Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.     

Evaluation of insulin resistance linkage to rat chromosome 4 in SHR of a Japanese colony

The spontaneously hypertensive rat (SHR) is a model of human insulin resistance syndrome. Quantitative trait loci for cellular defects in glucose and fatty acid metabolism have been mapped to an overlapping region of rat chromosome (RNO) RNO4 in SHR of the National Institute of Health colony, where a deletion in the Cd36 gene has been implicated as the causative mutation of insulin resistance. The present study has examined the potential presence of RNO4 linkage to a series of metabolic phenotypes in F(2) progeny derived from SHR of a Japanese colony (SHR/Izm) without the Cd36 mutation. Our data demonstrate that 'major' insulin resistance gene(s) are unlikely to exist on RNO4 in SHR/Izm and in vitro phenotypes measured in isolated adipocytes do not cosegregate in the F(2) population studied. Thus, it seems to be difficult to explain the underlying genetic mechanisms of insulin resistance by a single major gene on RNO4.     

Extensively high load of internal tumors determined by whole body MRI scanning in a patient with neurofibromatosis type 1 and a non-LCR-mediated 2-Mb deletion in 17q11.2

Deletions in 17q11.2 affecting the NF1 gene and surrounding regions occur in 5% of patients with NF1. The two major types of NF1 deletions encompass 1.4-Mb and 1.2-Mb, respectively, and have breakpoints in the NF1 low-copy repeats or in the JJAZ gene and its pseudogene. Deletions larger than 1.4-Mb are rare, and only seven cases have been reported so far. Here, we describe a 26-year-old NF1 patient with an atypical NF1 deletion of 2-Mb. In contrast to the 1.4-Mb deletions, which preferentially occur by interchromosomal recombination during maternal meiosis, the deletion described here occurred intrachromosomally on the paternal chromosome. The centromeric deletion breakpoint lies in an L1-element located 1.3-Mb proximal to the NF1 gene. The telomeric deletion boundary is located in a single copy segment between an AT-rich segment and an AluSx-element in intron 15 of the JJAZ1 gene. Structural analysis implies that non-B DNA conformations at the breakpoints destabilized the duplex DNA and caused double-strand breaks. Although the breakpoints of this 2-Mb deletion are not recurrent, it is conspicuous that one breakpoint is located in the JJAZ1 gene. Paralogous recombination between the JJAZ1 gene and its pseudogene causes the recurrent 1.2 Mb deletions. The genomic architecture of the NF1 gene region, influenced by paralogous sequences such as the JJAZ1 gene and its pseudogene, seems also to stimulate the occurrence of non-recurrent deletions mediated by non-homologous end joining. Patient 442 described here suffers from a very high burden of subdermal neurofibromas. Magnetic resonance imaging of the whole body revealed numerous internal tumors, mainly plexiform neurofibromas and spinal tumors. This demonstrates the value of whole-body MRI scanning in determining the total tumor load, which is an important aspect in genotype/phenotype correlations with regard to large NF1 deletions.     

Pharmacogenetic evidence that cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor gamma and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-type Cd36. In congenic and transgenic SHR with wild-type Cd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Does Cd36 gene play a key role in disturbed glucose and fatty acid metabolism in Prague hypertensive hypertriglyceridemic rats?

Close links between hypertension, hypertriglyceridemia, insulin resistance and other symptoms of metabolic syndrome was demonstrated in humans and experimental animals. Quantitative trait loci for defects in glucose and fatty acid metabolism, hypertriglyceridemia and hypertension were mapped in spontaneously hypertensive rats (SHR) on chromosome 4 and defective Cd36 gene was identified in this region. Here we investigated the polymorphism of Cd36 gene in Prague hereditary hypertriglyceridemic (HTG) rats, which represent another model of genetic hypertension and metabolic syndrome. These animals were compared with NIH-derived SHR and two different normotensive control strains (WKY, LEW). In spite of the fact that HTG and SHR rats had similar metabolic disturbances, genotype analysis of PCR products has shown that Cd36 mutation was not present in HTG rats. In conclusion, we have revealed that defective Cd36 is probably a candidate gene for disorded fatty-acid metabolism, glucose intolerance and insulin resistance in NIH-derived SHR, but other genes might play a role in pathogenesis of metabolic syndrome in Prague hereditary hypertriglyceridemic rats. This is in accordance with the absence of defective Cd36 gene in original SHR from Japan.     

