Download Polygenic inheritance and micro/minisatellites

Molecular Psychiatry (1998) 3, 21–31
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PERSPECTIVE
Polygenic inheritance and micro/minisatellites
DE Comings
Department of Medical Genetics, City of Hope Medical Center, Duarte, CA 91010, USA
While it has often been stated that the identification of the genes involved in complex polygenic traits may be extremely difficult, the principles learned in the past century about single
gene–single disease inheritance may not be relevant to polygenic inheritance. A new paradigm specific to complex disorders may be needed. It is proposed that micro- and minisatellite
polymorphisms play a role in the expression of many genes. As a result, these genes exist
in the population with many functional alleleomorphic variants. While each variant has only
a modest effect on a given phenotype, because the variants are common, and most quantitative traits are controlled by a number of genes, there is a reasonable probability that an individual will inherit a threshold number of functional variants beyond which there is an appreciable
effect on the phenotype. Twelve different aspects of such a new model for complex inheritance, some corollary implications, and three examples of its immediate application, are
presented with the hope that the model may allow an acceleration of the identification of the
genes involved in complex polygenic disorders.
Keywords: microsatellites; minisatellites; polymorphisms; psychiatric genetics; Z-DNA; behavior;
evolution
Since the re-discovery of Mendel’s Laws in the early
part of this century, the emphasis in human genetics
has been on single gene disorders inherited in a dominant or recessive fashion. In the past two decades a
large proportion of the genes for these disorders have
been identified, localized, cloned, and sequenced. As
the number of such genes remaining to be identified
has decreased there has been an increased interest in
the more common polygenic disorders. It has often
been suggested that the genes involved in these disorders will be far more difficult to identify. This difficulty is well illustrated by the psychiatric disorders.
Despite large numbers of linkage studies of manicdepressive disorder, schizophrenia, Tourette syndrome, panic disorder, autism, and others, with the
possible exception of bipolar disorder,1 there have
been few replicated findings.
Part of the problem may be the very success of the
studies of single gene disorders that are caused predominantly by exon mutations. In recessive disorders
such mutations result in a total cessation of gene function, and in dominant disorders the effects are also profound. This single gene–single disease model has often
led those studying polygenic disorders to focus on
identifying mutations in the exons of candidate genes,
either by sequencing2–4 or the use of special electrophoretic techniques.5
I propose here that the identification of the mutant
genes involved in polygenic disorders could be hinCorrespondence: DE Comings, MD, Department of Medical Genetics, City of Hope Medical Center, Duarte, CA 91010, USA.
E-mail: dcomingsKmindspring.com
Received 17 January 1997; revised and accepted 15 April 1997
dered by the attempts to transfer the model of single
gene inheritance to polygenic inheritance, and that a
new paradigm, specific to polygenic disorders, may be
needed to aid in the identification of the genes
involved in these common entities. The following is a
practical example of some of the issues involved.
In 1990 Blum et al6 reported an association between
the Taq A1 D2A1 allele of the DRD2 gene and severe
alcoholism. This polymorphism is 39 to the DRD2 reading frame. This was a controversial finding because
some confirmed it while others did not. Assuming that
alcoholism is a complex, multifactorial, polygenic disorder, and that the DRD2 gene is just one of many genes
that could be involved, this discrepancy in findings
was not surprising.
Given the role of dopamine as a modulator of many
different behaviors,7 we8 suspected that the D2A1 allele
might also be associated with other disorders as well
and in recent years numerous studies have reported a
significant association between alleles of the DRD2
gene and cocaine addiction, polysubstance abuse,
smoking, attention deficit hyperactivity disorder
(ADHD), Tourette syndrome, visual-perceptual disorders, conduct disorder, posttraumatic stress disorder,
pathological gambling, and compulsive eating.9,10
Despite these associations, largely as a result of the
application of the single gene–single disease model,
many have remained skeptical of the role of the DRD2
gene in human behavioral disorders because sequencing studies have failed to find any mutations in the
exons that could explain these findings. These findings
could be explained if the D2A1 allele was in linkage
disequilibrium with an unknown non-exon mutation
that played a role in the regulation of DRD2 function.8
Polygenic inheritance and micro/minisatellites
DE Comings
22
We and others have examined other genes (DRD1,
DRD3, DRD4, OB, TDO2, MAOA, TRH and HTR2A)
that also show significant associations between nearby
polymorphisms and different behaviors, despite an
absence of meaningful exon mutations.
