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J Inherit Metab Dis (2008) 31:580–598 DOI 10.1007/s10545-008-0984-9 REVIEW Garrod_s Croonian Lectures (1908) and the charter FInborn Errors of Metabolism_: Albinism, alkaptonuria, cystinuria, and pentosuria at age 100 in 2008 Charles R. Scriver Received: 26 June 2008 / Submitted in revised form: 15 July 2008 / Accepted: 16 July 2008 / Published online: 12 October 2008 # SSIEM and Springer 2008 Summary Garrod presented his concept of Fthe inborn error of metabolism_ in the 1908 Croonian Lectures to the Royal College of Physicians (London); he used albinism, alkaptonuria, cystinuria and pentosuria to illustrate. His lectures are perceived today as landmarks in the history of biochemistry, genetics and medicine. Garrod gave evidence for the dynamic nature of metabolism by showing involvement of normal metabolites in normal pathways made variant by Mendelian inheritance. His concepts and evidence were salient primarily among biochemists, controversial among geneticists because biometricians were dominant over Mendelists, and least salient among physicians who were not attracted to rare hereditary Ftraits_. In 2008, at the centennial of Garrod_s Croonian Lectures, each charter inborn error of metabolism has acquired its own genomic locus, a cloned gene, a repertoire of annotated phenotype-modifying alleles, a Presented at the 2008 SSIEM Annual Symposium in Lisbon, Portugal, 2–5 September 2008. Communicating editor: Verena Peters C. R. Scriver Departments of Human Genetics, Pediatrics, Biochemistry, and Biology, McGill University, Montreal, Canada C. R. Scriver Department of Medical Genetics, McGill University Health Centre, Montreal, Canada C. R. Scriver (*) Montreal Children_s Hospital Research Institute, 2300 Tupper Street, Montreal, Quebec H3H 1P3, Canada e-mail: [email protected] gene product with known structure and function, and altered function in the Mendelian variant. Introduction The fox knows many things, but the hedgehog knows one big thing Archilochus (680–645 BC) This fragment from antiquity will serve to introduce Garrod. It distinguishes a person with a unifying view of reality (the Fhedgehog_ in Garrod), yet alert to particular features and curious about their manifestations and origins (the Ffox_ in Garrod). Garrod_s unifying vision saw human chemical individuality in a setting of dynamic metabolism and heredity; he found particularity in the inborn errors of metabolism. Garrod_s Croonian Lectures are the hybrid (Fhedgefox_) product of his expertise. Garrod disguised as a hedgehog The Croonian Lectures, an honoured academic tradition in British science (Bearn 1993, pp. 76–80), were established under the terms of an endowment by William Croone, a founding member of the Royal Society of London and also a Fellow of the Royal College of Physicians. Croone (1633–1684) had planned to endow two lectureships: one to the Royal Society, the other to the Royal College of Physicians. However, at his death Croone had not yet provided the financial arrangements for the endowments. These were completed by his widow, in her will, in the year 1701; the invested income for the endowed lectures J Inherit Metab Dis (2008) 31:580–598 would come from the clear rent from the King_s Head Tavern in London! The first Croonian Lecture to the Royal Society was given in 1738; the first to the Royal College of Physicians in 1749. The Croonian Lectureship to the Royal College of Physicians has since acquired great distinction in the field of medicine. However, the important implications concerning genetics to be found in Garrod_s own Lectures were not further developed until the 1990s, with the exception of the Lectures given in 1971 (by John F. Brock: Nature, Nurture and Disease); in 1973 by Sir John Dacie (The Hereditary Haemolytic Anaemias); and in 1984 by David Weatherall when he chose human genetics as the overriding theme for his Croonian Lecture. Much of Garrod_s enduring reputation is embedded in his Croonian Lectures, which we now recognize as landmarks in medicine, biochemistry and human genetics (Bearn 1993, p. 78). Garrod_s Croonian Lectures were delivered to an audience at the Royal College of Physicians on four days in 1908 (18, 23, 25, and 30 June), and then printed in Lancet, as given, on 4, 11, 18, and 25 July 1908 (Garrod 1908); they were also reprinted, as a book, in 1909, under the historic title Inborn Errors of Metabolism (Garrod 1909b) (Fig. 1). The Lectures and the book are noteworthy for their insights: on the way a physician-scientist could approach a novel problem; for their significant observations that are case- (patient-) derived; for the evidence of a congenital onset in the clinical manifestations in these errors of metabolism; for the evidence for an hereditary basis (inborn and familial) and of recessive Mendelian inheritance (deduced from parental consanguinity); and for the evidence of dynamic metabolism revealed by a block in an otherwise normal pathway. While these features had been foreseen by Garrod in his paper of 1902 on The Incidence of Alkaptonuria (Garrod 1902), which he called Fa study in chemical individuality_, the Croonian Lectures of 1908 allowed him to assemble new evidence from other disorders that could be explained by a block at some point in the normal course of metabolism, due to a congenital deficiency of a specific enzyme. Of particular relevance, the inborn errors of metabolism led Garrod to draw novel Fphysiological conclusions from pathological conditions, rather than taking the more frequently adopted reverse approach_ (Bearn 1993, p. 80); an approach that would prefigure one taken half a century later by Beadle and Tatum (Olby 1974, pp. 143–145). Hindsight recognizes Garrod_s genius, but in his own lifetime, the recognition attributable to the concept of the inborn error of metabolism was rather subdued in 581 Fig. 1 Title page of The Croonian Lectures on the topic Inborn Errors of Metabolism, delivered to the Royal College of Physicians (London) in 1908. (Reproduced from the reprint of the Lectures initiated by Harris (1963), in the new publication series called Oxford Monographs on Medical Genetics) the communities of medicine, biochemistry and genetics. Garrod_s contribution to medicine did attract recognition and honours were bestowed accordingly. Yet his remarkable insight into the biochemical abnormalities in his particular patients—based as they were on his underlying view of metabolism as a continuous stepwise movement through intermediary products, each having only transient existence, which, when one step fails as an inborn event, affects the flow through the pathway or the network and causes an intermediate to accumulate and thus to identify a functional component in the process—did not immediately resonate as a new paradigm that would eventually attract a crowd of adherents not only in medicine, but also in biochemistry, and in genetics. The salience of Garrod_s ideas was greater among the biochemists than among the geneticists of the day; 582 and least among his medical colleagues, with the exception of the paediatricians (Olby 1974, pp. 131– 133). Garrod was proposing that the four disorders described in his Croonian Lectures were indeed congenital (present at birth) and also inborn (transmitted through the gametes), displaying themselves as an all-or-nothing phenomenon with the discontinuous distribution of a Mendelian trait, as perceived by Bateson, the biologist and geneticist, who was in communication with Garrod. We can now see how these inborn errors would support the conceptual link between Mendel_s factors (genes), and enzymes, when the latter became a major field of inquiry in biochemistry. Meanwhile, in medicine, the apparent rarity of the inborn errors of metabolism made them irrelevant to the medical profession and efforts to show their inheritance and congenital nature were of little help to the practice of medicine in Garrod_s lifetime. Moreover, the opinion that these disorders were Fnot of much moment_ because they were quite benign in the younger patient and thus were Fsports_, undermined what medical interest they might have had at the time (see p. 4 in the first Croonian Lecture). Accordingly, not much happened in the adoption of FGarrodian medicine_ in the first half-century following the Croonian Lectures. Garrod_s influence