Haplotypic Diversity Within the Ovine Calpastatin <Emphasis Type="Italic">(CAST)</Emphasis> Gene

Calpastatin (CAST) is a protein inhibitor that acts specifically on calpains and plays a regulatory role in postmortem beef tenderization and muscle proteolysis. Polymorphisms in the bovine CAST gene have been associated with meat tenderness, but little is known about how the ovine CAST gene may affect sheep meat quality traits. In this study, we selected two parts of the ovine CAST gene that have been previously reported to be polymorphic (region 1—part of intron 5 and exon 6, and region 2—part of intron 12), to investigate haplotype diversity across an extended region of the ovine gene. First, we developed a simple and efficient polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method for genotyping region 2, which allowed the detection of a novel allele as well as the three previously reported alleles. Next, we genotyped both regions 1 and 2 of the ovine CAST gene from a large number of sheep to determine the haplotypes present. Nine different haplotypes were found across this extended region of the ovine CAST gene and four haplotypes were identified that suggested historical recombination events within this gene. Haplotypes are typically more informative than single nucleotide polymorphisms (SNPs) for analyzing associations between genes and complex production traits, such as meat tenderness, but the potential for intragenic recombination within the ovine CAST may make finding associations challenging.     

Mutation Pattern Variation Among Regions of the Primate Genome

We sequenced three argininosuccinate-synthetase-processed pseudogenes (ΨAS-A1, ΨAS-A3, ΨAS-3) and their noncoding flanking sequences in human, orangutan, baboon, and colobus. Our data showed that these pseudogenes were incorporated into the genome of the Old World monkeys after the divergence of the Old World and New World monkey lineages. These pseudogene flanking regions show variable mutation rates and patterns. The variation in the G/C to A/T mutation rate (u) can account for the unequal GC contents at equilibrium: 34.9, 36.9, and 41.7% in the pseudogene ΨAS-A1, ΨAS-A3, and ΨAS-3 flanking regions, respectively. The A/T to G/C mutation rate (v) seems stable and the u/v ratios equal 1.9, 1.7, and 1.4 in the flanking regions of ΨAS-A1, ΨAS-A3, and ΨAS-3, respectively. These ``regional' variations of the mutation rate affect the evolution of the pseudogenes, too. The ratio u/v being greater than 1.0 in each case, the overall mutation rate in the GC-rich pseudogenes is, as expected, higher than in their GC-poor flanking regions. Moreover, a ``sequence effect' has been found. In the three cases examined u and v are higher (at least 20%) in the pseudogene than in its flanking region—i.e., the pseudogene appears as mutation ``hot' spots embedded in ``cold' regions. This observation could be partly linked to the fact that the pseudogene flanking regions are long-standing unconstrained DNA sequences, whereas the pseudogenes were relieved of selection on their coding functions only around 30–40 million years ago. We suspect that relatively more mutable sites maintained unchanged during the evolution of the argininosuccinate gene are able to change in the pseudogenes, such sites being eliminated or rare in the flanking regions which have been void of strong selective constraints over a much longer period. Our results shed light on (1) the multiplicity of factors that tune the spontaneous mutation rate and (2) the impact of the genomic position of a sequence on its evolution. Received: 10 February 1997 / Accepted: 21 April 1997     

Examination of the Intron in the meiosis-specific recombination gene REC114 in Saccharomyces

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes. Received: 16 October 1996 / Accepted: 10 February 1997     

