Group Title: BMC Genetics
Title: Refining associations between TAS2R38 diplotypes and the 6-n-propylthiouracil (PROP) taste test: findings from the Avon Longitudinal Study of Parents and Children
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Title: Refining associations between TAS2R38 diplotypes and the 6-n-propylthiouracil (PROP) taste test: findings from the Avon Longitudinal Study of Parents and Children
Physical Description: Book
Language: English
Creator: Timpson, Nicholas
Heron, Jon
Day, Ian
Ring, Susan
Bartoshuk, Linda
Horwood, Jeremy
Emmett, Pauline
Davey-Smith, George
Publisher: BMC Genetics
Publication Date: 2007
 Notes
Abstract: BACKGROUND:Previous investigations have highlighted the importance of genetic variation in the determination of bitter tasting ability, however have left unaddressed questions as to within group variation in tasting ability or the possibility of genetic prescription of intermediate tasting ability. Our aim was to examine the relationships between bitter tasting ability and variation at the TAS2R38 locus and to assess the role of psychosocial factors in explaining residual, within group, variation in tasting ability.RESULTS:In a large sample of children from the Avon Longitudinal Study of Parents and Children, we confirmed an association between bitter compound tasting ability and TAS2R38 variation and found evidence of a genetic association with intermediate tasting ability. Antisocial behaviour, social class and depression showed no consistent relationship with the distribution of taste test scores.CONCLUSION:Factors which could influence a child's chosen taste score, extra to taste receptor variation, appeared not to show relationships with test score. Observed spread in the distribution of the taste test scores within hypothesised taster groups, is likely to be, or at least in part, due to physiological differentiation regulated by other genetic contributors. Results confirm relationships between genetic variation and bitter compound tasting ability in a large sample, and suggest that TAS2R38 variation may also be associated with intermediate tasting ability.
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Research article
Refining associations between TAS2R38 diplotypes and the
6-n-propylthiouracil (PROP) taste test: findings from the Avon
Longitudinal Study of Parents and Children
Nicholas J Timpson*1,4, Jon Heron2, Ian NM Day', Susan M Ring',
Linda M Bartoshuk3, Jeremy Horwood', Pauline Emmett2 and George Davey-
Smith'

Address: 'Department of Social Medicine, Bristol University, Bristol, UK, 2ALSPAC, Department of Social Medicine, Bristol University, Bristol, UK,
3University of Florida, Dept. of Community Dentistry and Behavioral Science, Gainesvile, USA and 4The Wellcome Trust Centre for Human
Genetics, Oxford University, Oxford, UK
Email: Nicholas J Timpson* epnjt@well.ox.ac.uk; Jon Heron Jon.Heron@bristol.ac.uk; Ian NM Day Ian.Day@bristol.ac.uk;
Susan M Ring S.M.Ring@bristol.ac.uk; Linda M Bartoshuk lbartoshuk@dental.ufl.edu; Jeremy Horwood J.Horwood@bristol.ac.uk;
Pauline Emmett P.M.Emmett@bristol.ac.uk; George Davey-Smith george.davey-smith@bris.ac.uk
* Corresponding author


Published: 28 July 2007
BMC Genetics 2007, 8:51 doi: 10.1186/1471-2156-8-51


Received: I February 2007
Accepted: 28 July 2007


This article is available from: http://www.biomedcentral.com/1471-2156/8/51
2007 Timpson et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Previous investigations have highlighted the importance of genetic variation in the
determination of bitter tasting ability, however have left unaddressed questions as to within group
variation in tasting ability or the possibility of genetic prescription of intermediate tasting ability.
Our aim was to examine the relationships between bitter tasting ability and variation at the
TAS2R38 locus and to assess the role of psychosocial factors in explaining residual, within group,
variation in tasting ability.
Results: In a large sample of children from the Avon Longitudinal Study of Parents and Children,
we confirmed an association between bitter compound tasting ability and TAS2R38 variation and
found evidence of a genetic association with intermediate tasting ability. Antisocial behaviour, social
class and depression showed no consistent relationship with the distribution of taste test scores.
Conclusion: Factors which could influence a child's chosen taste score, extra to taste receptor
variation, appeared not to show relationships with test score. Observed spread in the distribution
of the taste test scores within hypothesised taster groups, is likely to be, or at least in part, due to
physiological differentiation regulated by other genetic contributors. Results confirm relationships
between genetic variation and bitter compound tasting ability in a large sample, and suggest that
TAS2R38 variation may also be associated with intermediate tasting ability.


