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HUMAN MUTATION 21:333^344 (2003)
DATABASES
PAHdb 2003: What a Locus-Specific
Knowledgebase Can Do
Charles R. Scriver,1n Mélanie Hurtubise,1 David Konecki,2 Manyphong Phommarinh,1
Lynne Prevost,1 Heidi Erlandsen,3 Ray Stevens,3 Paula J. Waters,4 Shannon Ryan,1
David McDonald,5 and Christineh Sarkissian1
1
Departments of Human Genetics, Biology, and Pediatrics, McGill University Health Centre, Montreal, Canada; 2Medical Genetics
Branch, NHGRI/NIH, Bethesda, Maryland; 3Department of Molecular Biology, The Scripps Research Institute, La Jolla, California;
4
Biochemical Genetics Laboratory Children’s and Women’s Health Center of British Columbia, Vancouver, British Columbia, Canada;
5
Department of Biological Sciences, Wichita State University, Wichita, Kansas
For the PKU Special Issue
PAHdb, a legacy of and resource in genetics, is a relational locus-specific database (http://
www.pahdb.mcgill.ca). It records and annotates both pathogenic alleles (n = 439, putative diseasecausing) and benign alleles (n = 41, putative untranslated polymorphisms) at the human phenylalanine
hydroxylase locus (symbol PAH). Human alleles named by nucleotide number (systematic names) and
their trivial names receive unique identifier numbers. The annotated gDNA sequence for PAH is typical
for mammalian genes. An annotated gDNA sequence is numbered so that cDNA and gDNA sites are
interconvertable. A site map for PAHdb leads to a large array of secondary data (attributes): source of the
allele (submitter, publication, or population); polymorphic haplotype background; and effect of the allele
as predicted by molecular modeling on the phenylalanine hydroxylase enzyme (EC 1.14.16.1) or by
in vitro expression analysis. The majority (63%) of the putative pathogenic PAH alleles are point
mutations causing missense in translation of which few have a primary effect on PAH enzyme kinetics.
Most apparently have a secondary effect on its function through misfolding, aggregation, and intracellular
degradation of the protein. Some point mutations create new splice sites. A subset of primary PAH
mutations that are tetrahydrobiopterin-responsive is highlighted on a Curators’ Page. A clinical module
describes the corresponding human clinical disorders (hyperphenylalaninemia [HPA] and phenylketonuria [PKU]), their inheritance, and their treatment. PAHdb contains data on the mouse gene (Pah)
and on four orthologous mutant mouse models and their use (for example, in research on oral treatment
of PKU with the enzyme phenylalanine ammonia lyase [EC 4.3.1.5]). Hum Mutat 21:333–344,
2003. r 2003 Wiley-Liss, Inc.
KEY WORDS:
Phenylalanine hydroxylase; phenylketonuria; PKU; database; genotype-phenotype; hyperphenylalaninemia; HPA
DATABASES:
PAH – OMIM: 261600; GenBank: NM_000277 (mRNA), U49897.1 (cDNA), AF404777 (gDNA), X51942
(mouse cDNA); Swiss-Prot: P00439 http://www.pahdb.mcgill.ca (PAHdb)
INTRODUCTION
So often neglected by the communities they serve,
databases are a legacy in and of science [Maurer et al.,
2000]. Science is an explanatory process. As a
particular domain of science develops, said Stéphane
Leduc cited in Keller [2002], there is first a stage of
explanation in classification and nomenclature (taxonomy) of its entities. Next, there follows inquiry into
mechanisms underlying the entities. The final stage is
to recreate or synthesize the entities. The science of
genetics recognizes mutation as both entity and
mechanism. Mutation can also be created. Mutation
r2003 WILEY-LISS, INC.
n
Correspondence to: Charles Scriver, Montreal Children’s
Hospital Research Institute, McGill University Hospital Centre, A717, 2300 Tupper Street, Montreal, Quebec H3H 1P3, Canada.
E-mail: [email protected]
Grant sponsors: Robert McDonald Gift; Quebec Network of
Applied Genetics/Fonds de la Recherche en sante¤ du Que¤ bec;
Canadian Genetic Diseases Network/Networks of Centers of
Excellence; Canadian Institutes for Health Research, formerly
Medical Research Council of Canada.
DOI 10.1002/humu.10200
Published online in Wiley InterScience (www.interscience.wiley.
com).
334
SCRIVER ET AL.
databases recapitulate the science in their various
ways and they can recreate it in silico. Databases have
thus become necessary resources in genetics. They are
repositories of the vast wealth of data being gathered
about individual genes and the genomes they inhabit.
PAHdb is one such legacy and resource.
PAHdb (http://www.pahdb.mcgill.ca) is an online
relational locus-specific knowledgebase [Scriver et al.,
2000] originating in, and still serving, the PAH
Mutation Analysis Consortium and other communities. This report is written by co-curators who
maintain PAHdb and work at five widely separated
locations, yet the internet connects us. Additional
databases related to the PAH gene are listed at the
head of this chapter.
Mutations at the human locus (symbol PAH;
MIM# 261600) affect the phenylalanine hydroxylase
enzyme (EC 1.14.16.1) and are a cause of the disease
phenylketonuria (PKU) or related forms of hyperphenylalaninemia [Scriver and Kaufman, 2001].