Micro/minisatellite polymorphisms in psychiatric
genetics
Minisatellites have been defined as repeat sequences of
up to 65 base pairs in length.11 Microsatellites consist
of shorter repeats variously defined as 2–5 bp in length.
For the purposes of the present paper, unless specifically stated, we will use the term micro/minisatellites
to cover both.
If psychiatric disorders are polygenic, and each gene
contributes to less than 10% of the variance of a given
quantitative trait, then association studies may provide
greater power than most linkage techniques.12
Although this technique can produce false positives
due to population stratification, this can be minimized
using the haplotype relative risk procedure13 with large
numbers of subjects.14 One of the major impediments
to the wider use of association studies is the lack of
availability of suitable polymorphisms at or near the
many candidate genes that have been cloned and
sequenced.15 Because of the high frequency of
micro/minisatellite repeats throughout the genome, we
and others have often used these polymorphisms in
association studies. Our initial assumption was that
like the neutral or silent single base pair polymorphisms, the micro/minisatellite alleles would be in
linkage disequilibrium with other ‘critical’ mutations
that affect gene function. However, after working with
these polymorphisms for several years we began to suspect that the micro/minisatellites themselves might be
the ‘critical’ mutations. There are two aspects of these
micro/minisatellite polymorphisms that are relevant –
their mutation rate and their size.
Mutation rate
The mutation rate of micro/minisatellite alleles is
higher than for non-repeat sequences.16 Lack of
exchange of flanking markers suggests the new
mutations are due to replication slippage or complex
conversion-like events.17 A high mutation rate could
make these polymorphisms less valuable for association studies since over many generations there would
be too much noise introduced into linkage disequilibrium relationships which require that two different
polymorphisms remain in phase for many generations.
However, if the micro/minisatellite mutations were
themselves the ‘critical’ alleles, they would actually be
more powerful for association studies, despite the
higher mutation rate.
Allele size
If linkage disequilibrium was involved in the association of micro/minisatellite alleles with specific phenotypes, each time a new mutation in the satellite
occurred there would be a specified chance it was
occurring on the same chromosome as the ‘critical’
alleles. However, on average there should be no trend
for the longer vs the shorter mutant alleles to preferentially be in linkage disequilibrium with these ‘critical’
alleles. However, if the size of the repeats played a role
in gene regulation (see below) there should be a trend
for an association of quantitative traits with groups of
different sized alleles rather than with specific individual alleles or allele sequences. If there is a major peak
of repeat gene frequency, both the shorter and the
longer alleles may be associated with phenotypic
effects.
In our published18–23 and unpublished results of
studies of behavioral phenotypes associated with
micro/minisatellite polymorphisms at different neuropsychiatric candidate genes we have found a significant association between the shorter or longer alleles
with various quantitative behavioral traits and mini- or
microsatellites at the following genes: MAOA, MAOB,
HTR1A, DAT1, DRD4, HRAS, HTT, OB, CNR1,
GABRA3, GABRB3, FRAXA, and NO. Some examples
are shown in Figure 1. Others have reported significant
phenotypic behavioral effects with specific size alleles
of the same polymorphisms of the DAT124,25, DRD4,26–
29
HRAS,30–32 HTT,33,34 INS35–38 and DBH39 genes.
While these studies do not rule out the presence of an
important role of single base pair changes in a subset
of these length variants, (see below and28,40,41), they are
consistent with the possibility that both length and
sequence can be involved, and that the different sized
micro/minisatellite alleles might have an effect on gene
function. This hypothesis would be much more plausible if different micro/minisatellite alleles could be
shown to have an effect on the rate of transcription or
translation of the genes. There are, in fact, many
examples of this.
Evidence that minisatellites affect gene function
The trinucleotide repeats
The most dramatic evidence for an association between
repeat sequences and disease comes from the involvement of long triplet repeats in a variety of neurological
disorders42 including fragile-X syndrome, Huntington’s
disease,43 myotonia dystrophica, Kennedy’s disease,
Friedreich’s ataxia,44 and others.42 At least five of these
disorders involve intronic GAG repeats producing
polyglutamine tracts in the amino acid sequence of the
respective gene products. A clue to the mechanism by
which polyglutamine tracts affect cell function was
provided by the observation that the hungingtin protein binds other cellular proteins, such as hungingtin
associated protein 1 (HAP1).45 Glyceraldehyde-3 phosphate dehydrogenase, a critical enzyme in glycolysis,
was also found to bind to the huntingtin polyglutamine
tracts.46 This enzyme is of particular importance for
neurological disorders because the brain relies almost
exclusively on glucose for its energy.