Background
Variation in the ability to taste PTC, PROP and related
compounds has been recognized as one of the classical
markers of population genetics. Since the seminal find-


ings of Blakeslee and Fox [1,2], the distribution of PTC/
PROP tasting has been extensively analysed. This varia-
tion has been noted to vary from the extremes of "taste
blindness" (a lack of sensitivity, or non-taster of PTC/


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PROP), to apparent "super tasting" (an extra sensitive
reaction to the bitterness of PTC/PROP) and has shown
marked variation in the distribution of such traits across
populations (taste blindness ranging from 3% in West
Africa, to 6-23% in China, 40% in India and around 30%
in North American Caucasian populations [3,4]).

Conventional assessment of bitter tasting ability has been
by PTC (phenylthiocarbamide)/PROP taste challenge and
response assessment. It was found that all bitter com-
pounds containing the thiocyanate (N-C = S) moiety elicit
bimodal patterns of response [5]. In addition to this it has
been shown that PTC taste responses are strongly corre-
lated with all of these compounds [6], as is the case for
PROP [3].

Initial genetic analyses presented indications of bitter
compound tasting ability as a complex genetic trait, but
provided little specific evidence as to possible causal, or
robustly associated genetic components [4,7-11]. How-
ever, specific positional cloning efforts have since identi-
fied a region of chromosome 7 (in the TAS2R38 gene),
which has shown patterns of haplotypic association
which are associated with specific measurements of bitter
tasting ability [12]. This has provided direct evidence of a
physiological link between genetic variation and tasting
ability and has prompted hypotheses as to the possible
relevance of bitter tasting for ultimate diet choice and
related health [13-15]. We note that the human genome
project has now identified more than twenty genes for bit-
ter taste [16], likely to also associate with health effects.

Existing studies assessing relationships between TAS2R38
haplotype variation and specific bitter tasting ability have
limited samples sizes and have largely been restricted to
the assessment of relationships between genetic variation
at the TAS2R38 locus and the binary measure "taster/non-
taster". Whilst there is evidence as to the existence of this
association, there has not to date, been large-scale replica-
tion or refinement of these observations. Furthermore,
whilst diplotypes of the TAS2R38 gene locus are thought
to prescribe one's ability to detect the bitter compounds
PROP and PTC (PROP representing a suboptimal, but
effective ligand for the TAS2R38 receptor), little its known
about the effects/association of haplotypes at intermedi-
ate/lower frequency or the cause of the distribution of bit-
ter tasting ability within haplotype defined groups.

Studies into the psychophysiology of PROP detection
have suggested that in addition to tasters and non-tasters,
there also "supertasters" of bitter compounds. Super-
tasters of PROP are believed to be tasters who also have
unusual tongue anatomy: a high density of fungiform
papillae, the structures that house taste buds. Supertasting
is not linked to variation et the TAS2R38 locus [15].


The present analyses are focused on "taster" or "non-
taster" as the only previous grouping of note related to
TAS2R38 locus variation. It is therefore not the existence
of super tasting that is in question, more the refinement of
the currently crude relationships recorded between
genetic variation at the TAS2R38 locus and the ability to
taste specific bitter compounds. It is predicted, that with a
study of this magnitude, the observation of the rare AA
haplotype at the TAS2R38 locus within the ALSPAC
cohort will allow investigation of its potential link to
intermediate tasting ability [17].

Such an investigation, together with the suggestion of sub-
jectivity in the rating of PROP taste test scores [18], also
raises the question as to the determination of the distribu-
tion of PROP taste test scores within genetically prescribed
tasting groups. Whilst physiological factors such as the
number of taste buds may be instrumental in the determi-
nation of this variation [19], it has been suggested that
psychological/behavioural factors play some role [20].

