Molecular basis of the Cd36 chromosomal deletion underlying SHR
defects in insulin action and fatty acid metabolism
Anne M. Glazier,
Timothy J. Aitman
Physiological Genomics and Medicine Group, MRC Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
Genomic and Molecular Medicine Group, MRC Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
Imperial College Genetics and Genomics Research Institute, Hammersmith Hospital, London W12 0NN, UK
Received: 7 September 2001 / Accepted: 3 October 2001
Abstract. The human insulin resistance syndromes—type 2 dia-
betes, obesity, combined hyperlipidemia, and essential hyperten-
sion—are genetically complex disorders whose molecular basis is
largely unknown. The spontaneously hypertensive rate (SHR) is a
model of these human syndromes. In the SHR/NCrlBR strain, a
chromosomal deletion event that occurred at the Cd36 locus during
the evolution of this SHR strain has been proposed as a cause of
defective insulin action and fatty acid metabolism. In this study,
three copies of the Cd36 gene, one transcribed copy and two
pseudogenes, were identified in normal rat strains, but only a
single gene in SHR/NCrlBR. Analysis of SHR genomic sequence
localized the chromosomal deletion event between intron 4 of the
normally transcribed copy of the gene and intron 4 of the second
pseudogene. The deletion led to the creation of a single chimeric
Cd36 gene in SHR/NCrlBR. The boundaries of the recombination/
deletion junction identified within intron 4 were surrounded by
long interspersed nuclear elements (LINEs) and DNA topoisom-
erase I recognition sequences. An 8-bp deletion at the intron 14/
exon 15 boundary of the second pseudogene abolishes the putative
splice acceptor site and is the cause of an aberrant 3Ј UTR previ-
ously observed in SHR/NCrlBR. We conclude that in SHR/
NCrlBR, the complex trait of insulin resistance and defective fatty
acid metabolism is caused by Cd36 deficiency, resulting from a
chromosomal deletion caused by unequal recombination. This
demonstrates that chromosomal deletions caused by unequal re-
combination can be a cause of quantitative or complex mammalian
The human insulin resistance syndromes include type 2 diabetes,
obesity, combined hyperlipidemia, and essential hypertension
(Reaven 1988, 1993, 1995; Kaplan 1989; Aitman et al. 1997a).
These disorders are genetically complex, and their genetic basis is
largely unknown. The spontaneously hypertensive rate (SHR) is a
model of these human syndromes and is characterized by defective
insulin-mediated glucose uptake and defective fatty acid metabo-
lism in isolated adipocytes (Reaven et al., 1989; Aitman et al.
1997b) and at the whole-body level (Iritani et al. 1977; Rao 1993).
Quantitative trait loci (QTLs) for SHR defects in glucose and fatty
acid metabolism, hypertriglyceridemia, and hypertension all map
to a single region on Rat Chr 4 (Pravenec et al. 1995; Bottger et al.
1996; Aitman et al. 1997b). Genetic analysis of an SHR inbred
strain derived from an NIH colony (SHR/NCrlBR) identified a
defective SHR gene, Cd36, at the peak of linkage to these QTLs.
SHR Cd36 cDNA contains multiple sequence variants, a divergent
3Ј untranslated region (UTR), and the encoded protein product is
undetectable in SHR adipocyte plasma membrane (Aitman et al.
1999). These data led to the conclusion that Cd36 deficiency is a
cause of insulin resistance, defective fatty acid metabolism, and
hypertriglyceridemia in SHR (Aitman et al. 1999; Pravenec et al.
Previous work indicated that there are at least two copies of the
Cd36 gene in normal rat strains, but only a single copy in SHR,
suggesting that a chromosomal deletion in the SHR lineage is the
cause of the Cd36 defect in this strain of SHR (Aitman et al. 1999).
Exonic sequence analysis indicated that the 5Ј end of the cDNA
was transcribed from the same transcription unit in all three strains,
but that the 3Ј end in SHR was transcribed from a normally un-
transcribed copy of the gene (Aitman et al. 1999). As Cd36 is
among the first QTL genes to be identified in a linkage study, we
wished to define the molecular basis of the apparent genomic
deletion in this rat strain.
In this report, we demonstrate that there are three copies of the
Cd36 gene in normal rat strains, define the breakpoints and mo-
lecular basis of the chromosomal deletion at the Cd36 locus, and
identify the basis of the divergent 3Ј UTR observed in SHR Cd36
cDNA. These molecular changes underlie the Cd36 defects in this
strain. This is the first report of a chromosomal deletion that un-
derlies a complex quantitative mammalian trait.
Materials and methods
SHR/NCrlBR (designated SHR), WKY/NCrlBR (WKY), and
Brown Norway/SsNOlaHsd (BN) rats were used in this study. SHR and
WKY rats were obtained from Charles River UK (Margate). BN rats were
obtained from Harlan UK Ltd (Bicester).
Isolation of large-insert genomic clones containing Cd36.
containing PACs were isolated at Research Genetics by hybridization with
a PCR-amplified genomic probe spanning intron 5 of the Cd36 gene. To
screen for BAC clones containing Cd36, rat BAC filters were obtained
from BACPAC resources (Department of Cancer Genetics, New York) and
hybridized to a
P-labeled PCR-amplified genomic probe spanning intron
5oftheCd36 gene. Both PAC and BAC clones were derived from BN
genomic libraries. Probes were labeled with the RediPrime random prime
labeling system (Amersham) and purified through a G50 Nick Spin Col-
umn (Pharmacia). Filters were pre-hybridized for 20 min with QuikHyb
solution (Stratagene), followed by hybridization to
P-labeled probes for
2 h at 65°C. Filters were washed in 0.1 × SSC, 0.1% SDS at 65°C prior to
autoradiography. Clone identity was verified with the Cd36 exon 6 HinfI
PCR-RFLP (Aitman et al. 1999). A HinfI restriction site is present at
nucleotides 640–645 of the SHR Cd36 cDNA, but absent in WKY cDNA.
Primers Hin3F, 5Ј-ACAKMYTTATCAAAGAGTCCAAGTCTTC-
TATGTTC-3Ј and Hin3R, 5Ј-ACCAACTGTGGTACTTATCG-3Ј amplify
across this RFLP, giving a 122-bp product. Primer Hin3F is degenerate,
containing K (G/T), M (A/C), and Y (C/T) to account for differences
between the SHR and WKY cDNA sequences. Primer Hin3F contains an
Correspondence to: A.M. Glazier; email: anne-marie.glazier@csc.
Mammalian Genome 13, 108–113 (2002).
© Springer-Verlag New York Inc. 2002