Other proposed mechanisms by which increasing
lengths of trinucleotide repeats affect gene function
include decreases in mRNA and protein production,47
Polygenic inheritance and micro/minisatellites
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increased DNA methylation,48 effects on nucleosome
assembly,49 decreased RNA polymerase efficiency,50
competition between DNA binding proteins and transcription factors,51 regulatory squelching,52 gene
silencing,53 and the formation of complex, non-B DNA
three-dimensional structures.50,54,55 Some of the
remarkable aspects of the above observations are that
the CTG repeat silenced the growth inhibitory
factor/metallothionein III gene promoter whether it
was upstream or at a distance downstream from the
promoter,53 the efficiency of nucleosome formation
increased according to the size of CTG repeats suggesting such blocks may repress transcription through the
creation of stable nucleosomes,49 and the reduction of
androgen gene expression remained proportionate to
the number of CAG repeats even over the range of normal alleles.47
While I have not included the extremely long triplet
repeats in the proposed hypothesis of polygenic
inheritance, these studies do introduce the concept
that the varying length of repeat alleles, even when
they are well into ‘normal’ range, may affect gene function through a wide variety of mechanisms.
The binding of nuclear proteins by minisatellites. The
HRAS gene
The first evidence that minisatellite repeats could
affect the function of neighboring genes by binding
transcription factors came from the studies of Krontiris
and colleagues of the minisatellite located near the
HRAS gene.56–58 This 28-bp repeat is located 1 kb 39 to
the HRAS gene.59 Rare alleles at this locus are significantly associated with a number of cancers.56 This
minisatellite
displays
transcriptional
enhancer
activity60,61 which occurs because it binds four members of rel/NF-kB transcriptional factors.56,58 Since
C → G exchanges occurred throughout the minisatellite, the increased enhancer activity of some alleles
could have been due to sequence differences producing
secondary structure variations, as well as length differences.57 Studies also showed that the enhancer activity
of the HRAS minisatellite could be dramatically upregulated by interaction with a transcription factor unrelated to rel/NF-kB.57 While the studies of Krontiris
were based on the unique sequence of rare variants, we
have shown that when the common HRAS alleles were
grouped simply on the basis of size, there was an effect
of the resultant genotypes on behavioral variables.19
IGH gene
In an extension of these studies, Trepicchio and Krontiris62 also examined the minisatellite located in the
DHJH junction of the human immunoglobulin heavy
chain (IGH) gene. This minisatellite, located 1500 bp
upstream of the JH genes, was found to bind a nuclear
protein factor present in a wide variety of cell types.
The binding site contained the mycHLH motif,
CACGTG, closely related to the USF/MLTF binding
site adjacent to the adenovirus major late promoter.
The binding of the nuclear protein factor resulted in
its sequestration to an inactive form, causing a suppression of transcriptional activation.
Insulin gene minisatellite
Another example of the role of repeats in gene regulation comes from studies of a 14-bp repeat minisatellite located 596 bp 59 to the human insulin gene. In family studies, Bennett et al35 observed that of the Class I
minisatellite alleles the most common 814 mobility
unit (mu) allele was infrequently transmitted to diabetic offspring, while the shorter 641–756 mu and the
longer 828 mu alleles were transmitted significantly
more often. While there were also numerous single
base pair RFLPs in the region, the studies indicated the
most significant effects were seen with the minisatellite
alleles. The primary involvement of the minisatellite
was supported by gene expression studies of pancreatic
RNA from several subjects with different class I alleles.
Kennedy et al36 examined the transcriptional effects
of alleles of this minisatellite in further detail. They
examined the shorter class I alleles (average 40 repeats)
and the longer class III alleles (average 157 repeats)
using transvection of plasmid constructs linked to CAT
or luciferase to assay transcriptional activity. This
showed dramatic differences based both on the length
and the sequence of the repeat. Alleles where the
repeat sequences differed by a single base pair substitution varied by as much as 10–260% of the rate of
transcription of the standard allele. The insulin minisatellite contains high-affinity binding sites for the transcription factor, Pur-1,36 and is capable of adopting a
number of variant DNA conformations.63,64 Recently
Vafiadis et al38 and Pugliese et al37 have shown that
the transcription levels of the INS gene in the thymus
correlate with alleleic variation of the VNTR-IDDM2
locus and suggest this plays a role in the selection of
insulin-specific T-lymphocytes which in turn play a
role in the pathogenesis of type-1 diabetes.