among geneticists was undermined by the limited awareness, at the time, of Mendelism at work in Homo sapiens, by ignorance concerning the physical nature of Mendel_s Ffactors_ (genes), and by the dominant influence of the biometricians of the day (until the Hardy–Weinberg law of inheritance was proposed). Thus, it was primarily in the field of biochemistry that Garrod was able to contribute to a new tradition after 1908, one that would override the established view of static biochemistry and replace it with the concept of dynamic metabolic pathways. On the other hand, biochemists, who were at least prepared to receive Garrod_s insights on the nature of metabolism and its pathways, were rather less captivated by the hereditary aspects of the inborn errors. One might also say that Garrod himself inhabited the older mind-set of Fphysiological chemistry_. But history shows that here was where the links between genetics and biochemistry in the human organism would eventually be forged (Beadle 1964). Until then, enthusiasm among the biochemists would focus mainly on the new enzymology (Olby 1974, p. 133). Beadle and Tatum (Beadle and Tatum 1941) are credited with the announcement of the one gene–one enzyme paradigm. But they were cautious in their initial emancipation of biochemical genetics and they J Inherit Metab Dis (2008) 31:580–598 spoke, not of Fone gene, one enzyme_, but only of gene and enzyme specificities that were of a similar order. Yet their work in Neurospora did lead them to perceive the relationships between gene, enzyme, and metabolism; and it then led them to recognize that Garrod had indeed foreshadowed them. In this long round about way, first in Drosophila, and then in Neurospora, we had rediscovered what Garrod had seen so clearly so many years before. By now we knew of his work and were aware that we had added little if anything new in principle. We were working with a more favorable organism, and were able to produce, almost at will, inborn errors of metabolism for almost any chemical reaction whose product we could supply through the medium, thus we were able to demonstrate that what Garrod had shown for a few genes, and a few chemical reactions in man, was true for many genes and many reactions in Neurospora. Beadle (1958): cited in (Olby 1974, p. 144) Garrod published a second edition of Inborn Errors of Metabolism in 1923 (Garrod 1923) (Fig. 2), to which he added two new inborn errors (congenital porphyrinuria and congenital steatorrhoea), in the hope that others would begin to recognize other metabolic conditions best explained by heredity. In 1935, he would be told about phenylketonuria (Bearn 1993, p. 145) but his hope to see or hear much more about the existence of other human Mendelian disorders was never fulfilled. Instead, Garrod would address the interesting concept of Fdiathesis_ (formulated by him as inborn susceptibility) and well illustrated by complex familial disorders that did not have an overt pattern of Mendelian inheritance. His ideas were again remarkably prescient; they were published in book form in 1931 (Garrod 1931b) under the title Inborn Factors in Disease, and republished with annotations in 1989 (Garrod 1931a) (Fig. 3). Garrod_s Croonian Lectures have, of course, never been neglected. They were republished by Harry (Harris 1963) in a book that included the famous 1902 paper on alkaptonuria and a long essay by Harris himself entitled The FInborn Errors_ Today. This book became the first in a new series of Oxford Monographs and Medical Genetics. Harris also created a new textbook, The Principles of Human Biochemical Genetics (Harris 1970), which grew through three editions each written by Harris. Eugene Knox (Knox 1958d) published a set of four essays in 1958, the 50th anniversary of Garrod_s Croonian Lectures, in which he reviewed growth in our knowledge related to the J Inherit Metab Dis (2008) 31:580–598 583 tests on blood and urine samples to discover chemical evidence associated with the clinical condition. In this way, he studied disease and found particular forms of evidence from which he assembled the larger vision. During the past 100 years, the evidence garnered from the four inborn errors of metabolism, described in his Croonian Lectures, has grown and been refined. That legacy of the Croonian Lectures is described here, in this essay, which should not be read as a comprehensive review of all the new information about the four disorders. Moreover the unifying view, revealed through the four inborn errors of metabolism (albinism, alkaptonuria, cystinuria, and pentosuria), as described by Garrod (and adumbrated by Knox (1958d)) has not been undermined by new evidence— only enhanced. One might say that the hedgehog has become wiser and better-informed thanks to the fox who has been providing the data. (Garrod presented Fig. 2 Title page of the second edition (1923) of Garrod_s Inborn Errors of Metabolism. This edition was dedicated to Garrod_s mentor and colleague, Frederick Gowland Hopkins original inborn errors. He could report only modest advances in our understanding of the four inborn errors: albinism was only beginning to be a full-time occupation for its diverse family of students; the enzyme deficiency in alkaptonuria would be first reported only in 1958; the physiological basis of cystinuria had been revealed only in the 1950s; pentosuria was more or less as it was when Garrod was alive. In 1960, the first edition of The Metabolic Basis of Inherited Disease appeared; it became a knowledgebase that grew through eight print editions (Fig. 4) and is now available as a continuously updated digital presence on the web (www.ommbid.com). Garrod disguised as a fox Garrod, the physician and pediatrician, examined patients himself, took family histories, and did simple Fig. 3 A photograph of Garrod at the time he was writing Inborn Factors in Disease (1931); this second book describes how heredity is relevant in the study of medicine and disease. Shown here is the dust jacket for the annotated reprinted version (1989) 584 J Inherit Metab Dis (2008) 31:580–598 Fig. 4 Garrod_s Inborn Errors of Metabolism has had many successors. Shown here is The Metabolic Basis of Inherited Disease (MBID), editions 1 to 6, 1961–1989, renamed as The Metabolic and Molecular Bases of Inherited Disease (MMBID) for the 7th and 8th editions (1995–2001). Thereafter, print editions have been replaced by an online format (OMMBID 2003) with continuous updating and editing. It reflects a revolution in publishing that would have served Garrod well the four inborn errors in alphabetical sequence. I have altered the sequence for reasons that will be apparent.) Alkaptonuria Garrod had an abiding interest in alkaptonuria. In 1899 (Garrod 1899) he described its relevant chemistry and familial occurrence; in 1901 (Garrod 1901) its congenital features and prevalent consanguinity in affected families; in 1902 (Garrod 1902) his evidence for human chemical individuality, for Mendelian recessive inheritance in a rare condition such as alkaptonuria and the evidence for involvement of a normal metabolic pathway as revealed through a pathological condition affecting it. Alkaptonuria emerged as the prototype for the inborn error of metabolism. The classic paper of 1902 has been republished, not surprisingly, at least three times (Garrod 1996, 2002; Harris 1963). There are several milestones in the story of alkaptonuria (Knox 1958d): in clinical chemistry, in pathology, in organic chemistry, and in the fields of internal medicine, biochemistry and statistical genetics; and it is a landmark in human genetics (Hogben et al 1932). While Garrod did use alkaptonuria to advance his concept of chemical individuality, he tended to view it as a rather benign medical condition: as a Fsport_ or a freak of nature. It is a view under revision (Phornphutkul et al 2002). The long view of the condition spans five centuries (O_Brien et al 1963), and begins even earlier with a backward glance at Harwa, the affected Egyptian mummy (Stenn et al 1977). Throughout its long history, there is ample evidence that the clinical phenotype of alkaptonuria is not all that benign. From Garrod_s fox-like vantage point, the salient characteristics of any metabolic sport (such as alkaptonuria) would include outlier chemical individuality, evidence of sibling involvement, and the