Download Scriver Charles R. Garrod`s Croonian Lectures (1908)

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
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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;
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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
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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)
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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
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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.)
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(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-
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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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