Comparative and genetic analysis of the porcine glucocerebrosidase (GBA) gene

The genomic sequence of the porcine (Sus scrofa) glucocerebrosidase (GBA) gene (5.7 kb), encoding glucocerebrosidase (glucosylceramidase; acid beta-glucosidase; EC 3.2.1.45), was determined and compared with human (Homo sapiens) GBA and GBAP (pseudogene). The porcine gene harbours 11 exons and 10 introns, and the genomic organization is identical with human GBA. The exon sequences, coding for signal peptide and mature protein, show 81% and 90% sequence identity, respectively, with the corresponding human GBA sequences. Short interspersed elements, SINEs (PREs), are present in introns 2, 4 and 7. There is no evidence of a pseudogene in pig. The deduced protein sequence of GBA consists of 39 amino acids of signal peptide (long form) and 497 amino acids of the mature protein; the latter shows 90% sequence identity with the human protein. Four polymorphisms were observed within the porcine gene: insertion/deletion of one of the two SINEs (PREs) in intron 2 (locus PREA); deletion of a 37- to 39-bp stretch in intron 4 (one direct repeat and 5′ end of PRE); deletion of a 47-bp stretch in the middle part of PRE in intron 4 (locus PREB); and single-base transition (C–T) in intron 6 (locus HaeIII–RFLP). GBA was assigned to chromosome 4q21 by FISH and was localized to the same region by linkage analysis and RH mapping, i.e., to the chromosome 4 segment where quantitative trait loci for growth and some carcass traits are located.     

Regulatory Evolution of a Duplicated Heterodimer Across Species and Tissues of Allopolyploid Clawed Frogs (<Emphasis Type="Italic">Xenopus</Emphasis>)

Changes in gene expression contribute to reproductive isolation of species, adaptation, and development and may impact the genetic fate of duplicated genes. African clawed frogs (genus Xenopus) offer a useful model for examining regulatory evolution, particularly after gene duplication, because species in this genus are polyploid. Additionally, these species can produce viable hybrids, and expression divergence between coexpressed species-specific alleles in hybrids can be attributed exclusively to cis-acting mechanisms. Here we have explored expression divergence of a duplicated heterodimer composed of the recombination activating genes 1 and 2 (RAG1 and RAG2). Previous work identified a phylogenetically biased pattern of pseudogenization of RAG1 wherein one duplicate—RAG1β—was more likely to become a pseudogene than the other one—RAG1α. In this study we show that ancestral expression divergence between these duplicates could account for this. Using comparative data we demonstrate that regulatory divergence between species and between duplicated genes varies significantly across tissue types. These results have implications for understanding of variables that influence pseudogenization of duplicated genes generated by polyploidization, and for interpretation of the relative contributions of cis versus trans mechanisms to expression divergence at the cellular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We have analyzed CYP21 gene sequences in 65 CAH families in Taiwan. All ten exons of the CYP21 gene were analyzed by differential polymerase chain reaction followed by single-strand conformation polymorphism electrophoresis and the amplification-created restriction site method. About 95% (123 chromosomes) contain mutations due to conversion of DNA sequences into its neighboring homologous pseudogene, CYP21P. Four novel mutations representing 5% of the total chromosomes have also been identified. The mutations were confirmed by sequencing an aberrant DNA fragment. These four mutations included a base change of the splicing donor site at intron 2 from GT to AT, a base substitution of C to T at codon 316, deletion of ten bases (TCCAGCTCCC) at codons 330–333 of exon 8, and duplication of 16 bases (CCTGGATGACACGGTC) at codons 393–397 of exon 9. The loss of the splicing donor site at intron 2 and the premature stop at codon 316 may result in aberrant splicing to reduce enzyme activity and a truncated protein with no enzyme activity, respectively. Likewise, both the duplication and the deletion forms create a frameshift and premature stop during translation. The resulting proteins lack the heme-binding domain and hence are expected to lose enzymatic activity. Since these mutations are not found in the neighboring CYP21P pseudogene, gene conversion should not be the cause of these novel mutations. Received: 20 April 1998 / Accepted: 30 May 1998     

A LINE element is present at the site of a 300-kb deletion starting in intron 10 of the PAX6 gene in a case of familial aniridia