likelihood that patients were the offspring of firstcousin parents. The hedgehog view of alkaptonuria then comes into play in the 1908 Croonian Lecture, where the old black-box view of metabolism gives way to metabolism as it might occur in connected compartments, and being the work of special enzymes set apart for each purpose. These new insights on biochemistry by Garrod were premature in the larger context of knowledge at the time and received little notice! Alkaptonuria revealed to Garrod that homogentisic acid is a normal metabolite in a normal pathway with normal enzymes at each of its steps. The now widelyheld view that metabolic pathways are dynamic and experience fluxes of metabolites undergoing change by interacting with enzymes (Kacser and Burns 1973; J Inherit Metab Dis (2008) 31:580–598 Kacser and Porteous 1987) is a major legacy of Garrod_s involvement with the chemistry of homogentisic acid in alkaptonuria. His awareness of autosomal recessive inheritance underlying the appearance of alkaptonuria in an individual is another manifestation of Garrod_s insight, after he had seized upon the rediscovery of Mendelism (Bateson and Saunders 1901). The Mendelian theme in alkaptonuria, still stated somewhat hesitantly in the second edition of Inborn Errors of Metabolism (Garrod 1923), gains real momentum after Garrod_s death when the Mendelian pattern of alkaptonuria expression in multiple pedigrees is made unambiguously apparent (Hogben et al 1932). Progress in alkaptonuria after 1908 The journey of discovery continued long after the Croonian Lectures. First there was evidence for the specific enzymatic deficiency in the tyrosine oxidation pathway; mapping and cloning of the corresponding gene became feasible; then there was further analysis of the natural history of alkaptonuria of the untreated patient; finally the prospect and relevance of a new therapy for alkaptonuria appeared (Kayser et al 2008). The core information about phenotype and genotype in alkaptonuria is in the public domain on the 585 internet and catalogued in Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/sites/ entrez?db=omim) under #203500 (alkaptonuria) and *607474 (homogentisate 1,2-dioxygenase; HGD); the corresponding enzyme classification is EC 1.13.11.5; the gene symbol is HGD (alternative symbols AKU or HGO); the cDNA nucleotide sequence is listed in GenBank under accession numbers U63008, AF000573 (HGO) and AF045167 (AKU). The pathway for phenylalanine/tyrosine oxidation has not changed significantly since it was described by Neubauer in 1909 (see references 7 and 8 in La Du 2001). Features of the normal HGD enzyme were first described in 1955 (Knox and Edwards 1955); the crystalline structure of HGD (Fig. 5) has since become known (Titus et al 2000). The enzyme degrades homogentisic acid by oxidative cleavage of the aromatic ring to yield maleylacetoacetic acid. The enzyme contains sulfhydryl groups and uses ferrous iron as its only known cofactor; ascorbic acid maintains the ferrous state of the iron moiety and there is no direct requirement for ascorbic acid in homogentisic acid oxidation (and ascorbic acid therapy will not play a significant role in the treatment of alkaptonuria for this reason). The enzyme deficiency in alkaptonuria was initially demonstrated in liver biopsy material from patients Fig. 5 Quaternary structure at 1.9 Å resolution of crystallized homogentisate dioxygenase (HGO) apoenzyme. (a) Ribbon diagram of the HGO protomer, which associates as a hexamer arranged as a dimer of trimers. (b) The HGO trimer viewed along a 3-fold axis. Most alkaptonuria causing missense alleles alter residues involved in contacts between subunits. Reprinted by permission from Macmillan Publishers Ltd: Titus GP et al., Nature Structural Biology 7: 542–546, copyright 2000; this source provides further images and discussion.) 586 (La Du et al 1958). Whereas homogentisate 1,2dioxygenase activity was missing in alkaptonuria liver, activities of all other enzymes in the tyrosine oxidation pathway were intact; the loss of HGD activity was not explained by the aberrant effects of an inhibitor or loss of any known cofactor. HGD activity was shown to be present in normal kidney but absent in kidney of the alkaptonuria patient (Zannoni et al 1962). The deficiency of HGD enzyme and its effect on the tyrosine metabolic pathway in alkaptonuria were also demonstrated in vivo using [14C]carboxy-labelled homogentisic acid (Lustberg et al 1969). Alkaptonuric patients excrete large amounts of homogentisic acid in urine yet they accumulate relatively less of the organic acid in prerenal pools such as plasma (Haffner 2006) because the renal tubule vigorously secretes the metabolite (Neuberger et al 1947). The renal secretory component appears to act as a physiological modifier of the metabolic phenotype and it may help to explain why onset of the clinical phenotype (ochronosis and arthropathy) is delayed, sometimes for decades, in the alkaptonuria patient, who is otherwise born with abnormal HGD function and profoundly altered homogentisate metabolism. Does the individuality seen in the onset and severity of clinical phenotype reflect a balance between various degrees of HGD enzyme deficiency (allelic heterogeneity) and efficiency of renal secretion of homogentisic acid? The genomic locus for alkaptonuria (AKU) harbours a single copy of the gene (HGD). The locus was mapped to chromosome 3q21-q23 by various means including homozygosity mapping (Pollak et al 1993), multipoint linkage analysis (Janocha et al 1994), and comparative mapping in mouse and human genomes (Montagutelli et al 1994). The candidate gene for homogentisate 1,2-dioxygenase was isolated and cloned in 1996 (Fernandez-Canon et al 1996) by an unusual approach involving probes derived from the corresponding HGD gene in the mould Aspergillus nidulans. The human gene was cloned by a second group (Gehrig et al 1997). The corresponding mouse gene was also cloned (Schmidt et al 1997). The human HGD gene spans õ60 kb of DNA and has 14 exons; it encodes a protein (49 973 Da; 445 amino acids) to form a functional hexamer (a dimer of trimers) with the required ferrous iron cofactor (Fig. 5). Examination of nucleotide sequences in various organisms shows that the HGD gene has been highly conserved during evolution. The HGD gene harbours alkaptonuria-causing mutations (Granadino et al 1997) which show extensive allelic heterogeneity (Beltran-Valero de Bernabe J Inherit Metab Dis (2008) 31:580–598 et al 1998, 1999; Kayser et al 2008). The mutant alleles are not randomly distributed in the gene and they cluster in a recurring CCC/GGG motif (BeltranValero de Bernabe et al 1999; Goicoechea De Jorge 2002); the motif is a mutational hotspot where a third of all alkaptonuria-causing mutations occur in the human gene. More than 60 different alleles associated with alkaptonuria are now known (Kayser et al 2008). Patients affected with alkaptonuria (an orphan disease) are to be found across the spectrum of human populations. The estimated prevalence of alkaptonuria in the human population is 3–5 per million (Hogben et al 1932; Knox 1958d) but that estimate may require revision. Alkaptonuria shows geographical and ethnic clustering, with conventional population-genetics explanations, in the Trencin district of Slovakia (Srsen et al 1978) and in the Dominican Republic (Goicoechea De Jorge 2002; Milch 1960). Garrod_s belief in the relatively benign nature of alkaptonuria, and of inborn errors of metabolism as a class, was supported by Osler, who saw the condition to be Fnot of much moment_ (Osler 1904). Garrod and Osler were both wrong about the benign nature of the clinical phenotype in alkaptonuria. Both rose, nonetheless, to be the Regius Professor of Medicine in the University of Oxford, Garrod following Osler to the appointment in 1920; Garrod retired as Regius Professor in 1927 and took up the writing of his now classic provocative essay on Inborn Factors in Disease (Garrod 1931b). The decidedly unbenign nature of alkaptonuria in the mid-adult-age and older patient is described, for example for Harwa, the Egyptian mummy (Stenn et al 1977), in Knox_ essay on alkaptonuria (Knox 1958d), in a classic review of the world literature on the disorder up to 1963 (O_Brien et al 1963), and again in an examination of the natural history of alkaptonuria in 