PKU reflects paradigms of both transformational
and translational knowledge. When the disease was
first recognized in 1934, it became the fifth in a series
of diseases known as ‘‘inborn errors of metabolism’’
[Garrod, 1908]. PKU thus consolidated an emerging
(transformational) view that Mendelian inheritance
could explain some forms of human disease. There has
also emerged an understanding (translational) that
the disease feature (mental retardation) of PKU was
preventable through early diagnosis and treatment. As
a result, our outlook on genetic disease in general
began to change. With time, PKU has emerged as an
explanatory prototype for human genetic disease that
links gene, mutation, enzyme, metabolism, and disease
effect [Scriver and Waters, 1999]. Accordingly, there
has been an exceptional opportunity to sample PAH
mutations in probands from populations around the
world wherever newborn screening was practiced. As
a result, PAH alleles and their attributes (polymorphic
haplotypes and populations) have become widely
known. PAHdb has thus emerged as a comprehensive
and useful prototype of the locus-specific database
[Claustres et al., 2002].
The origins, development, and design of PAHdb are
described in an earlier report [Scriver et al., 2000].
The database is built on four core elements: 1) a
unique identifier for each allele; 2) the source of the
information; 3) the context of the allele (e.g., the
species and name of the gene); and 4) the name of the
allele. PAHdb contains entities (mutations) and
annotates them with attributes. The Tables of
Mutations arise from these core elements. Additional
tables in the database provide information, from
in vitro expression analysis, on the functional effects
of mutations on enzyme integrity and function. The
database also visualizes in silico how mutations map
onto the 3D protein structure. A Curators’ Page
highlights unscheduled topics (e.g., discovery of BH4-
responsive PAH alleles) and novel data not readily
handled by the existing tables. Among other options,
PAHdb introduces visitors to the mouse Pah gene and
a mouse model of PKU. The clinical significance of
human PAH mutations is the subject of another
module. A counter logs visits (>30 hits/day) and
records the most recent date of curation. Table 1 is a
partial site map of PAHdb. The intellectual property of
PAHdb is copyrighted.
Because PAH alleles are named according to a
convention now widely accepted and used [Antonarakis et al., 2001], PAHdb can also be searched using
appropriate tools to retrieve and transfer its alleles to
an experimental ‘‘WayStation.’’ The WayStation will
be linked to a comprehensive data ‘‘Warehouse,’’ thus
making a repository of human genomic allelic
variation [Teebi et al., 2001]. When the WayStation
is established, PAH-related data could be submitted
either through it or directly to PAHdb.
PAH NUCLEOTIDE SEQUENCES: cDNA AND gDNA
Co-Curators: David Konecki, Mélanie Hurtubise, and
Manyphong Phommarinh
The human phenylalanine hydroxylase (PAH) gene
is embedded in the chromosomal region 12q23.2,
covering 1.5 Mbp, that contains PAH itself and five
other genes of known or unknown function [International Human Genome Sequencing Consortium,
2001; Venter et al., 2001]. The cDNA and full-length
genomic sequences are both visible online in PAHdb.
cDNA sequences have long been available [Kwok
et al., 1985; Konecki et al., 1992] but the genomic
sequence is recent (Konecki, D.S., unpublished;
deposited in PAHdb Nov. 2001).
The genomic sequence of the PAH gene and its
flanking regions spans 171,266 bp with ~27 kbp of 50
untranslated region (50 UTR) upstream from the
translation initiation site and ~64.5 kbp of 30
sequence downstream from the poly(A) site in the
last exon (exon 13). By convention in GenBank, the
first nucleotide of any sequence is numbered from its
50 end. However, to have gDNA nucleotides in
register with the older cDNA sequence (which has
long served PAH mutation nomenclature), the PAH
gDNA has been renumbered in PAHdb: the +1
nucleotide is the adenine of the translation initiation
site (ATG) in exon 1. Thus, gDNA exons, introns,
and the 30 UTR have positive numbers. The 50 UTR
has negative numbers. Table 2 lists the 13 exons in the
PAH gene and compares gDNA with cDNA positions.
Amino acid residues of the PAH protein that are
conserved in mouse, rat, and human are shown on a
protein sequence page directly under the cDNA
heading.
The 50 UTR of the human PAH gene contains cis
control elements [Konecki et al., 1992]. They are
PAHDB KNOWLEDGEBASE
TABLE 1.
A Partial Site Map of PAHdb (http://www.pahdb.mcgill.ca)
Page name (alphabetic order)
About
Consortium
Contact
Clinical
Haplotype
InVitro analysis
Molecular
Mouse
Mutation map
Other
PAHdbNewsletter
Search
Sequence
Submission
Survey
335
Content
Abstract; curators’ page; list of curators
List of members
Information
PKU for families, Link to GeneReviews
Polymorphic markers in PAH, Link toALFRED
See search page
Structural genomics of the PAH enzyme; link to Scripps Site
The mouse PKU/HPA models; mouse gene (link)
Mutations displayed on the PAH gene sequence
Copyright information;Various links (n = 18)
Print version of PAHdb (Dec. 2001)
The key page in PAHdb leading to:
In vitro expression analysis
Mutations
Mutations (statistics)
Mutation Associations
Clinical Authors
Genotype^phenotype correlations
CpG sites and mutability pro¢le
cDNA and genomic reference sequences, CpG sites, Alu sites exon^intron junctions
User registration, data submission
Hits counter, comments, etc.
shown on both the cDNA and gDNA sequences in
PAHdb.
