. Carriers had positive views of the education and testing experiences. Persons identified as 'non-carriers' were also surveyed (n = 135, response rate 41%). As in phase I, the majority (83%) again understood that a negative DNA test had not excluded them from possible carrier status. Students who participated in the informational component but were not screened served here as controls in the follow up survey (n = 208, response rate 53%). Their views were similar to those of the screened non-carriers, and similar also to those held by students, adults, pregnant women, couples, and CF relatives in other communities.

A polymorphic variant in the human HEXA gene is described. This gene encodes the alpha-subunit of hexosaminidase A, the enzyme which is deficient in Tay-Sachs disease (TSD). In individuals carrying the polymorphism there is a T-->C transition at position -6 in intron 13. The substitution creates a site for the restriction endonuclease Pst1. This variant has an unusual ethnogeographic distribution. It occurs on 1.4% of non-TSD carrier chromosomes in Ashkenazi Jews. All individuals ascertained carrying the Pst+ allele have ancestry in Lithuania, Belarus and Ukraine. By contrast, no individuals carrying the Pst+ allele have been detected among non-Jewish Lithuanians, Jews of Sephardic origin or in several other ethnic groups. Two unrelated non-Jewish families have been identified in which the Pst+ variant occurs. In both cases the variant occurs on a chromosome carrying a novel TSD mutation (G772C) association with the B1 phenotype. The Pst+ G772C chromosomes are of Scots-Irish descent.

SummarySeveral genes expressed in kidney and other tissues determine phosphate homeostasis in extracellular fluid. The major form of inherited hypophosphatemia in humans involves an X-linked locus (HPDR, Xp22.31-p21.3). It has two murine homologues (HypandGy) which map to closely-linked but separate loci (crossover value 0·4%–0·8%). Both murine mutations impair Na+-phosphate cotransport in renal brush border membrane; an associated renal disorder of 1, 25-dihydroxyvitamin D3(1, 25(OH)2D) metabolism has been characterized inHypmice. Whereas experiments with culturedHyprenal epithelium indicate that the gene is expressed in kidney, studies showing the development of the mutant renal phenotype in normal mice parabiosed toHypmice implicate a circulating factor; these findings can be reconciled if the humoral factor is of renal origin. The gene dose effect of HPDR,HypandGyon serum phosphorus values is consistently deviant and heterozygotes resemble affected hemizygotes. The deviant effect is also seen on renal phosphate transport; all mutant females(Hyp/HypandHyp/+) have similar phenotypes. On the other hand, there is a normal gene dose effect of HPDR in mineralized tissue; tooth PRATIO (pulp area/tooth area) values for heterozygotes are distributed between those for affected males and normals. The tooth data imply that the X chromosome locus is expressed in both renal and non-renal cells. The polypeptide product of the X chromosome gene(s) is still unknown.

Ed John Walton, Jeremiah A Barondess, Stephen Lock Oxford University Press, £40, pp 1038 ISBN 0 19 262355 9 The first edition of the Oxford Medical Companion was published in two volumes to positive reviews in, among others, the Lancet, New England Journal of Medicine, Nature, and Times Literary Supplement. The BMJ did not review it. The first edition provided definitions and facts (over 5000 entries); was a dictionary of brief international medical biographies (none of the 1000-odd subjects …

Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n = 6) and OI cell strains (n = 10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300 X magnification). Our results show that: (i) OI fibroblasts take longer (11-27 days, mean 20 days) than control cells (10-19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87 X 10(6) cells/P60, range 0.3-1.6 X 10(6] at stationary phase relative to control cells (2.2 X 10(6) cells/P60, range 1.7-2.6 X 10(6); F4,77 = 56.1, p less than 0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width : length ratio) was also abnormal in OI cells. (F4,730 = 37.6, p less than 0.01), types I and II OI cells have significantly increased ratios (p less than 0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.

The homozygous PRO/Re mouse has less than 1 percent of the very high proline oxidase activity that characterizes normal kidney cortex. In PRO/Re mouse the endogenous proline concentration is eight times normal in plasma and four times normal in kidney cortex cell, but 50 times normal in urine. The integrity of the membrane transport systems for proline uptake at the antiluminal surface of absorbing epithelium is retained in PRO/Re kidney, as determined by the slice method. Clearance studies in vivo under steady-state conditions indicate that the integrity of the luminal uptake system shared by glycine and proline, and serving proline absorption, is also intact. The exaggerated renal clearance of proline in PRO/Re mice (50 times normal) is explained when its raised intracellular concentration, caused by impaired proline oxidation, is considered. Backflux into urine flowing down the nephron will occur under these conditions, thus impairing net reclamation of proline in PRO/Re kidney. The findings reveal that membrane transport and intracellular metabolism of a substrate are, indeed, independent functions, but that metabolism of a substance can influence its transcellular transport.