58 contemporary (20th century) patients, age range 4–80 years (Phornphutkul et al 2002). Life-table analyses in the latter study found that joint replacement took place at a mean age of 55 years, renal stones developed at 64 years, cardiac valve involvement at 54 years, and coronary artery calcification at 59 years. Severity of disease manifestations was greater in men than in women and it accelerated after the age of 30. The late age-dependency of the clinical features of alkaptonuria, and its rarity, may explain why Garrod and Osler each considered it to be Fnot of much moment_. The causes and pathogenesis of the clinical phenotype, with destruction of connective tissues, are still unclear (Kayser et al 2008). In alkaptonuria, homogentisic acid slowly oxidizes to form benzoquinoneacetic J Inherit Metab Dis (2008) 31:580–598 acid and then to form melanin-like polymers; the latter are associated with the ochronotic pigmentation and ochronotic arthropathy. Naturally occurring animal models of alkaptonuria in monkey (Johnson and Miller 1993) and mouse (Kamoun et al 1992), and in a mouse model achieved by induced mutagenesis (Manning et al 1999; Suzuki et al 1999), may eventually reveal features about pathogenesis. But for now the major interest in these models lies in the opportunities they provide to study treatment modalities for alkaptonuria. The ochronosis and arthropathy of the disease appear to be related to a chronic excess of homogentisic acid in tissues. Various approaches, notably involving diets and ascorbic acid, to control levels of the organic acid and its derivatives have been tried and have failed to ameliorate the disease (reviewed in Kayser et al 2008). However, a new drug in the class being referred to as Forphan drugs_ (Haffner 2006) may change the natural history of alkaptonuria. The drug is the tri-ketone herbicide (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) known as NTBC or nitisinone (Orfedin). The drug is a potent reversible inhibitor of p-hydroxyphenylpyruvate dioxygenase. It is the treatment of choice in hereditary tyrosinaemia, where it blocks the tyrosine oxidation pathway and thus prevents excess formation of toxic metabolites including fumarylacetoacetate and its derivatives. Nitisinone was then tried in a murine form of alkaptonuria (Suzuki et al 1999), with striking effect on the formation and excretion of homogentisic acid. The mouse study was followed by a small study in human patients (Phornphutkul et al 2002; Suwannarat et al 2005). There was evidence for a dramatic amelioration in the chemical and metabolic phenotype in alkaptonuria; these preliminary studies have led to a long-term randomized clinical trial in 40 patients (NIH protocol 05-HG-0076), to be completed in 2009 (Kayser et al 2008). While the journey of discovery in alkaptonuria has been such a long one (Scriver 1996), it now seems likely that the patient with alkaptonuria will be a very important beneficiary. Cystinuria It will be clear, from all that has gone before, that we are still far from being in a position to formulate a satisfactory theory of cystinuria. Before this can be done, it will be necessary to accumulate many more data by patient investigation of individual cases, and, above-all, quan- 587 titative data. [italics added]. Thus, cystinuria, like alcaptonuria [Garrod_s spelling], may be classed as an arrest rather than as a perversion of metabolism. Garrod (1923), pp. 132 and 135 in Chapter VII: Cystinuria and Diaminuria Garrod was puzzled by the problem of cystinuria. He used this disorder as the third example in his Croonian Lectures (albinism and alcaptonuria were the first and second), to illustrate further the concept of Fan inborn error of metabolism_. One might describe the understanding of cystinuria at the time as follows: the fox did not know enough things; the hedgehog could not know the new Fbig_ thing—yet. Cystinuria is not the result of a deficient enzyme in a catabolic pathway, as Garrod might have supposed; in reality, it is the result of deficient function in a specific membrane protein, in kidney and intestine, serving the transmembrane movement of a selective group of amino acids (cystine, lysine, arginine, and ornithine) (Palacin et al 2008). Cystinuria, in its role as an inborn error of amino acid transport, points to the physiological process and the gene products underlying the mutant phenotype (Scriver and Tenenhouse 1992). Knox recognized the difference between an inborn error affecting an enzyme in a metabolic pathway and one affecting activity of a membrane transport system; he called the latter a Fhere-to-there_ase_ (Knox 1958c, p. 29). As in alkaptonuria, the historical journey of discovery in cystinuria has also been a long one (Knox 1958c, 1966). It begins in the era of Napoleon and Wellington with the identification of Fcystic oxide_ (cystine) in the bladder calculus of an English patient (Wollaston 1810). The first definitions of this rare medical condition (hereafter named cystinuria) came into being through observations, collected by Niemann, on 52 patients with urolithiasis in the 19th century (Knox 1958c, pp. 5–10). Niemann_s careful description of cystinuria would be recapitulated much later (Dent and Senior 1955) in terms that echoed Garrod_s description of an inborn error of metabolism: cystinuria was a condition present from birth, often present in siblings, and, apart from the urolithiasis, compatible with good health and longevity. In other words, cystinuria was a metabolic sport. The clinical nature of cystinuria was somewhat obscured from Garrod and others by another clinical discovery in 1903. It involved another disease of cystine metabolism (cystinosis, OMIM 219800), itself an unrecognized inborn error of metabolism, which would eventually reveal an often lethal disorder of intra- 588 cellular cystine transport and cystine accumulation. Greater familiarity with the clinical and biochemical phenotypes of cystinuria and cystinosis would, in time, distinguish one from the other. In 1905, Alsberg and Folin reported studies on sulfur metabolism in a patient with cystinuria, from which conclusions were drawn about the chemical identity and origin of the cystine in cystinuric calculi (Alsberg and Folin 1905). Cystine metabolism itself was apparently normal, but cystine was indeed excreted in excess by the kidney when it was infused parenterally (Wolf and Shaffer 1908); yet under normal circumstances, its plasma levels were not abnormally elevated in the cystinuria patient (Brown and Lewis 1932). From such seemingly contradictory evidence, the origins of the excess urinary cystine were not easily discerned. In 1908, these earlier findings were corroborated (Wolf and Shaffer 1908) and extended to reveal an excess of an Fundetermined nitrogen_ fraction in cystinuria urine, which, because it was attributable only in part to the excess of cystine itself, implied the presence of other amino acids or related metabolites in the urine of cystinuria patients. Because the analytical power available at the time was not sufficient to describe these components, it would be four more decades before the mysterious fraction was identified. By means of microbiological assays in 1947 (Yeh et al 1947), and then by means of quantitative ion-exchange elution chromatography in 1951 (Stein 1951), the undetermined nitrogen fraction in cystinuria urine was shown to comprise an excess of arginine, lysine, and ornithine along with the cystine. Because the plasma levels of these four amino acids were each normal in cystinuria (Fowler et al 1952; Stein 1951), it was surmised that a selective impairment of renal reabsorption apparently existed in cystinuria. Formal measurements of the renal clearance rates of the affected amino acids supported this hypothesis (Dent and Rose 1951; Dent et al 1954; Robson and Rose 1957). Because the impaired reabsorption of amino acids was selective, it was further proposed that the disorder of renal reabsorption involved a specific process capable of recognizing the substrates it used. By this tortuous path of discovery, cystinuria was eventually shown to be an inborn error of a specific process for renal reabsorption of certain amino acids. The answers might have come earlier if certain contingencies had been recognized for what they were. For example, Dr I. M. Rabinovitch (of Montreal) had