Here we analyse PROP bitter taste test scores collected in
the ALSPAC child cohort and their relation to known var-
iation at the TAS2R38 locus. Also, the availability of a
series of psychological/behavioural measures reflecting
factors thought to influence the ultimate response of a
child to this taste challenge, has allowed the investigation
of residual variation in bitter tasting ability. As such, we
aim to comment on the possible origins of the distribu-
tion of taste scores found within the prior defined haplo-
typic taste groups.

Results
Of the 13988 cases/pregnancies in the ALSPAC cohort,
samples from n = 9765 children and n = 8736 mothers
formed a working population of n = 12234 unrelated
individuals (all genotyped children and unrelated moth-
ers where children were not available, comprising 83% of
the cohort) available for haplotypic reconstruction. Of
this group, n = 8 had one missing genotype and n = 174
had missing data for two genotypes. Genotyping of the
TAS2R38 variants P49A and A262V yielded minor allele
frequencies of 0.40 and 0.45 respectively, Table 2. From
these genotyping results, haplotypic reconstruction
resolved the common haplotypes AV and PA at frequen-
cies of 0.55 (SE 0.0004) and 0.40(SE 0.0004) within this
population, Table 3. The rarer haplotypes AA and PV were
observed at frequencies 0.05 (SE 0.0003) and 0.001(SE
0.00008).

n = 4795 children completed a PROP taste test analysis
and were carried forward into further analysis. Figure 1
shows the distribution of PROP taste test scores in the
ALSPAC population. From the 168 children who com-
pleted the PROP taste test twice, the correlation of scores


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Table 2: Allele frequencies at variant loci TAS2R38 P49A and A262V in the ALSPAC sample

Nucleotide Amino Acid Allele Protein code Frequency in current study p*


49 G
C
262 T


Ala
Pro
Val
Ala


Comparative frequencies (Kim et al 2003)


0.60 (n = 10977)
0.40 (n = 7467)
0.55 (n = 10147)
0.45 (n = 8405)


* indicates p value for test of Hardy Weinberg Equilibrium (exact test [37])


between both tests was 0.62 (r2 = 0.39). This relationship
yielded a regression coefficient of 0.61 (0.49, 0.72). p =<
0.001. Figure 2 shows the distribution of differences that
exist between taste test scores for the 168 children over
these two time points. The mean time between the two
measurements for this group was 33.4 days.

The combination of PROP taste test data with haplotypic
information yielded a test population of n = 4178 chil-
dren. Of note, in analysis of PROP taste test scores by PC
haplotype, none of the reconstructed haplotypic values
fell below the cut off posterior reconstruction threshold
probability of 0.8.

The distribution of PROP taste test scores by haplotypic
combination (diplotype) is shown in Table 4. PROP taste
test score was observed to be lowest for the haplotypically
predicted non-tasters (AV/AV median 3.72, IQR [5.2]),
whilst predicted tasters had high PROP taste test scores
with the highest being for homozygous taster haplotypes
(PA/PA median 8.07, IQR [2.5]). Of note, whilst the car-
riage of the PA haplotype appeared to confer tasting abil-
ity throughout, the presence of the rarer heterozygote AV/
AA showed evidence for intermediate tasting ability.

Pairwise differences in mean PROP taste test score showed
expected patterns according to diplotype comparison
(Table 5). The largest difference in tasting response was
seen between diplotypes AV/AV (non-taster) and PA/PA
(homozygote taster), (4.35 95% CI [4.13, 4.56]). In con-
trast, the smallest difference between diplotypes AV/PA
(heterozygote taster) and AA/PA (heterozygote taster,
(0.10 95% CI [-0.26, 0.46]). The presence of the rarer hap-


lotype AA (when in comparison to either taster or non-
taster diplotypes) exhibited moderate pairwise differences
(e.g. AV/AA versus AV/AV pairwise difference of 1.65 95%
CI [1.21, 2.10] and AV/AA versus PA/PA pairwise differ-
ence of 2.70, 95% CI [2.26, 3.13]).