Msbp-4
A minisatellite-binding protein called Msbp-4 was isolated from mouse testis and tumor cells. Msbp-4 binding favored multiple copies of the PC-2 repeat
sequence, GGCAGGA, and showed sequence and
strand specificity.65,66 Other minisatellite binding proteins have also been reported.67
Effect of 39 UTR repeats on translation
Repeat sequences in the 39 untranslated regions of the
15-lipoxygenase (LOX) gene were shown to bind a 48kDa reticulcyte protein that specifically inhibited the
translation of LOX.68 Other examples of repeat
sequences playing a role in regulation of translation
have been reported.69
Evidence that microsatellites affect gene function
An important aspect of sequences containing alternating purines and pyrimidines, such as (CA)n microsatellites, is that they are capable of forming left-handed or
Z-DNA under physiological conditions.70,71 Z-DNA is
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24
Figure 1 Illustrative examples of the association between alleles of repeat polymorphisms at a variety of genes and quantitative
behavioral traits. (a) Monoamine oxidase A gene (MAOA) and VNTR polymorphism alleles divided into four groups. There
was a significant association with a number of quantitative variables relating to specific symptoms. The results are shown for
a manic symptoms score in 351 Tourette syndrome probands, their relatives and controls. There was a significant increase in
the score in subjects carrying the longest alleles.23 (b) Use of the same polymorphism in a group of substance abusers and
controls. There was again a significant association with the longest alleles and a drug abuse score.23 (c) Association between
a quantitative score for anxiety, based on the SCL-90 test, and the obesity gene (OB) microsatellite polymorphism alleles divided
into subjects homozygous for the ,208-bp alleles (left) and individuals homozygous or heterozygous for the $208-bp alleles
(right).20 (d) Association between the amplitude of the event-related potential (ERP) P300 wave (in uvolts) and the CNR1
cannabinoid receptor gene microsatellite polymorphism alleles divided into subjects homozygous for the $5 repeat alleles
(right) and individuals homozygous or heterozygous for the ,5 repeat alleles (left).21 (e) Association between the Brown Adult
ADD score and the GABAA B3 (GABRB3) receptor gene microsatellite polymorphism alleles divided into subjects homozygous
for $185-bp alleles (left) and those homozygous or heterozygous for the ,185-bp alleles. (f) Association between performance
IQ and the normal alleles of the FRAXA locus in random adults assessed for IQ.104
characterized by a zig-zag conformation of the phosphate backbone (Figure 2).72 The major groove is absent
in Z-DNA and the minor groove is much deeper than
in B-DNA, extending to the axis of the helix and exposing the bases.72
Hybridization studies have shown that the human
genome contains up to 100 000 regions of poly (CA)n
sequences, and that such sequences are common in all
eukaryotes including Drosophila and yeast.73 Studies
of 137 human DNA genes showed that Z-DNA forming
Polygenic inheritance and micro/minisatellites
DE Comings
Figure 2 Conformation of Z-DNA and B-DNA showing the
zig-zag nature of the phosphate backbone (dark spheres) and
the exposure of the base pairs (shaded spheres) in the deep
groove of Z-DNA (from Ref. 76 by permission).
sequences were non-randomly located with 35%
occurring at the 59 end of genes, often in promotor
regions, compared to 3% at the 39 end. Of the remaining sequences, 47% were found in introns vs 15% in
exons.74 The number of Z-DNA domains associated
with each gene ranged from 0 to 14. When the 13 000bp a-globin gene cluster, and the 73 326-bp of the bglobin cluster were examined, there was a dramatic
tendency for Z-DNA regions to cluster around sites of
initiation of transcription, and a virtual absence of ZDNA regions in intergenic sequences and at pseudogenes. These studies also showed that Z-DNA motifs
may be present up to 1.5 kb 59 upstream of the transcribed sequences.
Z-DNA and the regulation of transcription
A number of other studies have also suggested a role
of Z-DNA in gene function. DNAse I hypersensitivity
is associated with transcriptionally active chromatin.