proposed in 1934 that the deviant process in cystinuria reminded him of a similar process acting in renal glucosuria (cited in Patch 1934). Garrod himself J Inherit Metab Dis (2008) 31:580–598 saw discrepancies in the contemporary metabolic hypotheses put forward to explain cystinuria (Garrod 1909a) and he could have given a different Croonian Lecture on cystinuria if he had known more about the plasma levels of cystine and the composition of the undetermined nitrogen fraction in his patients_ urine. But these are speculations from the Fwhat if_ school of evidence. Better instead to celebrate how cystinuria influenced Garrod_s thinking than to speculate in the reverse direction. Accordingly, we celebrate the expansion of Garrod_s concept of Fthe inborn error of metabolism_ to see it encompassing functional systems other than metabolic pathways and enzymes—to include transmembrane transport systems encoded in membrane proteins. Cystinuria was clearly seen by Garrod to accommodate a Mendelian perspective. Family histories revealed consanguinity and segregation patterns revealed a Mendelian mode of inheritance. Cystinuria, like other Fsports_, was rare but it was not clear to Garrod how rare. Reliable prevalence data could not be ascertained until discrepancies originating in the different ways of measuring the prevalence of cystinuria were taken into account; rates will differ if one is counting only the population-frequency of urolithiasis, or measuring the frequency of chemical cystinuria, or identifying the precise amino acid excretion pattern that characterizes cystinuria. From the latter, cystinuria would be defined as a specific condition characterized by the presence of a particular urinary amino acid excretion pattern (Dent and Harris 1951), thus not confusing it with cystinosis, Fanconi syndrome, dibasicaminoaciduria and other apparent phenocopies. Garrod preferred to have quantitative data whenever possible (see. p. 132 in Garrod 1923). It was the quantitative studies, performed years later (Harris and Warren 1953; Harris et al 1955a, b) that revealed two inherited forms of cystinuria: one form, the more prevalent, had a completely recessive silent phenotype in the heterozygote; the other form, incompletely recessive, was partially expressed in the heterozygote (Harris et al 1955b). This broad classification was robust and remained useful (Crawhall et al 1969; Palacin et al 2008), but until Fcystinuria_ genes had been cloned and mutations identified, it would not reveal whether the phenotypes reflected allelic heterogeneity at one locus, or genetic heterogeneity affecting a specific heteromeric membrane transporter. A pause for some hindsight The present-day view of cystinuria as a Mendelian disorder affecting a specific and selective transmem- J Inherit Metab Dis (2008) 31:580–598 brane transport function located in the brush border membrane of epithelial cells, in proximal renal tubule and small intestine (and not as a disorder of the transsulfuration pathway of methionine and cysteine), emerged initially from the findings just described, which were then buttressed by an array of new findings summarized elegantly in a review by Segal and Thier (Segal and Their 1995). The review showed how necessary quantitative data emerged from studies both human and animal in origin; from measurements obtained both in vivo and in vitro, with a variety of methods including renal tubule microperfusion, measurement of solute uptake by the renal cortex slice and transport in the brush border membrane vesicle preparation; and from corresponding measurements of intestinal absorption and uptake in small-intestine mucosal biopsy samples. These studies revealed the following: interactions between amino acids on a carrier located in the luminal membrane of the epithelial cell; shared transport for dibasic amino acids and cystine on a carrier in kidney and intestine; topological heterogeneity of this and other carriers in the proximal nephron; independent transporters for cysteine and cystine; and further complexity revealed by the non-cystinuria Mendelian phenotypes called isolated hypercystinuria, selective hyperdibasic aminoaciduria (OMIM 222690), and lysinuric protein intolerance (OMIM #222700). When the inheritance of classic cystinuria was revisited by Rosenberg and colleagues, they saw evidence, in kidney and intestine, for three types of cystinuria families, a proposal still out with the jury but compatible with a later reclassification of cystinuria into type I and non-type I phenotypes embracing both allelic and locus heterogeneity.1 The molecular basis of cystinuria The inborn errors of amino acid transport have led to the identification of particular functions, the associated molecular agents, and they point to the corresponding genes (Scriver and Tenenhouse 1992). Cystinuria is an inborn error of cystine and dibasic amino acid transport in kidney and intestine (Camargo et al 2008; Palacin et al 2008). The variant function serves 1 Leon Rosenberg participated in many of the studies reviewed by Segal and Their, which generated the first in vitro evidence for the deficient transport function in kidney and intestine in cystinuria and evidence for a transport system embracing lysine, ornithine, and arginine but excluding cystine. (Professor Rosenberg was a participant in the Symposium about Garrod_s Inborn Errors of Metabolism.) 589 absorption of cystine, lysine, ornithine, and arginine in the small intestine, and reabsorption from glomerular filtrate in the proximal renal tubule in its convoluted segments (S1, S2) and the pars recta (S3). The process is complex, operating in specific ways at luminal (apical) and basolateral membranes of the epithelial cells, along the length of proximal renal tubule and intestine. As a result, there is heterogeneity in the process both at the level of tissues and in axial and radial dimensions of the renal tubule. Cystinuria, as a specific example, reveals how a transport process can transfer and conserve hydrophilic solutes in a mediated way across lipid barriers in plasma membranes. Heteromeric amino acid transporters (HATs) are the protein gene products for this role (Palacin et al 2005). The following is an overview of the process; details are given elsewhere in two excellent reviews (Palacin et al 2005, 2008). HATs possess interacting heavy (õ90 kDa) and light (õ50 kDa) subunits (Fig. 6). The former belong to the Solute Carrier family SLC3, the latter to family SLC7. Cystinuria (OMIM 220100) is a phenotype resulting from mutations either in the SLC3A1 gene at locus 2p16.3-p21 encoding the heavy HAT subunit named rBAT (OMIM 104614), or in the SLC7A9 gene at locus 19q13.1-q13.2 encoding the light HAT subunit named b0,+AT (OMIM 604144). SLC3A1 mutations usually confer the fully recessive (type I) cystinuria phenotype; SLC7A9 mutations are usually expressed as the incompletely recessive (non-type I) phenotype. The rBAT heavy HAT subunit is a type II integral membrane N-glycoprotein in the apical membrane with a single transmembrane domain, and an intracellular NH2 terminus and an extracellular COOH terminus. The b0,+AT light HAT subunit is hydrophobic, is non-glycosylated, and has 12 transmembrane domains, with both termini at intracellular locations. The light and heavy subunits are connected by a disulfide link in the extracellular domain (see Fig. 6; and discussion in Palacin et al 2005). The light subunit will park in the plasma membrane only when it can interact with its heavy (rBAT) subunit following protein synthesis; rBAT is normally produced in excess in renal cells. It locates itself primarily in the S3 segment of the proximal nephron, where it shows high affinity for substrate and fine tunes amino acid reabsorption (Palacin et al 2008). These facts are all relevant when interpreting the completely recessive (type I) phenotype in heterozygous cystinuria. The light subunit b0,+AT confers substrate specificity when the holotransporter is assembled. Without its heavy subunit companion, the transporter is not functional. The transporter activity is functionally coupled to an 590 J Inherit Metab Dis (2008) 31:580–598 Fig. 6 General features of a heteromeric amino acid transporter (HAT) comprising a heavy subunit (pink) and a light subunit (blue) linked by a disulfide bond (yellow). The human HAT affected in cystinuria has