Single nucleotide polymorphic (SNP) and restriction fragment length polymorphic (RFLP) sites,
currently used to create PAH polymorphic haplotypes,
are annotated on the genomic sequence. Other sites
at the PAH locus can be identified by using tools such
as the NEBcutter (www.neb.com).
Exonic regions in the human PAH gene are
~2.88% of the genomic sequence between the 50
+1 position down to the 30 poly (A) tract. Amplicon
primer sequences for all 13 exons are provided on the
cDNA sequence page. The shortest and longest exons
are 57 bp (exon 9) and 892 bp (exon 13), respectively.
The mean exon size is 170 bp. Three polyadenylation
signals [AATAAA] in exon 13 are annotated on the
gDNA sequence. The third site is used most
frequently. The shortest and longest introns are
556 bp (intron 10) and 17,874 bp (intron 2), respectively. Intron 3 is 17,187 bp in length. The mean
intron size is 6390 bp. These dimensions are typical for
mammalian genes.
The PAH genomic sequence consists of 40.7% GC,
slightly above the modal value (37–38%) for human
genes. RepeatMasker analysis shows the density of
interspersed repeats to be 42.2% in the PAH gene, a
value typical for a mammalian gene. RepeatMasker
data and a table of DNA variations, mostly SNPs, are
shown in PAHdb. Repetitive DNA is often the cause
of large genomic deletions and duplications [Antonarakis et al., 2001]. A search for Alu repeat elements in
the PAH gene was performed using NCBI BLAST
(www.ncbi.nlm.nih.gov/blast/). Alu repeat elements
are annotated on the gDNA sequence. Intron 2 has a
99% nucleotide identity with the Alu repeat element
between bp 17,273 and bp 17,546, which could
possibly coincide with the 50 deletion found in a PKU
family from Scotland [Sullivan et al., 1985]. Putative
Alu repeats are highlighted on the PAH genomic
sequence and CpG dinucleotide sites (n = 1198) are
annotated since these are potential sites for mutation
in the gene.
ALLELIC VARIATION
Co-Curators: Lynne Prevost, Manyphong Phommarinh, and Charles Scriver
PAHdb lists 439 human mutations classified as
potentially disease-causing [Cotton and Scriver,
1998]. Another 25 alleles are considered to be
non-pathogenic polymorphisms (but see K274E in
Expectations Confounded section, below) and there
are at least 16 alleles in the known STR and VNTR
regions of the gene. The PAH genomic sequence is likely
to contain many as yet unrecognized polymorphisms.
Pathogenic alleles
These are either displayed on a cartoon of the gene,
listed as entities by name in sequential order (50 to 30 )
in a core table, or listed in two expanded tables
containing attributes (e.g., unique identifier, associations with polymorphic haplotypes and populations, or
source of the data).
Polymorphic alleles
These are either annotated on the DNA sequences,
shown in a pictorial configuration on a cartoon of the
gene, or tabulated as configurations describing core
PAH haplotypes. There is a link to the ALFRED
database (http://alfred.med.yale.edu) where known
336
SCRIVER ET AL.
TABLE 2. ConversionTable of cDNA
Exon
1
2
3
4
5
6
7
8
9
10
11
12
13a
Exon Annotations to Genomic Nucleotide Numbers
cDNA
gDNA
Intron
Sequence
1^60
61^168
169^352
353^441
442^509
510^706
707^842
843^912
913^969
970^1065
1066^1199
1200^1315
1316^1359
1^60
4233^4340
22215^22398
39586^39674
50550^50617
61890^62086
64272^64407
65466^65535
70273^70329
72793^72888
73445^73578
76709^76824
78006^78897
1
2
3
4
5
6
7
8
9
10
11
12
61^4232
4341^22214
22399^39585
39675^50549
50618^61889
62087^64271
64408^65465
65536^70272
70330^72792
72889^73444
73579^76708
76825^78005
a
Exon 13 cDNA covers only the translated and termination codons; exon 13 gDNA extends 30 to include polyadenylation signals.
PAH polymorphisms are lodged for the study of
modern human populations [Kidd et al., 2000].
Haplotypes
Because two RFLP sites (EcoRI and EcoRV) have
yet to yield to PCR, the use of mini-haplotypes (STR
and VNTR alleles only) has been recommended
[Zschocke et al., 1995]. A more complex and
informative new set of haplotype configurations has
also been developed [Zschocke and Hoffmann, 1999].
The polymorphic PAH haplotypes are useful because
they facilitate the population genetics of PAH. They
also help to delineate identity, either by descent or by
state, of particular alleles (e.g., R408W [John et al.,
1990; Byck et al., 1994]). Haplotype markers have
also contributed to the discovery of large deletions in
the PAH gene [Zschocke et al., 1999; Gable et al.,
2003].