Fine structure mapping of the DNAse I hypersensitive
sites of the enhancer region of the SV40 minichromosome showed the presence of DNAase I cleavage
approximately 25 bp on either side of the segments forming Z-DNA.75 The position of these hypersensitive
sites may have been determined, in part, by Z-DNA
binding proteins.75 A survey of other transcriptional
enhancers showed that pairs of Z-DNA segments were
present in a number of transcriptional enhancers with
50–80 bp between the Z-DNA segments.70,75 Rich et al76
suggested that the spontaneous conversion of the ZDNA segments to B-DNA could produce a local
increase in negative supercoiling that may alter local
chromatin structure and facilitate entry of RNA polymerase into the molecule. Hamada et al77 also proposed that by reversibly interconverting between the B
and the Z forms in vivo the (CA)n sequences could distort DNA and result in the activation or inactivation
of genes.77
Banerjee et al78 examined the ability of various synthetic polymers to affect the transfection of herpes simplex virus thymidine kinase in LtK cells. Potential ZDNA forming polymers cotransfected with the tk gene
decreased the level of Tk+ transformed colonies, while
cotransfection with polymers that do not form Z-DNA
had no effect. They found the effect was dependent
upon the length of the microsatellites linked to the 39
or 59 end of the tk gene. Only the longer sequences,
capable of forming Z-DNA, had an effect. In a subsequent report, Banerjee and Grunberger79 found that
certain polynucleotides, capable of forming Z-DNA,
enhanced the transcription of the chloramphenicol
acetyltransferase gene when cotransfected into murine
L cells.
¨
Wolfl et al80 reported studies of the corticotropinreleasing hormone (CHR) gene with an intron that contained 100 bp of alternating purine/pyrimidine (CA)n
residues. These studies showed that the changes in ZDNA formation were linked to the rate of transcription
of the CHR gene and the action was gene specific. The
authors concluded that the presence of a Z-DNA motif
in the non-coding parts of all sequenced mammalian
CHR genes make it reasonable to consider that Z-DNA
may contribute to the regulation of the CHR gene.80
RNA polymerase cannot transcribe through ZDNA,81,82 and topoisomerase II and other proteins
show enhanced affinity for Z-DNA.76,83,84 A number of
nuclear proteins bind to Z-DNA but not to B-DNA.85
This may be related to the different conformation of
the two forms. The Z-DNA conformation exposes the
bases to interaction with proteins, while the B-form
does not (Figure 2). It has been suggested80,86 that ZDNA motifs at the 59 end of genes play an important
role in increasing the spacing of RNA polymerases and
that correct spacing is required to prevent transplicing
or erroneous splicing, thus maximizing the efficiency
of the RNA splicing process.
Five other studies have also indicated a role of (CA)n
repeats in gene regulation. First, recent studies have
shown that 59-UTR Z-DNA forming poly(TG)n tracts
play a role in the regulation of RNA editing at the GLuR
gene.87,88 These results suggest a significant role of
intronic sequences in controlling gene expression.87
Second, studies by Epplen and colleagues89 showed
that (CA)n microsatellites bind nuclear proteins in
humans and that binding vs non-binding is dependent
upon the number of repeats. They found that (CA)6 did
not bind nuclear proteins, (CA)13 bound with moderate
intensity, and (CA)25 bound nuclear proteins with the
greatest intensity. They suggested that simple repeats
could have subtle effects on gene expression via their
differential protein binding with respect to the genetic
background of multifactorial diseases. Third, a study
of 125 unrelated healthy individuals showed a signifi-
25
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26
cant (P , 0.0001) correlation between the serum levels
of the dopamine b-hydroxylase and homozygosity for
two different sized alleles of an associated (CA)n repeat
polymorphism.39 Fourth, a (CA)n repeat upstream of
the rat prolactin gene forms Z-DNA and inhibits transcription.90 Fifth, deletion of the (CA)n repeat at the
human cytosolic phospholipase A2 gene showed it had
an inhibitory effect on promoter activity.91
In summary, there are many examples where
micro/minisatellite sequences play a significant role in
gene regulation by altering the primary, secondary or
tertiary structure of DNA, by binding to transcription
or translation factors, or by affecting RNA editing. In
many studies the length of the repeat sequences affected the affinity for proteins involved in transcription or
translation, determined the probability that a sequence
would form Z-DNA, or determined whether an
enhancer sequence would adopt a conformation suitable for the entry of RNA polymerase. For example, in
the study by Schroth et al,74 using a computer algorithm to evaluate a Z-score indicating the potential to
form Z-DNA, the score was 0.6 for (CA)6, 1.3 for (CA)7
and 2.4 for (CA)8, ie the score doubled with each
additional dinucleotide. Modest variations in the
sequence also produced major changes in the Z-score.