a heavy subunit rBAT encoded by the SLC3A1 gene (locus 2p16.3) and a light subunit b0,+AT encoded by the SLC7A9 gene (locus 19q12-q13). (Taken from Palacin et al 2005 with kind permission from Physiology). amino acid antiporter activity. The intact rBAT/ b0,+AT carrier complex is maximally expressed in the epithelial cells of proximal renal tubule and small intestine. This heterodimeric transporter supports a physiological transport activity designated as the b0,+AT flux in the apical plasma membrane of epithelial cells in the nephron and small intestine. It mediates an electrogenic exchange (influx) of the dibasic amino acids and cystine with an efflux of neutral amino acids—the antiporter activity (Palacin et al 1998). The renal reabsorption of cystine is ultimately all accounted for by the rBAT/b0,+AT high-affinity transporter; the presence of other apical membrane carrier-mediated fluxes serving dibasic amino acids, yet to be identified, is exposed to notice in the cystinuria phenotype (Palacin et al 2008). Cystinuria_s molecular mysteries began to yield when mutation in the SLC3A1 gene affecting function of the rBAT heavy subunit was shown to be the cause of type I cystinuria (Calonge et al 1994). Expression analysis of the most prevalent SLC3A1 allele (p.M467T) revealed a trafficking defect preventing the rBAT subunit from reaching the plasma mem- brane. The non-type I cystinuria phenotype was then traced to mutation in the SLC7A9 gene (Font et al 2001) with alleles affecting the b0,+AT light subunit (Font-Llitjos et al 2005). It was then predicted that SLC3A1 alleles would segregate consistently with the type I cystinuria phenotype, and the SLC7A9 alleles would explain the non-type I phenotype. However, it was not so simple. A concerted effort by the International Cystinuria Consortium (ICC) (Dello Strologo et al 2002) offered a more stratified interdependent classification derived from findings in obligate heterozygotes and their relatives based on quantitative urine amino acid excretion phenotypes and associated genotypes. ICC used symbol A for the mutant SLC3A1 phenotype and symbol B for the mutant SLC7A9 genotype; most type I patients have an AA genotype; most non-type I patients have a BB phenotype. However, some AA or BB genotypes confer an unpredicted clinical phenotype, suggesting the presence of modifier events; and some rare patients are digenic (type AB) with partial (non-lithogenic) phenotypes (Font-Llitjos et al 2005; Harnevik et al 2003). The hypotonia-cystinuria syndrome, a rare congenital recessive phenotype with a microdeletion at chromo- J Inherit Metab Dis (2008) 31:580–598 some 2p21 affecting the SLC3A1 and PREPL genes (Jaeken et al 2006), illustrates another facet of genotype–phenotype associations in cystinuria. Whereas the renal phenotype causes urolithiasis in a subset of cystinuria patients, the intestinal phenotype (Rosenberg et al 1965) has no apparent clinical consequences because there are alternative sources (dipeptides) to meet nutritional requirements for the affected amino acids. For those at risk for urolithiasis there is either conservative therapy (high free water intake and alkalinization of urine) or more aggressive treatment, which is not risk-free, with oral sulfhydryl agents to create mixed disulfides that are more soluble than cystine (e.g. D-penicillamine and mercaptopropionylglycine). A little LPI detour: Lysinuric protein intolerance (LPI) (OMIM 222700) is not a variant of cystinuria (Simmell 2001). It is an independent disorder of the y+L exchanger (Palacin et al 2005) situated in the basolateral membrane of the proximal renal tubule and small-intestine epithelial cells, and in the plasma membrane of lung alveolar cells and white blood cells. The y+L solute flux is mediated by a heterodimeric transporter containing the light subunit y+LAT1 and heavy subunit 4F2hc. LPI-causing mutations occur in the SLC7A7 gene at locus 14q11.2 encoding the y+LAT1 light subunit. Impaired function of the transporter accounts for the complex, multisystem, and quite severe clinical phenotype in the LPI patient. The cystinuria story spans two centuries and it fulfils the major criteria for an inborn error of metabolism. Garrod was not able to discern the deviant function in cystinuria, but investigations of it later, by others, revealed that solute traffic through and between cells is a vital part of what he called dynamic metabolism, in which case, Garrod was indeed on the right path. Pentosuria: Garrod_s perfect Fsport_ Garrod_s fourth Croonian Lecture, delivered in 1908 (Garrod 1908), described a condition recognized by its persistent excessive urinary loss of a pentose sugar without any apparent clinical consequences. Garrod knew that the available facts about the identity of the sugar and the clinical significance of pentosuria were confusing, yet he would come to consider pentosuria, with its congenital onset and its positive family histories, as the least conspicuous (and least clearly defined) of the inborn errors of metabolism (Garrod 1923, page 591 172). Knowledge of the pentose sugars at the time was scant, a case of pentosuria had been reported in 1892, the optical inactivity of the sugar in pentosuria was a puzzle (Garrod 1908), and a pentose sugar identified as xylulose had been found in the human pancreas; but these were the outer limits of available information. Pentosuria was then found to involve the excretion of xylulose (Levene and La Forge 1914), the enantiomorph was L(+)-xylulose (Greenwald 1930), and the condition behaved as a congenital benign phenotype, inherited as a Mendelian recessive, apparently with high prevalence among Ashkenazi Jews (Lasker et al 1936) with an estimated allele frequency of 0.0127 (Lane and Jenkins 1985). Survival studies (Lasker 1955) would formally indicate that the condition was benign. Whereas Garrod had considered glucosamine as the possible source of the pentose (Garrod 1923, p. 192), it would eventually be shown (Horecker and Hiatt 1958) that L-xylulose was a normal metabolite occurring in the normal metabolic pathway of glucuronic acid oxidation (OMIM 260800) (Hiatt 2008). The mechanism by which an excess of pentose sugar could appear in urine was a matter of debate for some years, and because of technical limitations it was never clear whether the blood xylulose level was variant or normal in pentosuria (Knox 1958b, p. 394-5). This lack of significant information blurred the distinction between a mechanism of renal loss or an enzyme failure to explain the pentosuria. The problem resolved itself with the evidence that the basic defect in pentosuria was deficient activity, even absence, of L-xylulose reductase, an enzyme measurable in erythrocytes of normal and pentosuric subjects (Lane 1985; Wang and Van Eys 1970); a gene dosage effect was also apparent. The gene encoding the enzyme (symbol DCXR) was eventually cloned and mapped to human chromosome 17 (OMIM 608347). The NADP-linked enzyme (EC 1.1.1.10), acting as L -xylulose reductase (or L -xylitol dehydrogenase) exists in two forms in human tissues (Lane 1985); the major isozyme, missing in pentosuria, occurs in cytosol and mitochondria, while the minor form, retained in pentosuria, is limited to cytosol (Lane and Jenkins 1985). The enzyme acts in the glucuronic acid pathway where the carboxyl carbon of D-glucuronic acid is removed in a series of reactions to yield the pentose L-xylulose (Hiatt 2008). The pentose sugar is converted to D-xylulose via the transitional form, xylitol, and then phosphorylated, allowing D-xylulose 5-phosphate to enter the pentose phosphate pathway, followed by conversion to hexose phosphate. The pathway seems to be a biochemical fossil and may serve no essential function in human metabolism (but see below about 592 osmoregulation). Accordingly, pentosuria might be FGarrod_s perfect sport_. The pentosuria enzyme (L-xylulose reductase EC 1.1.1.10; OMIM 608347) is identical to diacetyl reductase (EC 1.1.1.5). It belongs in a superfamily of shortchain dehydrogenase/reductase enzymes, is highly expressed in kidney, and by virtue of its location may be a contributor to the pentosuria when metabolic runout of the sugar during reabsorption is impaired in the mutant phenotype. The human enzyme functions as a homotetramer (Nakagawa et al 2002); its crystal structure has been determined at 1.96 Å