From these observations, the generation of predicted tast-
ing ability groups was permitted and allowed the analysis
of the distribution taste scores between them, Figure 3.
Clear differences in the distribution of PROP taste test
scores were observed between the haplotypically pre-
scribed tasting groups, as confirmed by a rank sum test for
differences between these groups (z = -35.302, p =<
0.001). Furthermore, the creation of a third group defined
by the carriage of the rare haplotype AA (Figure 4), whilst
only observed in relatively small numbers (n = 207
excluding those with the common PA taster haplotype on
the basis of apparently dominant effects) revealed pat-
terns indicative of intermediate tasting ability when com-
pared to the main group distributions, this was again
confirmed by a rank sum test for differences between
these groups (z = -7.132, p =< 0.001) (Figure 2).

Analysis of quartiles of taste test score with respect to their
relationship with factors which could potentially explain
the spread of PROP taste test results within the haplotype
defined test groupings seen in Figure 3., showed no strong
evidence of trend effect. When assessed in both homozy-
gote taster predicted individuals and homozygote non-
taster predicted individuals (in efforts to avoid noise from
differing genetic background), social class, depression and
antisocial behaviour (thought to be potentially influenc-
ing child's reporting of taste sensation despite physiologi-


Table 3: Haplotype frequencies at variant loci TAS2R38 P49A and A262V in the ALSPAC sample


Haplotype P49A A262V


Observed Frequency

0.546
0.047
0.406


Comparative frequencies (Kim et al 2003)

0.47

0.49


Haplotype frequencies (common haplotypes representing -99% of all variation) are derived from a sample base n = 12234 (derived from n = 9399
children, n = 2661 mothers where child has no genotype data and 174 missing data points). Haplotypes for P49A/A262V genotypes obey Hardy
Weinberg Equilibrium (p = 0.8, chitesti [38]). The rare haplotype PV was observed in the ALSPAC population at a frequency of 0.001.



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et al [12], the common PA haplotype predicted those able
to taste the bitter compound PROP when at least one copy
was carried. Alternatively, homozygotes of the other com-
mon haplotype, AV, (haplotypes AV and PA accounting
for >90% of 2N) exhibited clear patterns of taste insensi-
tivity to the bitter PROP compound.

Importantly, the predicted intermediate tasting ability of
those carrying the rare AA haplotype [17] (in cases exclud-
ing heterozygotes with one copy of the common and
"taster" defining PA haplotype) appears to be demon-
strated in the ALSPAC cohort.


0 2 4 6
Taste Test Score


Figure I
Distribution of PROP taste scores in the
cohort by frequency. Taste test score deriv
eral labelled magnitude scale.



cal response) appeared to show no consistent
with the spread of taste test results, tables 6

Discussion and conclusion
As a study containing data on n = 4178 PI
scores and concurrent TAS2R38 haplotype
the ALSPAC cohort is the largest study to d;
the question of the haplotypic association
ability. This is the first instance of the appl
gVAS to children in this manner and has sh
a consistent approach to the measurement o
ity. Resolution of common haplotypes
TAS2R38 locus revealed variants adhering to
berg Equilibrium and for which frequencies
those reported elsewhere [12,21].

Analysis of the PROP taste test scores in
cohort has replicated previous findings o
between variation at the TAS2R38 locus and
ter taste test challenge. As predicted by the fi


8 10 As yet unreported, we were also interested in the presence
and patterning of variation in taste test scores within the
genetically prescribed tasting groups for PTC/PROP. We
performed an investigation into the distribution of PROP
mALSPAC taste test scores within genetically homogeneous groups
d from a gen- of tasters (homozygote PA individuals) and non-tasters
(homozygote AV individuals). This revealed that three
select factors (depression, social class and antisocial
behaviour) thought to influence the response of children
t relationship to bitter taste challenge did not appear to be associated
and 7. with the variation in taste test score in these groups.