GAD activity and GABA, the product of GAD action on L-glutamate, are both prominent in mature human renal cortex. GAD activity is low in fetal kidney but rises several fold preterm to establish the characteristic post-term specific activity. The ontogeny of the initial step in the GABA pathway parallels the need for kidney to accommodate acid-base regulation after birth. PLP coenzyme is required for GAD holoenzyme integrity. Fetal renal GAD was frequently undersaturated with PLP in our series of observations, raising the suggestion that maternal vitamin B6 nutrition is not always adequate.

We measured glycine release from ([2-3H]glycine)-labelled GSH and glucose formation from maltose incubated with rat kidney whole cortex homogenate, thin cortex slices or collagenase-treated tubule fragments. Liberation of glycine was inhibited (74-83%) by serine borate (20 mM), indicating a gamma-glutamyltransferase-dependent hydrolysis of GSH. In whole cortex homogenate, the GSH cleavage activity was 17.4 +/- 0.6 nmol GSH degraded/mg protein per min (mean +/- S.D.); cleavage activity by intact slices was 3.5 +/- 0.7 (P less than 0.001 relative to whole cortex homogenate) and in tubule fragments 9.4 +/- 0.8 (P less than 0.001). Homogenizing the tissue preparation increased cleavage rate in slices about 4-fold (12.4 +/- 2.9; P less than 0.005 relative to intact slice) but did not change the rate in tubule fragments (9.8 +/- 0.5). Maltose cleavage activity in whole cortex homogenate was 512 +/- 22 nmol glucose formed/mg protein per min, in slices 162 +/- 12, and in tubules 884 +/- 48. These findings imply that substrate in the incubation medium has a limited access to the luminal membrane of cortex slices but not of tubule fragments. They further imply that basolateral membrane is preferentially exposed in the slice preparation.

Ce texte presente les principaux resultats de travaux relevant de l'histoire sociale de la population, de la demographie historique et de la genetique des populations, realises depuis quelques annees par le Centre interuniversitaire de recherches sur les populations (SOREP). Ces travaux portent sur le Nord-Est de la province de Quebec (Canada), et plus particulierement sur la region du Saguenay. Le but de nos recherches est d'eclairer la dynamique de populations regionales, du 18e au 20e siecle, et d'en tirer des indications sur la structure et l'evolution des bassins genetiques. On s'interesse ici a quelques genes deleteres qui sont a l'origine de maladies graves dans ces regions du Quebec. L'article montre comment une demarche axee sur l'etude des faits de population peut soutenir des objectifs d'epidemiologie genetique et de prevention des maladies hereditaires en general. Il aborde aussi les problemes ethiques et juridiques souleves par une telle demarche, essentiellement appuyee sur l'exploitation d'un fichier de population.

Renal handling of phosphate in vivo and in vitro by the X-linked hypophosphatemic male mouse: Evidence for a defect in the brush border membrane. We have examined renal phosphate transport in vitro and reabsorption in vivo in hypophosphatemic, male mutant mice (Hyp/Y) carrying a gene believed to be homologous with that responsible for X-linked hypophosphatemia in man. Nor-mophosphatemic male littermates (+/Y) were used as controls. Fractional excretion of phosphate in Hyp/Y mice is increased (0.351 ± 0.073 [mean ± SD, N = 7] vs. 0.210 ± 0.0885 [N = 15] in +/Y mice [P < 0.01]) at serum phosphorus concentrations of 34.6 ± 7.6 and 56.8 ± 9.16 mg/liter, respectively (P < 0.001). Phosphate clearance exceeded creatinine clearance in Hyp/Y mice when their drinking water contained added phosphorus (2 g/liter). Urinary cyclic 3′5′-adenosine monophosphate concentrations increased in Hyp/Y mice when compared with +/Y littermates; but this finding, which is different from the human phenotype, was not accompanied by evidence of hyperparathyroidism in the Hyp/Y mice. Serum parathyroid hormone levels rose during phosphate-loading in Hyp/Y mice, as it does in the human patient. The phosphorus concentration in kidney cortex was similar in mutant and normal mice (Hyp/Y, 46.6 ±1.1 mmoles/g of protein; +/Y, 46.6 ± 1.0 mmoles/g of protein). Uptake of 32P-phosphate, at 0.1 and 1.0 mM in the medium, by slices of cortex or medulla, and labeling of intracellular inorganic and organic pools of slices observed first-order kinetics and was not significantly different in the two strains of mice. Modulation of calcium in the diet influenced these observations in both strains but did not disclose differences between them. Purified brush border membrane vesicles prepared from mouse kidney cortex homoge-nates accumulated phosphate by a sodium-dependent, arsenate-inhibited transport system. Total uptake of phosphate (sodium-dependent plus sodium-independent) by +/Y vesicles was 488 ± 23 pmoles/mg of protein per 60 sec. (mean ± SD) and 302 ± 10 pmoles/mg of protein per 60 sec by Hyp/Y vesicles (P < 0.001); sodium-independent uptake was similar in the two types of vesicles (79 and 78 pmoles/mg of protein per 60 sec, respectively). Uptake of D-glucose and relative purity of the brush border membrane fraction were not different in the two strains. The combined in vivo and in vitro data indicate that impaired net transepithelial transport of phosphate in the X-linked phenotype is the result of an intrinsic defect in net flux of phosphate at the brush border membrane.