For example, the synthetic sequence (CGCG)4 had a Zscore of 17 300, while the synthetic sequence (CGCC)4
had a Z-score of 5, or 3460 times lower than that of
(CGCG)4. In addition, the sequence (TGTG)4 had a Zscore of 2.4, while the sequence (TGCG)4 had a Z-score
of 135. This tetranucleotide sequence was also sensitive to length in that the Z-score for (TGCG)3 was 16.7,
or eight times lower than that of (TGCG)4. These
results, illustrated in Figure 3, support the potential for
alleles of varying length and sequence of polymorphic
micro- and minisatellite sequences to be associated
with variations in the level of expression of specific
genes.
A general hypothesis of polygenic inheritance
The various aspects of a micro/minisatellite hypothesis
of polygenic inheritance are listed as follows:
(1) Many micro/minisatellites have an effect on the
expression of the genes with which they are associated.
(2) Many genes are associated with one or more
micro/minisatellite polymorphisms.
(3) As a result of No. 1 and No. 2, many genes will
present with a range of functional alleleomorphic
variants. Those genes associated with several
micro/minisatellite polymorphisms, each with
multiple alleles, will present with an especially
large number of functional haplotypes. While
most genes will be associated with ±10–15% of
the average level of gene activity, the range may
be as high as ±40%, and if multiple
micro/minisatellite polymorphisms are involved
their effects can be additive.
(4) Significant functional variants are common in the
Figure 3 Z-DNA scores for repetitious DNA sequences based
on variations in length and sequence. Based on data of
Schroth et al.74
general population, with prevalences ranging from
1 to 100%. A 100% prevalence can occur if there
is a bimodal distribution of alleles consisting
entirely of hyperfunctional alleles (.10% of the
average) and hypofunctional variants (,10% of
the average).
(5) Individually these functional variants have only
a modest effect on the phenotype (0.5–8% of the
variance), and rarely cause disease, ie they are
generally not responsible for the classic one gene–
one disease autosomal dominant or recessive disorders.
(6) Since most of the micro/minisatellite polymorphisms are not in exons, the mutations in polygenic disorders are usually outside the exons and
often outside the transcribed sequences.
(7) Log score linkage studies lose power when more
than six genes are involved in a given disorder.12,92 Linkage studies are based on the
assumption that the presence of a given allele is
associated with the presence of the disorder and
the absence of the allele is associated with the
absence of the disorder. By contrast, in polygenic
inheritance the disorder is associated with the
presence of a threshold number of alleles of several different genes. As such, many members of a
pedigree may carry a given mutant gene but not
have the disorder, because of the absence of the
necessary threshold number of other mutant
Polygenic inheritance and micro/minisatellites
DE Comings
(8)
(9)
(10)
(11)
(12)
genes. Thus, many members of a family may carry
a specific gene and not have the disorder, and
other members of the family with the disorder
may not carry the mutant allele.93 Non-parametric
approaches to linkage94 are better suited to complex inheritance (but see95). However, when a
given gene accounts for less than 8% of the variance, a large number of parent–child sets or sibpairs must be examined.96 Association studies,
comparing the frequency of the mutant candidate
genes in severely affected probands to totally
unrelated, ethnically matched controls that are
free of the disease, can identify these small
effects.92,93,97
Both hyper- and hypo-functional alleles can have
an effect on the phenotype. For example, in psychiatric genetics, both an increase in the
expression of a given receptor gene leading to
receptor supersensitivity, or a decrease in the
expression of a receptor gene leading to receptor
hyposensitivity, could be associated with an altered phenotype.
A polygenic disorder occurs when an individual
inherits a threshold number of hypo- or hyperfunctional genes affecting the same phenotype.
Thus, if 20 different polygenes play a role in a
given quantitative variable, anyone with 10 or
more of these hypo- or hyperfunctional variants
would present with a significantly altered phenotype. The threshold number would vary according
to the degree of hypo- or hyper-functionality of
the alleles, and the severity of the altered phenotype would depend on the extent to which the
number of polygenes exceeded a critical threshold number.
Because micro/minisatellites are so common, the
probability of No. 9 occurring is relatively high.
Thus, polygenic disorders are much more common than single gene disorders. With the general
population frequency of many polygenes ranging
from 25% to over 50% the chance of inheriting a
threshold number is much higher than for the single gene disorders.
For a given micro/minisatellite the alleles can
have a negative or positive effect on the phenotype depending upon the genetic background of
other genes and on the nature of the phenotype.