On the basis of current observations, it is suggested that
ROP taste test physiological differentiation, likely derived from further
s, analysis in genetic factors [8,9,11,22], may form the basis of varia-
ate to address tion in taste score within groups previously defined as
with tasting "tasters/nontasters" by TAS2R38 haplotypes. These fur-
ication of the their possible physiological factors include not only alter-
own this to be native taste receptor complexes, but possibly factors
)f tasting abil- previously associated with the existence of "super tasting"
across the ability [23-26]. Importantly, this notes that (i) residual
Hardy Wein- variation in this particular taste test score, after previously
were close to recognized haplotype groupings, is likely to be due to
other physiological mechanisms and (ii) that there may
be a degree of phenotypic continuity in this trait recog-
the ALSPAC nised classically as bimodal in character. In addition to
)f association this, we observed that in the overall population, the clas-
response bit- sification of all individuals into the broad categories
endings of Kim "taster/nontaster" may not be appropriate. Evidence from


Table 4: Median PROP taste test scores (on a scale of 0-10 with 10 being the most intense taste response) by diplotype in the ALSPAC
cohort


Predicted PROP tasting ability

Non-taster
(Intermediate)
Taster
Taster
Taster


Median PROP Taste Score

3.72
5.37
7.48
7.58
8.07


Common haplotypic combinations shown above account for >99% of the sample population included in analysis. IQR represents interquartile range.
Derived from a sample base of 4178 (i.e. all those remaining with PROP taste test scores post haplotypic exclusion).



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Diplotype

AV/AV
AV/AA
AV/PA
AA/PA
PA/PA


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Figure 2
Distribution of differences between multiple taste
test assessments in the ALSPAC cohort. Distribution
of differences observed between general visual analogue
scale scores for the description PROP taste test results
within 168 repeat tested children from the ALSPAC cohort.
Mean difference between two measures is 0.24(0.16, 0.64), p
= 0.25.


this study suggests that the carriage of particular haplo-
typic combinations is associated with intermediate bitter
compound tasting ability.

Methods
The Avon Longitudinal Study of Parents and Children
(ALSPAC) is a geographically based cohort that recruited
pregnant women residing in Avon with an expected deliv-
ery date between ist April 1991 and 31st December 1992.
14 541 pregnant women were initially enrolled, with 14
062 children born. This represents 80-90% of the eligible
population [27,28]. Of these children, 13 988 were alive
at 12 months. From this, two sampling frames were to be
adhered to (i) the maximal population sample for haplo-


typic reconstruction (including children with DNA sam-
ples and mothers with DNA samples where a child's DNA
sample was missing) and (ii) the analysis data set includ-
ing just children with at least one genotype and concur-
rent PROP taste test results. This formed a maximal
baseline population of n = 12371 (reduced to n = 12234
with the loss of one sib from twin pairs) for which, in
order to allow investigation, ethical approval was
obtained from the ALSPAC Law and Ethics Committee,
and local research ethics committees.

PROP test
Paper disks impregnated with PROP have been shown to
be a crude but rapid way to test responses to PROP in large
groups [19]. As part of a face-to-face clinic session held
when the children were aged 10 years, a nutritionist inter-
viewed them about their diet. Following this, the nutri-
tionist proceeded to assess the subject's reaction to a bitter
(PROP) challenge using a general visual analogue scale
(gVAS) [29].

The disks were prepared by soaking circular pieces of filter
paper (Whatman #1) in a saturated solution of PROP (at
near boiling temperature) and then drying them. The
PROP crystallizes into the paper thus allowing the paper
to serve as a convenient way to permit a subject to taste a
limited quantify of PROP crystals. The PROP crystals go
into solution in the subject's saliva and produce a high
concentration of PROP at the taste receptor sites. The
paper produces bitterness approximately equivalent to a
solution of .0032 M, close to the highest concentration of
PROP that will remain in solution when PROP solutions
are refrigerated for storage. The purpose of using a high
concentration for screening is that PROP functions for
nontasters, medium tasters and supertasters diverge; thus
the highest practical concentration of PROP produces the
most accurate sorting.

In this test, the nutritionist explained to the child that they
were going to taste a piece of paper and would then mark


Table 6: Behavioural variables (number (%)) by quartile of PROP taste score for AV/AV homozygotes (non-tasters).