Seven infants (two French Canadian, four Ashkenazi Jewish, and one Greek) with massive selective hyperiminoglycinuria (proline, hydroxyproline, and glycine) were detected by urine screening in the second week of life. Follow-up investigations and family studies revealed that each subject had a benign condition, familial renal iminoglycinuria, an autosomal recessive condition. The family studies (Table 1 and Fig. 1) indicate the presence of at least two different mutant alleles segregating in this small group of probands. Hmozygotes of two forms and one genetic compound were identified. Quantitative studies revealed normal concentrations of proline and glycine in plasma (Fig. 2), normal maturation of creatinine clearance (as an index of glomerular filtration rate) (Fig. 3), and elevated renal clearance of proline and glycine (Table 2). Fractional excretion (CAA/CCR) of both proline and glycine in the probands was far in excess of that expected for the normal postnatal infant; FFPro and FEGly approached 100% of the filtered load on occasion (Fig. 4). A schedule of maturing tubular reabsorptive activity was apparent in the proband group. Proline reabsorption matured earlier than glycine reabsorption in the homozygotes (and the genetic compound) as it does in the normal infants (Fig. 5). Our findings suggest that three gene products serve net tubular reabsorption of imino acids and glycine in human kidney. One, affected by mutation in our patients, is responsible for a shared transport activity; a second with preference for proline, and not affected by the mutation, has an "early" schedule of postnatal maturation; and a third with preference for glycine, also not affected by the mutation, has a "late" schedule of maturation.

Summary: Parathyroid extract (PTE), dibutyrylcyclic AMP (dbcAMP), adenosine cyclic 3':5'-monophosphate (cAMP), calcitonin (CT), and calcium chloride were infused separately into anesthetized, sham-operated, or TPTX vitamin D-fed adult rats to examine the effect of these calciotropic agents on fractional excretion (FE) of α-aminoisobutyric acid (AIB), and phosphate anion (Pi). AIB is a nonmetabolizable amino acid. Inulin clearance, FEAIB, and FEPi were stable in the intact (n = 10) and TPTX rat (n = 10). TPTX decreased FEaib, and FEPi significantly (P < 0.001 for both). PTE and dbcAMP both increased FEaib in the intact rat (P < 0.001); failure to obtain this response in the TPTX animal was a key finding. PTE and dbcAMP increased FEPi (P < 0.001) in both the intact and TPTX animal. CT was the only agent (versus PTE, dbcAMP, adenosine cyclic 3‘:5’-monophosphate, and CaCl2) to increase FEAIB (P < 0.001) in the TPTX rat; furthermore, it was the only agent that did not increase FEPi in the TPTX rat although it had hypocalcemic and hypophosphatemic effects. Changes in inulin clearance or plasma concentration of AIB, following infusion of calciotropic agents, do not explain the unique responses in FEAIB in the TPTX rat. Our findings suggest that hyperaminoaciduria induced by parathyroid hormone and cyclic nucleotide in the intact animal may be mediated by CT. Hyperphosphaturia is not a necessary response to small-dose (25 milliunits/kg.hr) infusions of CT. Speculation: Intracellular calcium in renal epithelium may be the final determinant of hyperaminoaciduria in hyperparathyroidism.

Genetics: Voyage of Discovery for Everyman: (Presidential Address to The Society for Pediatric Research, April 29,1976, St. Louis, Missouri)