resolution (El-Kabbani et al 2004); and it has conserved residues for catalytic functions, subunit interactions, and coenzyme binding. Its prominent location in kidney and the evidence of evolutionary conservation invite speculation that it may actually play a role in osmoregulation (Nakagawa et al 2002). Two new non-pentosuria autosomal recessive disorders of pentose sugar metabolism have been discovered (Wamelink and Jakobs 2008), both involving the reversible part of the pentose phosphate pathway and unallied to essential pentosuria. These disorders are ribose-5-phosphate isomerase deficiency (EC 5.3.1.6; OMIM 608611) and transaldolase deficiency (EC 2.2.1.2; OMIM 606003). They are described in chapter 73S of the New Online Metabolic and Molecular Bases of Inherited Disease. Albinism That albinism is congenital and persists through life is self evident, the condition is as obvious as any structural malformation and its rarity in man is also evident. It stands to reason that an error of metabolism which persists from birth into adult, and even into advanced life, must needs be relatively innocuous (Garrod 1908) Garrod notes that there was evidence for recessive inheritance in the form of albinism on which he focused his comments. This form would later be designated as oculocutaneous albinism (OCA; OMIM 203100). Albinism nicely illustrated Garrod_s view that the Finborn error of metabolism_ was so often to be seen as a sport or a freak of nature, but in 1908 the phenotypic components of albinism were not well known and its ocular and brain components were not as well known as they are today. Garrod_s second essay on albinism, in the revised edition of Inborn Errors of Metabolism J Inherit Metab Dis (2008) 31:580–598 (Garrod 1923), is considerably longer than the first, recognizing as it does advances in knowledge about albinism stemming from an ongoing massive, and never completed, study of the condition in humans subjects (Pearson et al 1911). Garrod considered albinism to be a disorder either in the synthesis or in the maintenance of melanin pigment; or that it was a disorder of the cells in which melanin occurred. In the second edition of Inborn Errors, Garrod could suggest that the absence of pigment from skin and hair of albinos was attributable to the lack of a specific enzyme in the cells that were the normal seats of pigmentation (Garrod 1923, page 38). Knox (Knox 1958a), at the half century mark after the Croonian Lecture on albinism, saw the inclusion of albinism in the pantheon of inborn errors of metabolism as evidence of Garrod_s Fintuitive genius_. On the other hand, albinism continued to be a mystery at the 50th anniversary of the Lecture and one could only hope that Garrod_s prediction of a single enzymatic defect in albinism would eventually be confirmed (Knox 1958a). Whereas Garrod could propose a Mendelian explanation for one well-defined form of albinism, there exists a great variety of albino phenotypes and descriptors. Five autosomal loci, and one X-linked, each responsible for a form of albinism, have since been identified in human patients (King et al 2008). Accordingly, the possibility existed that within the albino collective there were multiple distinct hereditary forms that could intrude on Garrod_s understanding of albinism. He was rigorous and focused on a consistent single phenotype; one might add that he was lucky in his choice. Strong evidence for hereditary transmission of a form of albinism (presumably the oculocutaneous form) did appear (Davenport and Davenport 1910; Davenport 1916) soon after Garrod_s Croonian Lecture on albinism was published. However, there were persons who did not agree with Garrod, or with anyone who proposed a Mendelian origin for albinism; one of those critics was Karl Pearson (Pearson et al 1911, part IV). Pearson was an influential person and he held the prominent position as Director of the Galton Laboratory at University College London. He and his colleagues produced a massive monograph on albinism (Pearson et al 1911) which harboured findings used in the debates by those who did not support Mendelism as an explanation for albinism. Pearson_s own position was clear: FThere is no definite proof of Mendelism applying to any living form at present_ (see Church 1909, p. 155). Today_s readers of Pearson_s statement must find it hard to believe that he J Inherit Metab Dis (2008) 31:580–598 represented a large community of opinion. Garrod_s influential Croonian Lectures helped to turn that tide of opinion. It is reasonable to suppose that Pearson was finding evidence in albinism to support the view that heredity acts through continuous or quasi-continuous (Galtonian) variation rather than through the discontinuous (Mendelian) variation associated with mutation of large effect. But Pearson_s study combined (lumped) many different forms of albinism, and it was confounded by its failure to stratify (split) the whole into its component types of albinism. When Hogben (1931) deconstructed Pearson_s data, he noticed consanguinity at work in albinism and could point to evidence for autosomal recessive inheritance of various albino forms. Autosomal recessive inheritance of complete albinism (the OCA form) was later confirmed in clinical observations (Trevor-Roper 1952) which showed that albinism would have to be accounted for by both locus and allelic heterogeneity. Students of Garrod_s legacies will not regret a visit to Knox_ revealing essay on albinism (Knox 1958a), where one is reminded by an excellent arbiter of debate that limitations of theory may not be revealed when the facts are too few. The second half-century after the Croonian Lecture has witnessed a gathering of facts and a transformation in our understanding of albinism. Melanin pigment in skin, hair, and eyes represents the most visible marker of human (chemical) variation (King et al 2008). Hypopigmentation and variation in pigmentation are highly heritable markers of human chemical individuality (Antonarakis and Beckmann 2006). Albinism is an extreme variant. Albinism is heterogeneous both genetically and phenotypically. Garrod presumably saw a patient with the distinctive type IA (tyrosinase-negative) form of oculocutaneous albinism (OMIM 2003100, OC1A), in which case his concept that albinism was attributable to the lack of an enzyme involved in the synthesis of melanin would be proved correct; the enzyme in the OCA phenotype is tyrosinase. The albinism phenotypes are a group of hereditary congenital hypopigmentation disorders affecting skin, hair, and eyes: autosomal in OCA (OMIM 203100); Xlinked in ocular albinism (OA) (OMIM 300500). Melanocytes produce melanin and the process of pigmentation is complex, with the involvement of many steps during development and of many genes and proteins, as proposed long ago (Childs 1970) yet ignored for too long. The rate-limiting enzyme in the OCA disorder of pigmentation, and the corresponding pathway of melanin synthesis, is tyrosinase (EC 593 1.14.18.1), which hydroxylates tyrosine to form DOPA quinone in the initial steps of a pathway leading to mature melanosomic melanin. Classical albinism, the form of interest to Garrod, is now clearly seen to be a deficiency of tyrosinase activity: complete in OCA type IA (OMIM 203100, 606933); partial in OCA type IB (OMIM 606952, 606933). The tyrosinase gene (TYR) maps to chromosome 11q14-21. Meanwhile, the vast array of albinotic phenotypes is being steadily dissected and explained at genetic, molecular, and cellular levels (King et al 2008). Post-Croonian perspectives, or what the hedgefox might know No quality is so universal, in the appearance of things, as diversity and variety (from Michel de Montaigne_s essay: Of experience.) Garrod presented facts in his Croonian Lectures— facts the fox knew well. Among them was evidence that Mendelian inheritance was at work in human families; and that metabolism in human beings resembled a flowing weir rather than an isolated set of still ponds. Missing, of course, from the assembly of facts available to Garrod, were details about Mendel_s factors (genes) and modern insights on the close relationship between genes, enzymes, and metabolism. At the centennial of Garrod_s Croonian Lectures, there are many new facts to assemble in what might be called The Post-Croonian Fox Reports. The FFox Reports_ will show that the four charter inborn errors of metabolism (albinism, alkaptonuria, cystinuria, and pentosuria) have each seen their genomic locus mapped, the gene cloned, its mutations identified and annotated, a relevant protein gene product isolated and characterized, and the metabolic consequences of variant gene expression delineated. Such facts have served the interests of biochemists and geneticists; belatedly they are also serving medical interests. The generic inborn error of metabolism is not always a Fsport_ (as assumed by Garrod and Osler) and, more likely than not, there are consequences for health. The corresponding need for diagnosis, counselling and treatment has given rise to an expert community of biochemical and medical geneticists which now serves a large community of patients put at risk by their orphan diseases (a new name for the inborn errors), while using Web-based data/knowledge bases that can serve everyone. These derivates of the original Fox Reports are new and impressive and they evolve in ways unknown to Garrod. 