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Characterization & evolutionary analysis of human CD36 gene.

Background and objectives: Understanding evolutionary genetic details of immune system genes responsible for infectious diseases is of prime importance concerning disease pathogenecity. Considering malaria as a devastating disease in the world including India, detail evolutionary understanding on human immune system gene is essential. The primary aim of this study was to initiate work on one such gene, the human CD36 gene responsible in malaria pathogenesis.

Methods: DNA sequences of the human CD36 gene was retrieved from public domain and fine-scale details were characterized. Both comparative and evolutionary analyses were performed with sequences from six other taxa (5 mammalian one avian) where CD36 homologs are present. Different statistical analyses were also performed.

Results: Differential distribution in number and length of exons and introns was detected in CD36 gene across seven taxa. The CpG islands were also found to be distributed unevenly across the gene and taxa. Neighbour-joining tree was constructed and it was observed that the chimpanzee and human are diverged at the CD36 gene relatively recently. The chicken, Gallus gallus was found to be diverged from rest of the taxa significantly. Also copy number variation was observed across different taxa.

Interpretation & conclusions: Comparative genomic study of a human immune system gene CD36 show relationships among different taxa at the evolutionary level. The information can be of help to study genetic diversity in malaria endemic zones and to correlate it with malaria pathogenecity.

Key words CD36--comparative genomics--evolution--malaria--phylogenetics

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Comparative genomic analyses play an important role in understanding differential organizations of genome across different taxa and in understanding how genomes have evolved over time. With the availability of whole-genome sequence information of an array of taxa from different branches of the tree of life, it has now become easy to compare genes and genomes across different taxa. This approach also helps in finding hitherto unknown genes, regulatory networks, and regions of the genome that are important in biological functions and thus determining the precise role of evolution in genomes. This whole process should start with computational approach, first in characterization of genes in a particular species of importance and looking at homologous DNA sequences that are conserved across different taxa, followed by comparison of length of genes and introns and exons (in nucleotides), number of introns and exons, distribution of CpG islands, etc. Such types of studies provide comprehensive information that helps in understanding how genes are affected by different evolutionary forces. Further, determination of gene copy number variations is also to some extent responsible for bringing evolutionary changes which accounts for a substantial amount of genetic variation (1). Current efforts are directed toward a more comprehensive cataloguing and characterization of genes that might provide the basis for determining how genomic diversity impacts biological function, evolution, and common human diseases (2). This is of special importance, since genetic control and prevention strategies could be undertaken once complete knowledge on character and evolutionary pattern of disease-related genes in genome are at hand.

To this respect, the human CD36 gene, located on chromosome 7 and encoded by 15 coding regions (including one un-translated region) (3) (Fig. 1) is of utmost importance, as it serves as a major receptor for the human malaria parasite Plasmodium falciparum (4). It has been reported that inhibition of the immune response to platelet-mediated clumping of parasite-infected erythrocytes is strongly associated with severe malaria and that CD36 gene expression is required for such clumping (5). Moreover it has been reported that a non-sense mutation (T188G) in CD36 gene is also associated with protection to severe malaria (6). CD36 is not only involved in the sequestration of the parasite, but could also play a role in the innate and acquired immune response to malaria infection (7). This makes the CD36 gene very important as human malaria is concerned, and demands detailed fine-scale genetic evolutionary understanding.

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We therefore conducted a study for detailed characterization of the CD36 gene and to establish evolutionary relationships among different taxa following computational genomic approaches. Specifically, we determined various aspects of introns particularly the length, number and relationship to CpG islands across different taxa, etc. Evolutionary relationships based on CD36 gene were inferred among all the studied taxa through phylogenetic analysis. We also determined number of copies of CD36 gene in each individual taxon and compared its variation across these different taxa.