Homozygote non-taster (AV/AV)

I

2

3

4

linear test, p


Manual social class (n = 1055)

129
(48.86)
130
(47.79)
119
(45.25)
116
(45.31)
0.3


Antisocial activities (n = I 190)

55
(18.46)
26
(8.61)
49
(16.23)
42
(14.58)
0.7


Depressed tendency (n = I 174)

83
(28.04)
75
(25.34)
68
(23.05)
88
(30.66)
0.01


Note, reference groups are not shown. 1, 2, 3, 4 represent grouping by quartiles of PROP taste test (general labelled magnitude scale).



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Table 7: Behavioural variables (number (%)) by quartile of PROP taste score for PA/PA homozygotes (tasters).


Homozygote taster (PA/PA)


3

4

linear test, p


Manual social class (n = 646)

69
(42.07)
65
(36.93)
71
(46.10)
71
(46.71)
0.2


Antisocial activities (n = 721)

29
(15.85)
31
(15.82)
23
(13.37)
32
(18.82)
0.6


Depressed tendency (n = 710)

38
(21.47)
32
(16.58)
40
(23.39)
53
(31.36)
0.6


Note, reference groups are not shown. 1, 2, 3, 4 represent grouping by quartiles of PROP taste test (general labelled magnitude scale).


how strong they thought the sensation of the taste was on
a line. The child was asked to name their senses and was
prompted if he/she could not. The nutritionist then asked
the child to describe the loudest, most intense sound they
had ever heard and the brightest, most intense light they
had ever seen. They then pointed to the scale on the
datasheet (a 10 cm line), explaining that the left end of the
scale represented no sensation and the right end repre-
sented the most intense sensation, explaining again the
most intense sensation as the loudest sound or brightest
light that they had just described. The child was then
asked to point on the line where a whisper and where a
shout would go on the scale. If children understood the
instructions, a whisper would be rated as less intense than
a shout which, in turn, would be rated less intense that the
loudest sound. Once children rated the sounds in the cor-
rect order, they were asked to place a disc of PROP paper
on their tongue and move it around for about 10 seconds.
The child was then given a pen and asked to make a mark
on the line. Once the child had left the room the nutri-
tionist measured how far from the left the child had
marked (10 cm being the maximum).

Of note, the instructions regarding the whisper were used
to check that the child understood how the scale worked
and which end represented the largest magnitude of stim-
ulation in regard to the senses.

PROP taste test consistency
Within the focus at 10 years of age follow-up screens, a
small subset of the ALSPAC cohort who under took taste
tests were re-invited to perform the test again (i.e. a repeat
of the clinic at 10 years). Of 237 children assigned re-invi-
tation (approximately 3% of the total sample set) 203
children completed the test the first time and 196 the sec-
ond time. In total, 168 children performed the test twice.
These provided data for the assessment of internal consist-
ency for the PROP taste test. Consistency was assessed by
analysis of the correlation between test scores at both
measurements and by the regression of repeat test data.


Genotyping
Genetic variants were selected on the basis of previous lit-
erature indicating their association with PTC taste test
scores. SNPs to be investigated here are the A49P and
V262A variants of the TAS2R38 gene on Ch7 (rs713598
and rs1726866 respectively). Both of these are non-syn-
onymous protein change causing variants the former lead-
ing to a Proline/Alanine change at amino acid position
145, whilst the latter leads to a Alanine/Valine change at
amino acid position 785. As such, these are taken as likely
to be functionally significant in relation to the action of
the TAS2R38 gene (although the actual implication of
these variants are unknown) [12].

DNA, from cord blood or peripheral blood was extracted
and processed as described previously [30]. SNPs were
genotyped using the KASPar chemistry which is a compet-
itive allele specific PCR SNP genotyping system using
FRET quencher cassette oligos[31]. All genotyping was
performed by KBioscience [32].

Psychosocial factors
Social class was derived using OCPS classifications [33]
with information taken from a self completed question-
naire administered to the mothers at 32 weeks of preg-
nancy. This information yielded a 6-level variable I, II, III
non-manual, III manual IV, V, which was dichotomised
centrally for a manual/non-manual split.