MutationTypes
Pathogenic PAH alleles, as predicted from the DNA
sequence, comprise point mutations causing missense
in translation (63%), most of which are likely to affect
protein folding and assembly leading to secondary
effects on enzyme function (see In Vitro Expression
Analysis, below). Other PAH mutation types include:
small deletions, 13%; confirmed splice, 11%; silent
(putative), 7%; nonsense, 5%; small insertions, 1%;
large deletions are rare.
Some PAH point mutations, previously classified as
missense or silent, generate alternative splice sequences (see Expectations Confounded section,
below). A web site (http://exon.cshl.edu/ESE/) provides tools to assist in the recognition of new splice
donor or acceptor nucleotide sequences generated by
such point mutations.
Frequencies
The absolute aggregate frequency of pathogenic
PAH alleles rarely exceeds 0.01 in human populations
and only rarely does the absolute frequency of any
particular allele exceed 0.01 (see Table in Zschocke
[2003] for source data). The relative frequencies of
PAH alleles follow a recognizable pattern for human
locus-specific mutations [Weiss, 1996]. Regardless of
the number of pathogenic alleles at the locus, only a
few (o10) represent the major portion (75%) of those
in the population. The majority of probands (~75%)
have compound mutant PAH genotypes.
Population Associations
The history of human populations contains the
history of their alleles and vice versa. PAH alleles have
contributed to an understanding of the genetic
configurations typical of European populations
[Cavalli-Sforza and Piazza, 1993], for example:
Germany [Zschocke and Hoffmann, 1999]; Denmark
[Guldberg et al., 1993a]; Sicily [Guldberg et al.,
1993b]; and, possibly, for their ancient origins in some
populations [Zschocke et al., 1997]. The global
European profiles of those PAH mutations (n=29)
that exceed 3% individual relative frequency in at
least two different populations is now known
[Zschocke, 2003]. The effects of range expansion,
genetic drift, and founders on genetic structure of
overseas populations is illustrated in detail by alleles at
the PAH locus, for example: Iceland [Guldberg et al.,
1997]; Quebec [Carter et al., 1998; Scriver, 2001];
and the USA [Guldberg et al., 1996].
Identity by State or by Descent?
Several PAH mutations, identical by state in their
nucleotide change, occur on two or more different
haplotype backgrounds. For example R408W, the
most prevalent pathogenic PAH allele, occurs prominently on haplotypes 1 and 2 in European-derived
populations [John et al., 1990; Treacy et al., 1993;
Eisensmith et al., 1995; Tighe et al., 2003]. Because a
CpG dinucleotide is involved, R408W has the
potential to be a recurrent mutation and identical
only by state on haplotypes 1 and 2 [Byck et al., 1994;
Tighe et al., 2003]. R408W also appears on haplotypes
PAHDB KNOWLEDGEBASE
of the filter paper screen blood spots [Altland et al.,
1982], that the PAH locus is unusually mutable or will
serve as a sentinel for human genome mutability.
PAH c.1066-11 g->a (IVS10nt-11)*: II
%
2
Haplotype
10
* mutation site
-
+
-
+
+
-
*
-
-
IBD
+
+
+
-
*
-
-
IBD
+
+
+
-
*
-
-
IBD
•
30-90
6
+
-
v.rare
39
-
+
•
25
+
-
-
+
36
34
+
-
+
-
2 (Italy) 9
+
+
-
+
+
-
*
-
-
(IBD)
(Spanish Gypsies)
rare
+
+
-
*
*
-
+
337
(IBD)
?
A scheme to explain the presence of a major PAH
mutation (c.1066^11g4a IVS10nt-11, PKU-causing) on six
di¡erent haplotypes. Relative frequencies of the di¡erent mutant haplotypes are shown in the left hand column. With the
possible exception of a recurrent mutation, the putative mechanisms are indicated on haplotype 9. All other forms are
identical by descent from the haplotype 6 con¢guration. Intragenic recombination leads to the mutation on H39 or H10
and RFLP site mutations put it on H34 or H36. Figure from
Scriver and Kaufman [2001], with permission.
FIGURE 1.
other than 1 and 2. In these instances, the R408W
allele could be identical by descent because mutation
at a polymorphic PAH marker is the more likely
explanation for the variant haplotype association.
Eleven different PAH mutations in Germany, each
occurring on more than one haplotype, have been
examined for evidence of identity by state, recurrence,
or an alternative explanation [Zschocke and Hoffmann, 1999]. Figure 1 illustrates possible genealogies
(mutation lineages) for the IVS10nt-11 mutation
(c.1066–11g>a) which is prevalent in southern
European populations.
Some pathogenic PAH mutations, including
R408W and IVS10nt-11, occur at quite high frequencies in particular populations. Selective advantage in
the heterozygote is one possible explanation and the
case for this hypothesis is being argued elsewhere in
this issue [Krawczak and Zschocke, 2003]. Recurrent
mutation and founder effect/genetic drift are alternative explanations.
