UNCTIONAL STUDIES: TYPING mRNA IN F1 CROSSES
TO STUDY GENE REGULATION
Polymorphisms that are transcribed into the coding region or 3UTR
of agene can have profound effects on the production of a functional
protein or on regulation of the RNA or protein encoded by a gene (12).
One such transcribed polymorphism affected susceptibility to asthma in
a mouse genetic model, a deletion in the coding region of the gene
resulted in C5-deficiency that correlated with susceptibility (68). In
humans, a polymorphism in the TCF7 gene, C883A, was associated with
type 1 diabetes (69). In addition to direct functional effects, a
transcribed polymorphism can be used to analyze transcriptional
regulation of a gene. If two mouse strains differ in the allele present in a
transcribed gene, the F1 generation derived from a cross of these
strains can be used to study cis- vs trans-regulation (70) (Fig. 2). In F1
mice, the transcription factors, polymerases, and other transactingfactors that will affect transcription of the RNA in question are
derived equally from each parental strain. If the mRNA level is
regulated by transacting factors, then the F1 mice should have equal
levels of the two alleles represented in the mRNA transcribed by this
gene, (allele 1/allele 2) = 1. If however, the mRNA expression is
controlled in a cis manner, in other words, if this gene controls its own
expression, then the proportion of allele 1 to allele 2 will not equal 1
(70). The following example is from an animal model for T-helper cell
differentiation. The expression of TCF7 mRNA is about threefold
higher in the B10.D2 mouse strain than Balb/c. An SNP was identified
in the second exon of the TCF7 gene. After converting the mRNA to
cDNA, the ratio of cDNA of one genotype to the other was determined
by quantitative, allele-specific PCR (Fig. 3). In this case, the ratio of
allele 1 to allele 2 was not equal to one, indicating that the
transcriptional control is caused by cis-acting factors. 94 Wang et al.
SNP Discovery and Genotyping 95 Cowles et al. (70) analyzed allelespecific transcription in F1 mice and in different tissues for these mice.
This allowed them to study cis-regulated expression in conjunction with
tissue-specific expression. Gene regulation plays an important role in
mammalian biology, so the interest in gene regulation will continue to
increase, and SNP-based assays allow easy analysis of transcriptional
regulation. 6. CONCLUSIONS The number of human SNPs available
in the public databases continues to grow at a very rapid rate. More
importantly, the quality of the information Fig. 2. The messenger RNA
(mRNA) level for TCF7 is controlled in a cis-acting manner. (A) A
depiction of breeding two inbred parents to generate a first filial (F1)
generation. The F1 mouse receives equal contribution from each parent.
(B) The level of two alleles (C/G) of the TCF7 single nucleotide
polymorphisms in F1mouse. The F1 mouse would have equal C or G
allele from both parents that is transcribed into mRNA. If the RNA
level is controlled in a cis-dependent manner, the levels would be
different from 50/50 as seen in this figure. available for each SNP is
improving even faster. Between June and November of 2003, the
number of uniquely mapped human SNPs grew from approx 4.1 to 5.8
million, whereas the number of experimentally validated SNPs grew
from approx half a million to an impressive 2.4 million. The number of
SNPs with a known allele frequency increased at a much slower rate,
but as data from the international haplotype map project becomes
available in 2004, this should change dramatically. For the individual
researcher, additional human SNP discovery will only be conducted on
limited regions for which a higher SNP density may be required. For
these purposes, existing SNP discovery technologies would be sufficient.
For most model organisms, however, the SNP coverage is still too low.
Except for the commonly used inbred mouse strains, only a handful to a
few thousand SNPs have been deposited in NCBI dbSNP for other
organisms. For example, dbSNP build 118 only contains two reference
SNPs for the chimpanzee Pan troglodytes. As additional genomes from
other organisms are sequenced, and in silico computational biology
takes off, generation of extensive polymorphism data will become a
priority. Impressive progress has been made in the area of SNP
genotyping in recent years, yet, in terms of throughput and pricing,
whole-genome scans of sufficiently large numbers of cases and controls
for association studies continue to be beyond the reach of all but a few
institutions. Although the price per single genotype has decreased to
close to $0.01 for some technologies, the comprehensive or actual price
of genotyping in real life situations including labor costs, consumables,
and up-front instrumentation investments are more realistically in the
range of $0.1–$1. In addition, such prices are most often only achievable
in sustained ultra-high-throughput operations processing 10–100
thousands of genotypes per day. At a comprehensive price of $0.05 per
genotype, generating 100 thousand genotypes per day would entail
yearly expense of roughly 1 million dollars. Similarly, a100,000 SNP
whole-genome scan of 500 cases and 500 controls would cost $5 million.
As with sequencing, progress in SNP genotyping prices and throughput
has been incremental rather than exponential in recent years.
Promising developments in the field of genome sequencing bear close
monitoring; if it does indeed become possible, as some have projected,
to sequence a whole mammalian genome for a few thousand dollars,
these developments may make targeted genotyping of select
polymorphisms unnecessary.Finally, progress is being made in terms of
understanding the functional relevance of SNPs and other
polymorphisms. New and improved experimentaland in silico methods
for determining and predicting the biological 96 Wang et al.SNP
Discovery and Genotyping 97 nction of polymorphisms transcribed into
RNA (as discussed here), as well as intronic regulatory SNPs, are
urgently needed.