Material & Methods

Nucleotide sequences of CD36 gene of seven different taxa Mus musculus (GenBank Acc. No.NM_007643.3), Rattus norvegicus (GenBank Acc. No.NM_031561.2), Pan troglodytes (GenBank Acc. No.XM_519573.2), Macaca mulatta (GenBank Acc. No.NM_001032913.1), Homo sapiens (GenBank Acc. No.NM_001032913.1), Canis familiaris (GenBank Acc. No. XM_533140.2) and Gallus gallus (GenBank Acc. No.NM_ 001030731.1) were downloaded from National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/) during August/ September 2007. Out of seven taxa considered, six were mammalian and one avian (G. gallus). Characterization (total gene length, intron and exon number and length, ratio of coding nucleotides to total gene size, details of exons) of CD36 gene was done following information at the NCBI website. For sequence analysis, different statistical and web resources were used. Multiple sequence alignments and construction of neighbour-joining phylogenetic tree were performed using MegAlign, a part of Lasergene (DNASTAR software, Madison, USA, http://www.dnastar.com). Individual branch lengths were calculated using phylogeny option in the statistical program VEGAZZ (downloaded from http://www.vegazz.net). Consensus sequences of CD36 gene were searched in individual taxa. For this, the 'BLAST' option at the NCBI website was used. The hits showing more than 80 per cent homologous DNA sequences were considered as copies of CD36 gene in those particular taxa. Pearson's correlation coefficients were calculated using 'Analyze-it' (http://www.analyse-it.com/) software, an add-on to the MS Excel software. For all statistical analysis, P<0.05 was considered as level of significance. Determination of CpG islands in the introns of CD36 genes was conducted using the website http://www.hgmp.mrc.ac.uk/Software/EMBOSS/ Apps/cpgplot.html. This program defines, by default, a CpG island as a region where the GC level is over 50 per cent, the calculated observed/expected (O/E) CpG ratio is over 0.6 and these two conditions hold for a minimum of 200 continuous nucleotide bases. Short and long introns are defined according to description provided by Gazave and co-workers (8), where it was suggested that introns of more than 1029 bp nucleotides be considered as long and less than 1029 bp as short introns. Similar guidelines were followed in the present analysis.

Results

Characterization in all seven taxa revealed a variable size of CD36 gene [minimum of 29058 nucleotide base pair (bp) in M. mulatta and maximum of 74804 bp in H. sapiens]. A maximum of 15 exons were found (including untranslated regions of exons) in three taxa (H. sapiens, P troglodytes, and C. familiaris) and a minimum of 12 exons (including untranslated regions of exons) in M. mulatta (Table). This gene is located in different chromosomes in different taxa and the exon-intron ratio varies widely across taxa. It was evident that C. familiaris had the highest ratio and H. sapiens had the lowest, signifying that in H. sapiens, the non-coding nucleotides are at abundance. The details of exon-intron structure of CD36 gene (including the UTRs in blue colour) in each taxa is shown in Fig. 2. The size of CD36 gene in each taxa and relative size of exon and intron are shown in Fig. 3. The figure revealed the differential size distribution of both exons and introns across all the taxa. It was evident that the non-coding nucleotides constituted a major portion of the total nucleotides of the CD36 gene and that the size of the gene is somehow dependent on the size of the introns in all taxa. This claim was further substantiated by the observation of statistically significant positive correlation (r = 1, P<0.001). Further, the percentage of coding nucleotides in the CD36 gene varied across taxa; the highest was detected in M. musculus (9.3% of the total nucleotides) and lowest in H. sapiens (2.9%; Fig. 4).

In order to test the hypothesis that the first intron of a gene is usually bigger in size in comparison to the rest, the size of the first introns of CD36 gene was determined in each taxon. All the introns of each taxa were plotted separately based on sizes (Fig. 5). The length of first intron was largest for almost all the taxa except C. familiaris and M. musculus. The second largest intron was found to be the third one, in general.

It was suggested that the first introns contain about ten-fold number of CpG islands (8). To find if this contention is valid in the CD36 gene, CpG islands were determined in the first, long and short introns (8) in all the taxa. The figure (Fig. 6) depicts the CpG islands in the first introns separately for all the taxa. The occurrence of CpG islands varied across different taxa for both the long and short introns (Figure not shown). Contrary to the expectation (8), GC poor regions were found in short introns in most of the taxa (Fig. 6).

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With a view to understand phylogenetic relationships among all the seven taxa at CD36 gene, an un-rooted neighbour joining (NJ) tree was constructed (Fig. 7). The length of each branch is also shown in the NJ tree which is highly variable across taxa. It was clearly seen in the NJ tree that P. troglodytes and H. sapiens were closely related to each other and belonged to one clade. The avian sequence from G. gallus fell in a completely separate branch. Also, M. musculus and M. mulatta sequences seemed to have diverged from a single common ancestor in recent past.