De Novo Alleles
These are rare occurrences in the PKU and related
phenotypes. M1I and IVS3nt-6 have each been
reported once as de novo alleles, once from Norway
[Eiken et al., 1996] and E76G has been reported once
from Taiwan [Chen et al., 2002]. Another occurrence
(unnamed) is reported once from Southern Germany
[Aulehla-Scholz and Heilbronner, 2003]. The apparent rarity of de novo alleles could be a feature of
ascertainment. Such alleles will not be recognized if
the parental alleles have not also been analyzed, a
practice that is not uniform. Accordingly, one cannot
say that these few de novo alleles have revealed mutation hotspots. Nor does it seem, from the monitoring
Insertion/Deletion Mutations
Small deletions or insertions occur in the PAH
gene. Large deletions affecting coding regions are rare.
Some deletions involve a single exon. More frequently, multiple exons are affected. (For a literature
review, see Gable et al. [2003]). The relative
frequency of large PAH gene deletions is apparently
low (o1%) but this may reflect poor ascertainment.
Most of the large recognized deletions were discovered
when a disease-causing point mutation could not be
identified by current methods of mutation analysis or
when a haplotype configuration lacked one or more of
its polymorphic alleles. Large insertions or gene
duplications have not yet been reported.
50 UTR Alleles
Polymorphic alleles occur in the 50 UTR of the PAH
gene that are without apparent phenotypic effect
[Svensson et al., 1993]. A large deletion (3767bp,
nt-4173 to –407 in the Konecki sequence) has been
identified [Chen et al., 2002]. The deletion removes a
liver-specific enhancer element harboring a major
hepatocyte nuclear factor-1-binding site. It severely
impairs PAH gene transcriptional activity conferring
functional hemizygosity on the affected proband.
Expectations Confounded
Several PAH point mutations are mentioned here
to show how they challenged easy interpretation.
Experimental evidence defied the sequence-based
predictions that initially categorized them as either
disease-causing or silent. These mutations highlight
the relevance of using a combination of RNA
processing analysis, in vitro expression analysis,
molecular modeling, and other means [Terp et al.,
2002], to identify the mechanism of mutation effect.
The prevalent Y204C (c.611A>G) allele in exon 6
is associated with PKU, yet when the protein is
expressed conventionally in vitro, the mutation
appears non-pathogenic. RT-PCR analysis of illegitimate transcripts, however, revealed the creation of
a novel splice site by this allele causing loss of
protein function. The mutation was therefore given
a new trivial name: E6nt–96A>G [Ellingsen et al.,
1997].
Several alleles, including Q304Q (c.912G>A)
[Guldberg et al., 1996], T323T (c.969A>G),
K398K (c.1194A>G) [Zschocke and Hoffmann,
1999], and V399V (c.1197A>T) [Chao et al.,
2001], initially called silent, are either shown to, or
are likely to, promote exon skipping. In each case
the adjacent nucleotide sequence becomes an
important context to help explain the mutation
338
SCRIVER ET AL.
effect [Ellingsen et al., 1999]. Prediction of such
effects from consensus sequence algorithms (viz
http://exon.chsl.edu/ESE/) is still imperfect and
experimental studies on RNA processing are still
necessary. A growing awareness [Cartegni et al.,
2002] that exonic alleles in human genes can affect
pre-mRNA splicing clearly encompasses the PAH
gene where P281L, R408Q, G272X, and Y356X
(and perhaps many other apparent missense,
nonsense, and silent mutations) are associated
with exon skipping [Ellingsen et al., 1999].
The K274E (c.820A>G) allele [Gjetting et al.,
2001a] initially appeared to be a cause of PKU.
However, in vitro expression analysis showed
normal enzyme function. Further genotype analysis
at the PAH locus, followed by in vitro expression,
revealed that a rare pathogenic allele (I318T,
c.953T>C) was also being transmitted in cis with
K274E. The latter is a polymorphism (frequency
~0.04) in the source population (African–American). PAHdb lists variant PAH alleles paired in cis
(see cis Mutation Table in PAHdb). Three of those
pairs, submitted earlier by C. Aulehla-Scholz
directly to PAHdb, are now published [AulehlaScholz and Heilbronner, 2003].
BH4-responsive PAH Alleles
Following the discovery of patients with a variant
form of hyperphenylalaninemia responsive to pharmacological doses of tetrahydrobiopterin [Kure et al.,
1999], at least 21 corresponding primary mutations in
the PAH gene have been proposed as BH4-responsive.
This new phenotype is listed in the Mutation Tables in
PAHdb and illustrated on a Curators’ page showing
where the putative alleles map on the protein.
Detailed molecular explanations for BH4-responsiveness have been proposed [Erlandsen and Stevens,
2001].
MOLECULAR MODELING
Co-Curators: Heidi Erlandsen and Ray Stevens
PAHdb hosts a module (http://www.pahdb.mcgill.
ca/molecular.html) for molecular modeling of the
PAH protein. It shows the derived three-dimensional
structure of the enzyme (PAH) (Fig. 2) and predicts
the structural effects of some currently known PAH
mutations.