A practical example is the observation by
Gelernter et al25 of an association between
cocaine-induced paranoia and the 9 allele of the
dopamine transporter gene (DAT1) gene. By contrast, Cook et al24 and Comings et al18 observed an
association between the 10 allele of the DAT1
gene and attention deficit hyperactivity disorder.
One might conclude from such divergent reports
that one or the other observation must be incorrect. However, there is a reasonable probability
that both are correct, and that different alleles of a
given micro/minisatellite may be associated with
different phenotypes.
A reasonable approach to the study of polygenic
disorders is to choose candidate genes and examine the effect of alleles of the nearest
micro/minisatellite polymorphisms on relevant
quantitative traits.
Implications of the micro/minisatellite paradigm
for polygenic inheritance
The above proposed characteristics of polygenic
inheritance carry a number of additional implications.
These are as follows:
Many genes can be involved in the polygenic inheritance of some trait If many micro/minisatellite polymorphisms have the potential of altering the rates of
transcription or translation of the gene they are closest
to, and if many genes are associated with at least one
micro/minisatellite, then many and possibly most
genes have the potential to contribute in some degree
to the polygenic inheritance of the phenotypes they
control.
Exon mutations may provide only a minor contribution
to polygenic inheritance Many studies of the potential role of specific genes in psychiatric or other disorders start with a search for mutations in the exons of
a candidate gene. When such studies are negative it is
often concluded that the gene has nothing to do with
the disorder being examined. If the majority of the
mutations involved in complex, polygenic traits are in
non-exons, such conclusions would be invalid. The
present hypothesis does not exclude a role for exon
mutations that have only a minor effect on the function
of the gene, it only emphasizes that these studies can
be negative despite the presence of functionally
important variants of that gene.
The study of the additive effects of several genes will
be informative If on average, each candidate gene
contributes to only 0.5–8% of the variance of a given
quantitative trait, depending upon the phenotype and
the size of the study, the results may be of modest, borderline or negative significance. However, the examination of the additive or epistatic effect of two or more
candidate genes may show a much more significant
effect. We have observed this for the additive effect of
the DRD2, DbH and DAT genes,18 the DRD1 and DRD2
genes,98 the OB and DRD2 genes,20 and other gene combinations (unpublished).
Simply comparing allele frequencies in controls vs subjects may be inadequate Many association studies
simply examine the frequency of specific alleles in subjects compared to controls. However, when a diagnosis
is based on a complex set of symptoms, a given allele
may show a significant association with several of the
quantitative traits involved in the diagnosis, but not
the diagnosis itself. For example, if schizophrenia is a
polygenic disorder, the alleles of a specific candidate
gene may show a significant association with negative
symptoms (eg affective flattening), which are present in
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Polygenic inheritance and micro/minisatellites
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28
some but not all cases, but no association with positive
symptoms (eg delusions or hallucinations), or with the
diagnosis of schizophrenia itself. Limiting the analysis
to simple dichotomous diagnoses, rather than the
examination of sub-syndromal quantitative traits, may
miss genes that make an important contribution to a
polygenic disorder. Heterosis may also allow a gene to
have a significant effect on the phenotype in the
absence of difference in the frequency of individual
alleles in controls vs patients.99
Evolution may not need to wait for mutations, they are
always there One of the tenets of evolution is a
dependence upon the occurrence of just the right rare
mutations that instead of being deleterious to the species, improve it, and allow selection to produce a new
and better adapted species. This leaves open the question of how such rare events ie mutations that affect
the right gene, are advantageous instead of deleterious,
happen at the right time, and have occurred often
enough to provide for the rich diversity of species. The
concept of random drift helps some but that seems best
suited to explain the accumulation and fixation over
time of neutral base pair and amino acid changes.
The concept of micro/minisatellite polymorphisms
providing many alleleomorphic functional variants of
each gene eliminates the need for this improbable
sequence of events. Thus, new mutations or alleles do
not have to occur, they are already present, and in great
abundance, and affecting most genes. It is not necessary for that rare advantageous mutation to just happen
to occur at the right time and in the right gene since
all genes come in a rich array of functional variants.
What is necessary to drive evolution is an environmental or ecological change to fuel the selection of a given
set of polygenes better suited for the new environment.