Since it is known that many immune system genes are present in multiple copies in the genome and CD36 gene is one of the very important human immune system genes, we were interested in finding whether multiple copies of this gene are present in each taxon. Thus the whole genome of each taxa was scanned separately for extra copy of CD36 gene. In all the taxa, more than one copy of CD36 gene was found, except M. musculus. However, there seemed to be a great variation in copy number across taxa (Fig. 8). A minimum of only one extra copy of CD36 gene was found in M. musculus, whereas a maximum of 55 copies were detected in the avian taxa, G. gallus. Comparing only the six mammalian taxa, C. familiaris seemed to possess the highest number (37 copies) of CD36 gene. On the basis of copy numbers, H. sapiens (11 copies) appeared to bear somehow a close resemblance to M. mulatta (10 copies) and P. troglodytes (7 copies). Similarly, M. musculus (single copy) and R. norvegicus (two copies) appeared to be close to each other in this characteristic. Further, in order to understand if the copy numbers in some respect relates to the size of gene, intron and exon, correlation coefficients (r) were calculated. In all cases the r values were found to be negative but not statistically significant (data not shown).

Discussion

Our study on characterization and comparative analysis of CD36 gene in different taxa revealed several interesting features, both on basic understanding and evolutionary perspectives of this gene. This gene is known to be responsible, although partly, for human malaria pathogenecity (3-7). The size of the gene, introns, exons, number of exons and introns in each taxon, size of individual introns and exons and the ratio of coding to the non coding regions varied considerably across taxa. Further, location and exon-intron ratio of this gene also varied across taxa. In humans, the non coding nucleotides are in abundance (9,10), signifying the fact that human CD36 gene contains more non coding nucleotides in comparison to other studied taxa. Although the fact that whether accumulations of non coding DNA has helped in increasing the overall genome size (11) is still debated, our findings, though restricted to a single gene, seemed to corroborate this hypothesis. This is further reflected from the overall percentage of coding nucleotides in each taxa, where H. sapiens contained the least percentage of coding regions in CD36 gene. Further, detection of a statistically significant positive correlation between intron length and gene length signified this fact.

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It has been postulated that the first intron of a gene is usually different from rest of the introns both in size and GC content. Usually the first introns are larger and contain substantial amount of GC nucleotides (8). In the CD36 gene, a very similar pattern of this intron was observed. Also, in general, a larger size of introns was observed at the third position in almost all the taxa which is a unique observation. However, in general longer introns were found in almost all the taxa. Further, CpG islands seemed to be abundantly distributed in the first introns of almost all the taxa analysed here. Since we observed a comparatively lower CpG content in short introns, this might explain that short introns harbour a relatively lesser proportion of regulatory elements than long introns (8). Hence, finding high number of long introns and more CpG islands in the long introns of CD36 gene suggests presence of more repetitive units (8). Since it is believed that genes containing short introns are generally highly expressed (12) and we detected long introns in CD36 genes, reflects the fact that CD36 might not be a very highly expressed gene (13). However, to further bolster this fact about CD36 gene, experimental evidences will be needed.

Phylogenetic analysis at a particular gene throws light on the evolutionary relationships among different taxa and also evolutionary paradigm of the gene. The phylogenetic analysis could clearly show that H. sapiens and P. troglodytes are very closely related to each other at CD36 gene and a recent divergence at this gene from a common ancestor might be certain. These two taxa were previously compared between themselves and amongst other taxa for other genes and it was shown that human and chimpanzee functional DNA were more similar to each other than either is to other apes (14). Although very identical findings were observed in terms of exon and intron numbers and gene length between M. musculus and R. norvegicus, at the phylogenetic level they were found to be placed in different branches. Thus, the CD36 gene seems to have evolved very differently than the rest of the genes across taxa. It became clear from the present study that the CD36 gene has evolved much to its extent across different taxa that have been studied here.

A wide range of copies of CD36 gene was found to be present in different taxa ranging from single to 55 copies. Although no precise information is available to correlate these two characters, the copy number variations found in the seven taxa reflected the fact that these might somehow be related to malaria pathogenesis. For example, the avian taxa (G. gallus) had the highest copy numbers of CD36 gene, inspite of reported malarial incidences. However in dog, C. familiaris, malaria incidence has not been reported at all but it also has a high copy number of CD36 gene. In other five primates (including humans), where malaria incidences are frequently reported, CD36 gene had very few copies in their respective genomes. However, detail functional analysis (particularly related to dosage dependence) would be necessary to evaluate this contention. Since copy number variations could be related to differential evolutionary paradigm and genetic diversity of the concerned gene (2), detail genetic diversity studies across taxa and populations of each individual taxon would be necessary to ascertain a definite correlation between the CD36 gene copy number and malaria pathogenesis.

In conclusion, this study provided new insights and comprehensive understanding of CD36 gene in different taxa. Considering that CD36 gene is related to malaria pathogenecity and disease severity, further molecular, evolutionary and genetic diversity studies would be needed to understand this gene and its role in malaria.