The human enzyme exists in a pH-dependent
equilibrium of homo-tetramers and homo-dimers
[Martinez et al., 1995] and like the two other human
aromatic amino acid hydroxylases (tyrosine hydroxylase and tryptophan hydroxylase), it has three
domains: an N-terminal regulatory domain (residues
1–142); a catalytic domain (residues 143–410); and a
C-terminal tetramerization domain (residues 411–
452). X-ray crystallography has been used previously
to determine the 3D structures of truncated forms of
phenylalanine hydroxylase [Erlandsen et al., 1997;
Fusetti et al., 1998; Kobe et al., 1999], but because it
is difficult to crystallize the full-length PAH subunit,
no primary full-length tetrameric PAH structure has
yet been analyzed. However, two truncated forms
have been characterized. These include a dimeric
form containing regulatory and catalytic domains
[Kobe et al., 1999] and a tetrameric form containing
catalytic and tetramerization domains [Fusetti et al.,
1998]. From these two structures, and from a higherresolution dimeric double-truncated form of the PAH
enzyme [Erlandsen et al., 1997], it has been possible
to derive a composite full-length structural model
[Erlandsen and Stevens, 1999] (Fig. 2).
The regulatory domain of PAH contains an a–b
sandwich with an interlocking double bab motif. The
N-terminal autoregulatory sequence (ARS, residues
19–33) extends over the active site in the catalytic
domain [Erlandsen et al., 1997]. The tetramerization
domain contains two b-strands forming a b-ribbon
and a 40 Å long a-helix. The four a-helices (one from
each monomer) pack into a tight anti-parallel coiledcoil motif in the center of the tetramer structure
[Fusetti et al., 1998]. The region containing the
catalytic domain has a basket-like arrangement with a
total of 13 a-helices and 8 b-strands. The active site,
located in the center of the catalytic domain, is a
pocket that is 13 Å deep and 10 Å wide [Erlandsen
et al., 1997]. Adjacent to the active site is a channel
that is 16 Å long and 8 Å wide by which substrate may
access the active site [Anderson and Flatmark, 2001].
As isolated, PAH crystals contain an active site Fe(III)
iron atom [Martinez et al., 1991; Erlandsen et al.,
1997] located 10 Å below the surface of the protein
on the floor of the active site at the intersection of the
channel and the active site pocket. The Fe(III) atom
is coordinated to H 285, H 290, and one oxygen atom
in E 330. The cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and the L-Phenylalanine substrate bind close to the iron at the active site
[Erlandsen et al., 2000; Anderson and Flatmark,
2001; Anderson et al., 2002].
A set of 269 PAH missense mutations (see The
PAH Mutation Analysis Consortium Newsletter,
December 2001, online in PAHdb), 23 nonsense
mutations, and 10 silent mutations were used for
molecular interpretation. Most missense mutations
map onto the region between and including exon 5
(PAH residue 148) and exon 12 (PAH residue 438):
57 of these mutations are located in the regulatory
domain sequence (residues 1–142); 231 mutations
target the catalytic domain (residues 143-410); and 14
are located in the tetramerization domain sequence
(residues 411–452) (Fig. 3). A summary of genotype/
phenotype/structural interpretations for these mutations has been published in Erlandsen and Stevens
[1999].
PAHDB KNOWLEDGEBASE
339
FIGURE 2. Three views of the tetrameric form of the composite model of phenylalanine hydroxylase. The regulatory domain
(residues 19-142) is colored orange, the catalytic domain (residues 143^410) is colored gray, and the tetramerization domain
is colored blue.The active site iron is shown as a yellow sphere. A: Front view; B: side view, seen in the plane of the paper along
the x-axis as compared to A; C: side view, seen in the plane of the paper along y-axis as compared to A. Figure from Erlandsen
and Stevens [2001], with permission.
Ca-trace of one monomer of the full-length model of phenylalanine hydroxylase.The trace is colored yellow for residues not associated with any PKU mutations, and the active site iron is shown for reference as a green sphere.The region colored in red consists of residues that have PKU mutations associated with them. The regions colored blue have residues that
show a high predicted frequency of mutation, and the regions colored green have residues that show a high calculated frequency of mutation (PKU database).The four residues colored purple are the residues with both the calculated and predicted
frequency of mutation being high (Arg158, Arg252, Arg261, and Arg408). Figure from Erlandsen and Stevens [1999], with
permission.
FIGURE 3.
The structural information for PAH has helped to
formulate predictions [Erlandsen and Stevens, 1999;
Kobe et al., 1999] about the likely effects of yet
unclassified or newly discovered missense mutations.
Furthermore, with the aid of recent crystal structure
analysis using cofactor and substrate analogs bound at
340
SCRIVER ET AL.
the active site [Erlandsen et al., 2000; Anderson and
Flatmark, 2001; Anderson et al., 2002], the newly
discovered BH4-responsive PKU/HPA genotypes can
be mapped onto the PAH structure to seek molecular
explanations for their BH4-dependent clinical responses [Erlandsen and Stevens, 2001].
lationships [Okano et al., 1991] and, in broad terms,
such correlations exist [Kayaalp et al., 1997; Guldberg
et al., 1998; Desviat et al., 1999]. There are, however,
significant exceptions. Thus, IVE data are not always
robust predictors of the in vivo phenotype and there
are some partial explanations for the inconsistencies
[Scriver and Waters, 1999].
