Transcriptomic analysis of rat QTL linked to hypertension by exomic sequencing and micro array gene expression analysis

1. Work in progress
• Using available Rat cDNA sequence for all (but one) of the
putative genes within the SISA 6000 region, oligonucleotide
primers have been designed to enable screening of these
genes for expression differences in the kidney of the rat
strains of interest, using RT-PCR methodology. So far, 12 of
the loci have been tested in the 25 week and 6 week
WKY/SHR strains.
• In tandem with this work – full length rat cDNA sequencing
is being undertaken to identify possible sequence variations
between the rat strains. So far, 7 of the loci have been
investigated for cDNA sequence variations, with the Calc-a
sequence exhibiting a synonymous point mutation between
the strains.
Genomic analysis of a hypertension QTL on Rat chromosome 1
Clemitson J, Dixon RJ, Haines S, Hall L, and Samani, NJ Department of Cardiovascular Sciences, University of Leicester
Introduction
By analysing crosses between the spontaneously
hypertensive rat (SHR) and the Wistar-Kyoto (WKY) rat, we(1,2)
have previously shown that a region in the mid-portion of rat
chromosome 1, contains quantitative trait loci (QTLs)
affecting blood pressure (BP). This region has been captured
in reciprocal congenic strains derived from SHR and WKY.
Further work, has indicated that the major proportion of the
BP effect of these loci is mediated through the kidney(3).
The region containing one of these BP QTLs, has now been
narrowed down further and isolated in a congenic sub-strain,
named SISA 6000 (Figure 1). As this region is < 4 Mb in size,
it is now feasible to directly search for the BP susceptibility
genes within the region.
Methods
• Translation of Genetic Map to Physical Map.
Genetic markers defining the region of interest, were
positioned on the physical Rat genome sequence, by
using BLAST and BLAT searches of the Rat genome
sequence within three genome browsers (Ensembl,
UCSC and NCBI), with genetic marker associated
sequence data.
• Creation of Comparative Gene Map.
Rat genome annotation from the three genome browsers
was examined and integrated for the SISA 6000 region.
The annotation of the complementary syntenic regions
in Mouse and Human was also investigated within the
three genome browsers and integrated with Rat SISA
6000 annotation, to create a putative gene list with
various degrees of confidence in accordance with
annotation evidence (Figure 2).
References
(1) Frantz, S., et al. Hypertension (1998), 32, p639-646
(2) Frantz, S., et al. Hypertension (2001), 38, p216-221
(3) Clemitson, J.R., et al. Hypertension (2002), 40, p292-297
FIGURE 2. Comparative gene map of SISA 6000 region
Results Summary
• The SISA 6000 QTL has been positioned on the Rat
genome sequence.
• This genomic region lies within a conserved syntenic
segment between Rat, Mouse (chromosome 7) and Human
(chromosome 11).
•The Rat SISA 6000 genomic region is 3.9Mb in size.
• Using a comparative genomics approach to identify the
majority of the genes within this QTL minimal region - a
comparative gene map was created (Figure 2).
• The comparative gene map suggests 49 putative genes lie
within this region.
• The orientation of this conserved segment (gene list) is the
same in Rat but reversed in Mouse & Human.
Gene list (concensus SISA6000 list)
Calc-b
Calc-a
NOVEL - NM_024514
NOVEL - ENSRNOG00000011367
Pde3b
NOVEL - RNOR01008459.2542.7655
NOVEL - RNOR01008461.14636.41822
NOVEL - LOC293167
NOVEL - ENSRNOG00000011405
NOVEL - ENSRNOG00000011736
Psma1
Copb1
NOVEL - LOC293168
NOVEL - LOC293169
NOVEL - LOC293170
R-RAS2
F-SPONDIN
NOVEL - RNOR01008488.143.251
NOVEL - LOC293171
NOVEL - LOC293172
NOVEL - LOC293173
NOVEL - LOC293174
NOVEL - LOC293177
NOVEL - LOC308888
NOVEL - ENSRNOG00000014315
NOVEL - ENSRNOESTG00000001427
Pth
NOVEL - LOC308889
NOVEL - LOC308890
Arntl
NOVEL - LOC308891
NOVEL - LOC308892
NOVEL - LOC308893
NOVEL - LOC308894
NOVEL - ENSRNOG00000014885
NOVEL - ENSRNOG00000015087
NOVEL - LOC293178
TEAD-1
Parva
NOVEL - LOC293180
NOVEL - ENSRNOG00000016244
NOVEL - LOC308895
Dkk-3
NOVEL - LOC308896
NOVEL - LOC308897
NOVEL - RNOR01008624.1581.2180
NOVEL - LOC293181
NOVEL - LOC308898
NOVEL - LOC293182
KEY
Homologs found in Human & Mouse
Homologs found in Mouse only
Rat predicted gene with RAT EST support
Rat predicted gene with mammalian EST homology support
Rat EST Transcripts - transcript predictions made by Ensembl using EST evidence alone
Gene found in Human only - falls within Gap in Rat / Mouse
KEY
Homologs found in Human & Mouse
Homologs found in Mouse only
Rat predicted gene with RAT EST support
Rat predicted gene with mammalian EST homology support
Rat EST Transcripts - transcript predictions made by Ensembl using EST evidence alone
Gene found in Human only - falls within Gap in Rat / Mouse
MOUSE RAT HUMAN
FIGURE 1(a). Dissection of SISA1 congenic region on Rat
chromosome 1
SHR
SISA 1
SISA 6000
175 185 195
SISA 9000
Blood Pressure (mmHg)
SHR genotype
WKY genotype
SISA 1
congenic
region
SISA 6000 SISA 9000
FIGURE 1(b). Blood pressure measurements in congenic
substrains at 20 weeks
Chromosome1