For the assessment of depression the children were given
a series of envelopes with statements written on them
about how they might have been feeling or acting in the
previous two weeks. This was done at the same clinic ses-
sion. Depression was assigned in cases where 6 or more
indicators of depression existed. This represented the top
quartile of the population distribution. The statements
used have been taken from the Short Mood and Feelings
Questionnaire [34], which has been designed to provide a
rapidly administered questionnaire for use in epidemio-
logical studies.




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Table 5: Pairwise differences of mean PROP taste test score by diplotype in ALSPAC


AV/AV


AV/AA


AV/PA


AA/PA


PA/PA


Non-taster Intermediate
*
2.11 (1.68,2.53)
2.10 (1.67, 2.75)
2.70 (2.26, 3.13)


Non-taster Taster (Het)
Intermediate Taster (Het)
*
0.10 (-0.26, 0.46)
0.59 (0.42, 0.76)


Non-taster Taster (Het)
Intermediate Taster (Het)
Taster (Het) Taster (Het)
*0.49 (0.1
0.49 (0.1 1, 0.86)


Non-taster Taster (Horn)
Intermediate Taster (Horn)
Taster (Het) Taster (Horn)
Taster (Het) Taster (Horn)


Pairwise difference of mean PROP taste score displayed with predicted tasting status in opposing matrix space. Estimates and 95% Cl derived from
categorical regression of taste test score by diplotype with robust SE.


Lastly, antisocial behaviour was defined by the presence
of any 1 of 11 antisocial activities report by the child dur-
ing the same clinic session, covering a range of behaviours
including stealing, cruelty to animals, smoking, drinking,
taking drugs and a series of dummy questions. Questions
were derived from the self-reported antisocial behaviour
for young children questionnaire [35]. A variable was
then derived indicating whether any such activity had
been carried out.

For the purpose of analysis of the distribution of taste test
scores by social class, antisocial behaviour and depres-
sion, tasting scores were organised into quartiles.

Statistical analysis
Table 1. summarises haplotypes and predicted taster sta-
tus identified in relation to previous literature (i.e. an
individual's status as either a 'taster' or 'non-taster' of bit-


0 5 10 0 5 10
Taste Test Score


Figure 3
Distribution of PROP taste test scores by genetic
prediction of tasting ability in the ALSPAC cohort.
Test groups here correspond to 0 = non-tasters as defined
by the homozygous carriage of the TAS2R38 haplotype AV; I
= tasters as defined by the homozygous or heterozygous car-
riage of the TAS2R38 haplotype PA. Ranksum analysis for
comparison of random points from these two distributions
yields p =< 0.001.


ter compounds). Haplotype names 'PAV' and 'AVI' refer to
recognized 'taster' and 'non-taster' states respectively [12]
and are specifically derived from their protein coding
sequences. In the context of this study, haplotypes 'AVI'
and 'PAV' have been renamed AV and PA respectively. An
individual was therefore designated a 'non-taster' if they
carried two copies of AV and a 'taster' if they carried at
least one copy of PA.

Previous results [12] demonstrated the existence of the
variant site TAS2R38 V296I (34 bases from TAS2R38
V262A) in total linkage disequilibrium (LD) with
TAS2R38 V262A in a European population. In light of
this, it was felt appropriate that the genotyping of
TAS2R38 P49A and TAS2R38 A262V alone would allow
the effective tagging of common haplotypes in this region.

Having collected genotype data, haplotypes were con-
structed using the programme PHASE (version 2.02, [36],


0 5 10 0 5 10
Taste Test Score


Figure 4
Disrtibution of PROP taste test scores by the rare
AA haplotype in the ALSPAC cohort. Test groups here
correspond to I = any carriage of the rare AA haplotype,
excluding carriers of the common PA taster haplotype, i.e.
predicted intermediate tasters; 0 = all other individuals.
Ranksum analysis for comparison of random points from
these two distributions yields p =< 0.001.