IN VITRO EXPRESSION ANALYSIS
Co-Curator: Paula J. Waters
PAHdb contains data on in vitro expression analysis
(IVE) of 81 different naturally occurring human
mutations (227 individual records, see Table in
PAHdb under IVE-Human). It also contains information on 12 artificially created mutations in the human
nucleotide sequence (13 individual records, IVEArtificial) and on 41 artificially created mutations in
the rat Pah gene (42 records, IVE-Rat). The search
option on the Home Page leads to a search page
containing all the relevant links. IVE-Human is listed
under User-defined queries. IVE-Rat and IVE-Artificial
are listed under pre-queried data. Another link on this
page leads to IVE-Commentary that describes how to
use and interpret the IVE Human table.
There are three reasons to put IVE data on PAHdb:
1) to provide evidence that the mutation alters
protein function; 2) to document the severity of
mutation effect; and 3) to describe the mechanism of
effect. Only 81 different human PAH mutations, from
the more than 400 known mutations, have yet been
studied by IVE. Two reviews [Waters et al., 1998;
Waters, 2003] discuss the advantages and disadvantages of the different systems that are available for
mutation analysis by IVE and highlight key overall
findings from IVE. Prediction of mutation effects
using IVE data is complemented by the analysis of
PAH structure by both molecular modeling [Erlandsen and Stevens, 1999] and spectroscopy [Teigen
et al., 1999].
Most PAH mutations are predicted from the DNA
sequence to be missense alleles. The corresponding
amino acid substitutions frequently affect folding and
assembly of the protein, leading to decreased cellular
PAH activity as a result of the enzyme’s degradation.
Only a minority of the disease-causing mutations
directly affect the kinetic properties of the enzyme
(e.g., D143G [Knappskog et al., 1996]). Some
mutations appear to have combined effects on both
sutrate or cofactor binding [Leandro et al., 2000;
Gjetting et al., 2001b] and on protein folding [Waters
et al., 2000; Gamez et al., 2000].
The BH4-responsive PAH mutations await detailed
analysis by IVE to determine whether they are kinetic
mutants affecting BH4 binding or alleles that affect
protein folding and assembly where BH4 at high
concentration might act as a chemical chaperone.
Expression analysis of PAH mutations also has the
potential to shed light on genotype/phenotype re-
CLINICAL RESOURCES
Co-Curator: Shannon Ryan
PAHdb provides visitors with a ‘‘Phenylketonuria
Resource Booklet for Families’’ and an overview of the
treatment for PKU. It is intended only as a
supplementary resource for patients, family members,
and other interested persons who wish to become
familiar with PKU and the associated vocabulary.
PAHdb does not replace necessary patient–care-giver
relationships in optimal treatment of PKU. PAHdb
also provides links to other sites containing information on PKU (e.g., www.espku.org). Access to
resources in languages other than English is also
provided (e.g., French, German, Spanish). Content of
these sites varies. Some sites give details of everyday
life with PKU and are intended for the lay person.
Others are intended for the health-care professional.
GeneClinics/GeneTests, a peer-reviewed internet resource (www.geneclinics.org), contains a document
authored by the PAHdb co-curators. The latest
version includes information, for example, on BH4responsive PAH phenotypes and on current diagnostic
and molecular tests.
MOUSE MODELS
Co-Curators: Christineh N. Sarkissian and David
McDonald
PAHdb describes mouse lines carrying mutations
in the orthologous Pah gene (GenBank accession
# X51942, cDNA) and their use in the development
of a potential new therapy for PKU with phenylalanine ammonia lyase enzyme (EC 4.3.1.5) [Shedlovsky
et al., 1993; Sarkissian et al., 2000; Sarkissian et al.,
1999]. Mouse and human PAH enzymes show
homology and evolutionary divergence [Ledley et al.,
1990]. Conserved amino acid residues (92% of the
sequence) are indicated in the database. The PAHdb
mouse page contains a list of published articles, mouse
links, descriptions of ongoing research, and animal
husbandry.
Three mouse Pah mutations have been artificially
created: Pahenu1 [McDonald et al., 1990]; Pahenu2; and
Pahenu3 [Shedlovsky et al., 1993]. The Pahenu3 allele is
now historic. With the currently existing mutant
alleles, mouse phenotypes can be produced that
range in severity from mild HPA (Pahenu1 homozygosity) to a more pronounced HPA (Pahenu1/Pahenu2
PAHDB KNOWLEDGEBASE
compound heterozygosity) to classical PKU (Pahenu2
homozygosity).
The Pahenu1 allele is a c.364T>C missense mutation (V106A) in exon 3. The V106 residue is
conserved among mammalian species [McDonald
and Charlton, 1997]. The allele has no human
counterpart. The mutation probably affects protein
folding, stability, and assembly of the enzyme, as is the
case with neighboring human missense alleles
(A104D, S110L) in the regulatory domain of the
PAH subunit [Erlandsen and Stevens, 1999]. The
Pahenu2 allele (c.835T>C) is a missense mutation
(F263S) in exon 7 [McDonald and Charlton, 1997].