As an example, the evolution from primates to Homo
sapiens required many different changes, including
standing up-right, thumb-finger apposition, the development of speech centers, the expansion of the frontal
lobes, and many others. There is evidence that a number of different hominid species were simultaneously
and independently undergoing these changes.100 To
accept that multiple advantageous new mutations of
many different genes just happened to occur in the
right temporal order, and in several different lineages,
is difficult. However, if all the necessary mutations
were already present, and environmental changes
selected for the polygenic sets that were most advantageous, the parallel progression in multiple lineages
is more readily understandable. This is not meant to
imply that exon mutations in some critical genes do
not also play a role in evolution, only that the presence
of many functional alleles associated with micro-minisatellites may be a better explanation.
Microsatellites and minisatellites may both involve ZDNA motifs While Z-DNA forming (CA)n repeats represent the major class of microsatellite polymorphisms,
computer programs designed to identify Z-DNA motifs
show that a wide range of sequences, in addition to
dinucleotide repeats, also form Z-DNA.74 If these
longer sequences were involved in minisatellite repeat
polymorphisms, they could also play a significant role
of gene regulation. The suggestion that minisatellites
(and Alu repeats101) are the evolutionary progeny of
microsatellites11 further testifies to their interrelatedness.
Immediate practical applications
A hypothesis is most useful if it has immediate heuristic value. The following are examples of such value.
(1) Resolution of the statistical problem of highly polymorphic alleles One of the disadvantages of the use
of highly polymorphic micro/minisatellite polymorphisms in association studies is that when each of the
large number of alleles, and even larger number of
potential genotypes is examined, the power of the
study may be so seriously compromised as to render it
useless. However, if the size of the repeats is the critical variable, even the most complex of polymorphisms
can be reduced to three genotypes consisting of homozygosity for the shorter alleles, homozygosity for the
longer alleles, and heterozygotes. We have found this
approach of value for studies of the role of a number of
genes (OB, MAOA, MAOB, CNR1, GABRA3, GABRB3,
DBH, FRAXA, and NOS1) in behavioral traits.
(2) The identification of polymorphisms for candidate
genes using genomic clones While many of the potentially interesting candidate genes have been cloned and
sequenced, for most, no polymorphisms have been
reported in the Genome Data Base, making association
studies impossible. Based on the present hypothesis, if
the sequence is known, one method of identifying useful micro/minisatellites is to obtain large genomic
clones carrying the gene of interest from commercial
sources. These can then be screened for repeat
sequences which may be highly informative in association studies.
(3) The identification of polymorphisms for candidate
genes using Z-DNA screening The present model suggests that the repeat sequences capable of forming ZDNA will prove to be the most valuable polymorphisms in studies of polygenic inheritance. This suggests that screening known sequences using the Zhunt-II program,74 could provide important clues about
the location of the most informative polymorphic
regions. To test this, we utilized this program to examine the sequence of NOS1.102 Based on the results of
studies showing aggression in knock-out mice, without
the NOS1 gene,103 we had examined various behavioral
traits in humans using a repeat polymorphism we
identified by visual examination of the sequence of the
NOS1 gene.102 Since some associations were observed,
we wondered if: (a) the polymorphism we were using
would be identified by the Z-hunt II program; and (b) if
there were other regions in the gene that might contain
informative polymorphisms. Both predictions were
Polygenic inheritance and micro/minisatellites
DE Comings
true. Based on the assumption that the various lengths
of the NOS1 (CA)n alleles would play a role in the
function of the NOS1 gene, we divided the alleles into
two groups consisting of the shortest 50% of the alleles
(,199 bp) and the longest 50% of the alleles ($199 bp).
Preliminary studies suggest some associations with
behavioral phenotypes. We then screened the published sequence of the NOS1 gene for Z-DNA regions
using the Z-hunt-II algorithm.72 This showed that the
polymorphism we were using was one of three regions
of high Z-DNA content. A second region with an even
higher Z-score identified a new previously undetected
polymorphic region.
These three examples illustrate in practical terms
how the predictions of the model may lead to the acceleration of the identification of the genes involved in
polygenic disorders. The use of this model in conjunction with the sequencing data soon to be available from
the Human Genome Project may contribute greatly to
our knowledge about polygenic disorders through an
increased ability to identify the presence of
micro/minisatellites in proximity to the known candidate genes.
Acknowledgements
I thank PS Ho, Oregon State University, Corvallis, OR
for performing the Z-hunt-II screening. Supported in
part by the National Institutes of Drug Abuse grant
RO1-DA08417 and Tobacco Related Research Disease
Program grant 4RT-0110.
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