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AV/AV
AV/AA
AV/PA
AA/PA
PA/PA


*
1.65 (1.21, 2.10)
3.76 (3.57, 3.95)
3.86 (3.48, 4.24)
4.35 (4.13,4.56)


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Table I: Prediction of tasting ability by TAS2R38 haplotype


Kim et al haplotype Present Study Label P49A rs713598 A262V rs1726866 [V2961]

AV [1] AV G T [A]
AA[V] AA G C [G]

PV [1] PV C T [A]
PA FV1 PA C C rG1


Taster Status


Non-taster if homozygote
Small effects on non-taster status when heterozygote
AVI/AAV
unknown
Taster if homozygote/heterozygote


(Haplotypes taken from Kim et al [8]. Haplotype labels are derived from amino acid coding patterns. Square brackets indicate exclusion of variant in
present study due to linkage disequilibrium with included variants.)


http://www.stat.washington.edu/stephens/phase.html).
This software employs a Bayesian method for the recon-
struction of chromosomal phase using genotype data and
generates counts and frequencies of observed haplotypes.
The underlying method in this approach is a Markov
Chain-Monte Carlo procedure in which the probability of
preceding observations (in this case unambiguous phase
information) allows population genetic inference about
unresolved haplotypic phase. Having run phase, posterior
probabilities of phase reconstruction were employed to
allow the incorporation of a haplotype accuracy cut off for
further analysis. For the purpose of taste test analysis, this
was constrained at 80% accuracy for the reconstruction of
PTC haplotypes.

Initial analysis considered median (due to skewed distri-
bution of PROP taste test scores) PROP taste test scores by
the haplotypic combination (diplotype) carried by those
in the ALSPAC sample. Based on Kim and colleagues [12],
our prior hypothesis was that bitter tasting ability would
differ according to taster status as defined by haplotypic
complement homozygouss AV = non-taster, heterozygous
or homozygous PA = taster, other = excluded). This model
formed the basis of test groups in taste test scores were
included for analysis. To assess the distribution of PROP
taste test results for the rarer AA haplotype, a further
grouping was generated as defined by any carriage of the
AA haplotype (excluding the common and taster predict-
ing PA haplotype) versus the rest of the sample. This
grouping was largely designed on the basis of the findings
of Bufe et al [17] and was deigned to assess the potentially
intermediate tasting ability of those carrying the rare AA
haplotype.

Due to the expected bimodal distribution of PROP taste
test results, the non-parametric rank sum approach to test-
ing for differences between the properties of test groups
was used in this analysis. This also prompted the use of
robust standard errors in the presentation of mean pair-
wise differences in taste test score by diplotypes along
with their 95% CI. Test statistics for the analysis of quar-
tiles of PROP taste test with social class, depression and
antisocial behaviour were generated by via simple trend
analysis.


All analyses were performed in STATA version 8.2.9 and
SPSS version 9.12.

Competing interests
The authors) declare that they have no competing inter-
ests.

Authors' contributions
NT performed the statistical analysis and coordinated the
writing of the paper and acts as guarantor

JH sourcing and piloting of materials and assisted in
project management of data collection

INMD inputs to improvements phase of analyses and
manuscript

SR production of DNA bank and overseeing provision of
DNA and linking genetic data

LB was involved in original data collection and contrib-
uted to paper revision

PE lead the team that administered the PROP challenge
having taken advice from LMB on the correct procedure,
she was fully involved in the writing of the manuscript.

GDS analysis plan and revising manuscript

Acknowledgements
We are extremely grateful to all the families who took part in this study,
the midwives for their help in recruiting them, and the whole ALSPAC
team, which includes interviewers, computer and laboratory technicians,
clerical workers, research scientists, volunteers, managers, receptionists,
nurses and particularly the nutrition team who administered the PROP
challenge. The UK Medical Research Council, the Wellcome Trust and the
University of Bristol provide core support for ALSPAC. This publication is
the work of the authors who also serve as guarantors for the contents of
this paper. NT is funded by a MRC studentship. JH is funded by MRC/Well-
come. INMD group is funded by UK MRC, BHF, and University of Bristol.
LB is funded by the National Institute of Deafness and other Communica-
tive Disorders (NIDCD); from grant DC 000283.

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