This allele has a human counterpart (F263L). Residue
F263 is located close to the Fe atom in the active site
of PAH enzyme and a substitution here is most likely
detrimental to activity (ligand binding) as well as
proper folding of the protein [Anderson et al., 2002].
The substitution is also directly adjacent to amino
acids involved in pterin-binding on the human
enzyme [Erlandsen and Stevens, 1999; Erlandsen
et al., 2000]. Although corresponding phenylalanine
residues are affected in human and mouse, the cause
of HPA would be different because of the different
(serine and leucine) substitutions. Either substitution
results in a severe clinical phenotype in the particular
host, reaffirming the importance of this residue in the
structure/function of the PAH protein.
The Pahenu1/Pahenu2 compound heterozygote strain
was developed [Sarkissian et al., 2000] as a model of
most human PKU-causing genotypes which are
heteroallelic compounds [Kayaalp et al., 1997]. The
metabolic phenotype in this mouse is intermediate to
that of Pahenu1 and Pahenu2 homozygous mice [Sarkissian et al., 2000]. Enzyme activity in the heteroallelic
mouse is less than the predicted average activity of
Pahenu1 and Pahenu2 homozygotes, suggesting that
negative complementation may exist in the heteroallelic tetrameric enzyme.
The archived Pahenu3 mutation (c.1126+2t>g
transversion at the exon 11-intron 11 boundary)
activates two cryptic splice donor sites and has a
complicated effect [Haefele et al., 2001]. There is no
known corresponding human allele.
As described in PAHdb, these PKU mouse models
have thus far proven their utility providing, for
example, insights into: teratology among the progeny
of mutant females; learning and behavior; brain
metabolic pools; short- and long-term effects of
neurochemical alterations; cerebral protein synthesis;
brain myelination and CNS glial cell plasticity;
cognitive defects; and experimental approaches to
treatment of PKU.
DISCUSSION
The Curators of PAHdb welcome suggestions for
improvements and notifications of errors or omissions
341
in its content. The current version of PAHdb has been
transferred onto a COMPAQ server running Red Hat
Linux version 7.3. This server also hosts four other
locus-specific mutation databases and the catalogue
for the Repository of Mutant Human Cell Strains. It
also carries the WayStation [Teebi et al., 2001] for the
project to create a repository of all human genomic
allelic variation, notably its pathogenic alleles.
Data can be submitted to PAHdb by registered
users. The submission script, until recently difficult to
use, has been improved. The web interface, running
on Apache webserver, and the PAHdb software are
also being improved.
PAHdb can accommodate population-related mutation reports and the data from corresponding articles
in this issue of the Journal will appear in PAHdb.
Data in the two reports documenting BH4-responsive
PAH alleles in this issue will also be accommodated.
Their data will be linked to the BH4 database
(www.bh4.org).
Large deletions are rare in the PAH gene (see Gable
et al. [2003] in this issue of the Journal). Whether an
allele actually affects phenotype, or whether the
phenotype can be predicted from the genotype,
remains an important general inquiry—no less so at
the PAH locus. The present guidelines [Cotton and
Scriver, 1998], useful as they are, are no longer
sufficient. We illustrate the problem here with several
examples. Because in vitro expression analysis can
provide data useful to this inquiry, PAHdb devotes a
large module to them. However, such data are not
available for the majority of PAH alleles. Accordingly,
molecular modeling is another feature of PAHdb
because it may provide insights on the likelihood of
the mutation having an effect on the enzyme and thus
on phenotype. In this initiative, PAHdb serves as a
prototype for other Mendelian diseases. Data in
PAHdb were used in another approach to the inquiry
about causation [Terp et al., 2002], namely, an
assessment of the functional significance of an amino
acid substitution as it might be understood from its
biophysical properties.
The mechanisms by which mutations affect cellular
function have always been of fundamental interest.
PAHdb acknowledges investigations which reveal that
many missense alleles seem to produce their effect
through misfolding, causing instability and aggregation
of the PAH monomer and/or aberrant oligomerisation
(see Waters [2003] in this issue of the Journal). If
misfolding is a prevalent pathogenic mechanism in
Mendelian disease, might there be diffusable chemical
chaperones to play therapeutic roles? Is tetrahydrobiopterin playing such a role for some of BH4responsive PAH alleles?
PKU is a relatively prevalent variant Mendelian
phenotype. Only a few PAH alleles achieve high
relative frequencies in distinct populations and the
mechanisms underlying prevalence and non-random
342
SCRIVER ET AL.
distribution of PAH alleles are of abiding interest
[Scriver et al., 1996]. The hypothesis of selective
advantage in the heterozygote (over-dominant selection) has a certain appeal [Krawczak and Zschocke,
2003]. The debate for and against selective advantage
continues to be aired and is not yet closed.
ACKNOWLEDGEMENTS
The March of Dimes, HUGO, and the Human
Genome Variation Society have been supportive
forces. The present curators thank colleagues active
at earlier stages of PAHdb: David Côté, Ken Hechtman, Liem Hoang, Jaroslav Novak, Piotr Nowacki,
Saeed Teebi, and Ziggy Zeng. The authors thank
Johannes Zschocke and Saeed Teebi for their comments on the manuscript for this article.
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