Acknowledgment

The authors thank Dr Surbhi Pal Chaudhary for help in the initial stage of work and Dr U. Sreehari for help in Fig. 6.

Received November 12, 2007

References

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(2.) Freeman JL, Perry GH, Feuk L, Redon R, McCarroll SA, Altshuler DM, et al. Copy number variation: new insights in genome diversity. Genome Res 2006; 16: 949-61.

(3.) Fernandez-aiz RE, Armesilla AL, Sanchez-Madrid F, Vega MA. Gene encoding the collagen type I and thrombospondin receptor CD36 is located on chromosome 7q11.2. Genomics 1993; 17 : 759-61.

(4.) Aitmau TJ, Cooper LD, Norsworthy PJ, Wahid FN, Gray JK, Curtis BR, et al. Malaria susceptibility and CD36 mutation. Nature 2006; 405 : 1015-6.

(5.) Omi K, Ohashi J, Patarapotikul J, Hananantachai H, Naka I, Looareesuwan S, et al. CD36 polymorphism is associated with protection from cerebral malaria. Am J Hum Genet 2003; 72 : 364-74.

(6.) Pain A, Urban BC, Kai O, Casals-Pascual C, Shafi J, Marsh K, et al. A non-sense mutation in CD36 gene is associated with protection from severe malaria. Lancet 2001; 357 : 1502-3.

(7.) Serghides L, Smith TG, Patel SN, Kain KC. CD36 and malaria: friends or foes? Trends Parasitol 2003; 19 : 461-9.

(8.) Gazave E, Marque's-Boner T, Fernando O, Charlesworth B, Navarro A. Patterns and rates of intron divergence between humans and chimpanzees. Genome Biol 2007; 8 : R21.

(9.) Bird CP, Stranger BE, Liu M, Thomas DJ, Ingle CE, Beazley C, et al. Fast-evolving non-coding sequences in the human genome. Genome Biol 2007; 8 : R118.

(10.) Ponting CP, Lunter G. Signatures of adaptive evolution within human non-coding sequence. Hum Mol Genet 2006; 15 : R170-5.

(11.) Sironi M, Menozzi G, Comi GP, Bresolin N, Cogliani R, Pozzoli U. Fixation of conserved sequences shapes human intron size and influences transposon-insertion dynamics. Trends Genet 2005; 21 : 484-8.

(12.) Castillo-auis D CI, Mekhedov SL, Hartl DL, Koonin EV, Kondrashov FA. Selection for short introns in highly expressed genes. Nat Genet 2002; 31 : 415-8.

(13.) Versteeg R, van Schaik BD, van Batenburg MF, Roos M, Monajemi R, Caron H, et al. The human transcriptome map reveals extremes in gene density, intron length, GC content and repeat pattern for domains of highly and weakly expressed genes. Genome Res 2003; 13: 1998-2004.

(14.) Khaitovich P, Hellmann I, Enard W, Nowick K, Leinweber M, Franz H, et al. Parallel patterns of evolution in the genomes and transcriptomes of humans and chimpanzees. Science 2005; 309 : 1850-4.

Reprint requests: Dr Aparup Das, Scientist 'D', Evolutionary Genomics & Bioinformatics Laboratory, National Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi 110 077, India e-mail: aparup@mrcindia.org

Gauri Awasthi, Aditya P. Dash & Aparup Das

Evolutionary Genomics & Bioinformatics Laboratory, National Institute of Malaria Research (ICMR) New Delhi, India
Table. Properties of CD36 gene in seven taxa studied

 Properties of CD36 gene

Taxa Chromosomal Total gene Total exon length
 location length (in bp) (in bp)

Mus musculus 5 53681 3016
Rattus norvegicus 4 53743 2551
Pai: troglodytes 7 39392 2201
Macaca mulatta 3 53681 3016
Homo sapiens 7 74804 2048
Canis familiaris 18 32945 2267
Gallus gallus 1 43316 2331

 Properties of CD36 gene

Taxa No. of exons with No. of exons Intron/Exon
 UTR without UTR ratio

Mus musculus 15 12 0.059
Rattus norvegicus 14 12 0.049
Pai: troglodytes 15 12 0.059
Macaca mulatta 11 10 0.043
Homo sapiens 15 14 0.030
Canis familiaris 15 12 0.073
Gallus gallus 14 12 0.056

Bp, nucleotide base pairs; UTR, un-translated regions
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Author:Awasthi, Gauri; Dash, Aditya P.; Das, Aparup
Publication:Indian Journal of Medical Research
Date:May 1, 2009
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