594 On earlier occasions at SSIEM, I revisited the theme of Finborn errors_ to discover what it can tell us about personal and public biochemical genetics (Scriver 2001); about the unavoidable process of our own ageing (Scriver 2002); and about our phenomes and how they emerged from our genomes (Scriver J Inherit Metab Dis (2008) 31:580–598 2004). These were initiatives to discover some of the big things—known to the hedgehog. Context is always important when new knowledge is transforming existing perspectives. A few Fhedgefox_ examples will suffice on this centennial occasion. Each has a wide context summarized in the famous apho- Fig. 7 A Fclustered heat map_ allows organization of data to be analysed, visualized, and interpreted. The image replaces long linear assemblies of data on computer paper. The 2D display serves various types of data; molecular, protein expression, metabolite concentration, DNA copy number, and more. This image includes data on drug activity in different cancer cell types. Expression levels are coded red for high level and blue or green for low level. The need for novel representations of data in post-genomic biology is an ever-present challenge. (Taken from Weinstein JN. A post-genomic visual icon. Science 2008; 319:1772–1773.) J Inherit Metab Dis (2008) 31:580–598 rism of Theodosius Dobzhansky: Nothing makes sense in biology except in the light of evolution. The double helical structure of our own DNA is known, as is the role played by specific base pairing for copying our genetic material and how it gives expression to mutations in the DNA code. The (Human) Genome Project(s) has/have since ascertained the complete nucleotide sequence of representative genomes while trying to understand the origins of genome architecture and how it supports a morbid genome map. Full genome sequences from members of Species, Families, and Kingdoms, are being interrogated to reveal the origins of living organisms on planet Earth, while addressing yet another problem: how phenotypes emerge from genotypes when genomes speak biochemistry, not phenotype. To the latter end, various Fomes_ are investigated such as the transcriptome, the newly recognized small RNA transcriptomes, the proteome, and so on. The model organism of particular interest (H. sapiens) will use the ENCODE project (Encyclopedia of DNA Elements) and its databases to decode genotypes and phenotypes (dbGaP); ENCODE will assist large-scale, genomewide association studies (GWAS). Clear definitions of (disease) phenotypes will help and Mendelian phenotypes remain useful as mentors. The Human Variome Project, which addresses variations in alleles and genome architecture, is acquiring its own encyclopedic catalogue of sequence variants indexed to the human genome sequence . All of this uses an impressive array of analytic and informatic technologies (see Fig. 7) able to capture the evidence of individuality, perhaps for eventual use in Fpersonalized medicine_. It will support a science of the individual (Childs et al 2005). Medicine may not be an exact science, which is an inconvenient truth, and every illness and case history, like every person, is different; we respond accordingly, and we participate in a science of the individual. Whereas Garrod could recognize chemical individuality through the powerful lens of an inborn error of metabolism, individuality is also revealed, for example, in the more subtle polygenic phenotype contained in normal plasma amino acid values, where the distribution of values contains subsets reflecting individuality in the amino acid metabolome (Scriver et al 1985). Moreover the latter becomes a factor, for example, in setting the threshold against which a variant Hartnup genotype may or may not become manifest as the Hartnup disease phenotype (Scriver et al 1987). Garrod used four charter inborn errors of metabolism as evidence for Mendelian inheritance of variation (chemical individuality) in the human species. These disorders each had recessive metabolic phenotypes, a 595 feature that led to enquiries into the molecular basis of dominance and recessiveness (Kacser and Burns 1981; Kacser and Porteous 1987), and to interrogation of the processes in diploid organisms that buffer the effects of genetic variation on phenotype (Hartman et al 2001). That the buffering is distributed in scale-free gene and disease networks (Goh et al 2007) invites systems biology to meet natural variation when mapping genes to their function and while investigating the genotypeto-phenotype problem (Benfey and Mitchell-Olds 2008). The shift towards concern for multifactorial common disease, initiated by Garrod in 1931, need not be taken at the expense of Mendelian disorders when the latter can help us to understand the infrastructure of complex traits (Antonarakis and Beckmann 2006; Scriver and Waters 1999). Metabolism was seen by Garrod to be dynamic and complex in its components. The Mendelian disorders that buttressed his argument in the Croonian Lectures were rare and seemingly contradictory sources of this insight. Garrod took his argument further and proposed an understanding of more prevalent disease forms; he proposed Finborn factors in disease_ to explain susceptibilities to and the expression of these diseases (Garrod 1931a). Is it any surprise that after a century of awakening to Garrod_s insights, and to the enumeration and interpretation of nearly two thousand Mendelian disorders (all of which are not simple and reflect their own forms of complexity (Scriver and Waters 1999), attention shifts to the more prevalent diseases that beset us? If it is understood that the organism is a complex phenome comprising robust efficient modules forged by natural selection during evolution of life on planet Earth (Oltvai and Barabasi 2002), it will be a natural progression to see disease phenotypes as reflections of incongruities in the gene and disease networks that we are beginning to discover (Goh et al 2007). The challenge is to know the components and what they are doing in the modules and networks—and how to offset their impact on our health and adaptation when they are variant. And in that context, there are a couple of old important questions, poised to keep us alert and concerned: What can I know? And with that knowledge, what ought I do? (Immanuel Kant) Acknowledgements The formative, enduring, and FGarrodrelated_ influences on the author are sources of this essay; they include a McLaughlin Travelling Fellowship (1958–60), a Markle Scholarship (1961–66), Career Awards from the Medical Research Council (Canada), several mentors (Alexander Bearn, 596 Barton Childs, David Cusworth, Charles Dent, F. Clarke Fraser, Harry Harris, Bert La Du, Roland Westall); many scientific colleagues (Claude Laberge, Gerard Bouchard, Ken Morgan, Leon Rosenberg, and colleagues in the Quebec Network of Genetic Medicine, and the Canadian Genetics Diseases Network); and current funding from Le Fonds de la recherche en santé du Québec, the National Institutes of Health (USA). Infrastructure at the Montreal Children_s Hospital Research Institute and McGill University facilitated the work for this article. Lynne Prevost, Jacques Mao, and Christineh Sarkissian transformed written words and sketches into a digital text and Powerpoint. 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