We have identified a 300-kb germline deletion in 11p13 in a family with aniridia but no Wilms’ tumor. Cloning and sequencing of the breakpoint revealed that the deletion starts in intron 10 of the PAX6 gene and removes the C-terminal part of the proline-serine-threonine rich domain, leaving both DNA-binding domains intact. The PAX6 gene is joined head-to-head to a LINE-1 (L1) element. The L1 is truncated at the 3′ end, removing part of ORF2. Sequencing of the L1 element shows that it does not encode a functional transposase and is therefore probably not an active element. These data suggest that the L1 element is normally present at the site of the distal deletion endpoint in 11p13. No extensive sequence homologies are detected at the deletion junction points; however, the PAX6 gene as well as the L1 element have runs of T nucleotides at this position, indicating that the deletion occurred by nonhomologous recombination. Several consensus recognition sequences for topoisomerase I flank the deletion site in both sequences, suggesting an involvement of this enzyme during the deletion-recombination process. Received: 22 November 1995 / Revised: 25 March 1996     

Low frequency of deletion alleles in patients with steroid 21-hydroxylase deficiency in a Mexican population

Steroid 21-hydroxylase deficiency is caused by mutations in the CYP21 gene. Approximately 95% of mutant alleles are generated by recombination events between the acitve gene CYP21 and its highly homologous pseudogene, CYP21P. Deletion alleles are generated by unequal crossing over, while point mutations are the result of gene conversion events. Deletions account for 20–25% of the 21-hydroxylase deficiency alleles in most populations studied. We have looked for deletions among 53 unrelated Mexican patients with steroid 21-hydroxylase deficiency and found that deletions represent less than 1% of the disease alleles. These findings suggest that nearly all mutant alleles in our patient population contain point mutations and that the low representation of deletion alleles among clinically diagnosed patients may be due to missing detection of salt wasters, mainly males, who may die during the neonatal period. Received: 17 November 1995 / Revised: 29 February 1996, 12 April 1996     

Comparative Analysis of Evolution in a Rodent Histone H2a Pseudogene

Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding' regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the 5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent gene conversion by functional H2a genes. Received: 1 April 1997 / Accepted: 12 June 1997     

Expression of theQ8/9 d gene by T cells of the mouse

TheQ genes, specifying Qa antigens and situated in the extended part of the major histocompatibility complex (MHC) of the mouse, comprise a subgroup of MHC class I genes whose significance and function are still largely unknown. In screening a cDNA library made from the BALB/c inducer T-cell line Cl.Ly1-T1, we isolated 11 clones representingQ8/9, but none representingQ6 orQ7. Confirmatory evidence is given that theQ8/9 gene originated from fusion of the 5′ region of theQ8 gene with the 3′ region of theQ9 gene at a recombination site or hot spot in the vicinity of intron 4. Contrary to previous impressions thatQ8/9 is an inert pseudogene, we find that theQ8/9 gene can be functional and encode a Qa-2,3 antigen. One variety of the 11 Q8/9 clones isolated lacked exon 5, which encodes the transmembrane domain of class I glycoproteins, and thus may account for secretion of a soluble form of Qa-2,3 antigen thought to be released by activated T cells.     

Completion of the mouse aggrecan gene structure and identification of the defect in the cmd-Bc mouse as a near complete deletion of the murine aggrecan gene

Mouse cartilage matrix deficiency (cmd), an autosomal recessive phenotype caused by absence of aggrecan, maps to Chromosome (Chr) 7 and is caused by a 7-bp deletion in exon 5 generating a premature stop codon (Watanabe et al. 1994). Another spontaneous mutation with the same locus and phenotype, cmd-Bc, has now been defined as the complete loss of exons 2 to 18, resulting in a significantly shortened mRNA (1.2 kb). The upstream breakpoint is in intron 1, 18.8 kb 3′ of exon 1; the downstream breakpoint lies 10.5 kb past the final aggrecan exon 18. The deletion is flanked by sequences homologous to topoisomerase I and II cleavage sites and a 7-bp direct repeat, suggesting the defect resulted from a nonhomologous recombination event. Additionally, the size of the first intron and the intron-exon structure between exons 12 and 14 were determined, establishing the length of the murine aggrecan gene as 68.6 kb. This report completes the structural analysis of the murine aggrecan gene, defines a second null mutation, and reinforces the importance of aggrecan in development. Received: 20 May 1999 / Accepted: 26 July 1999     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.