Min Zhuo

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.

Identifying Candidate Genes for Type 2 Diabetes Mellitus and Obesity through Gene Expression Profiling in Multiple Tissues or Cells

Type 2 Diabetes Mellitus (T2DM) and obesity have become increasingly prevalent in recent years. Recent studies have focused on identifying causal variations or candidate genes for obesity and T2DM via analysis of expression quantitative trait loci (eQTL) within a single tissue. T2DM and obesity are affected by comprehensive sets of genes in multiple tissues. In the current study, gene expression levels in multiple human tissues from GEO datasets were analyzed, and 21 candidate genes displaying high percentages of differential expression were filtered out. Specifically, DENND1B, LYN, MRPL30, POC1B, PRKCB, RP4-655J12.3, HIBADH, and TMBIM4 were identified from the T2DM-control study, and BCAT1, BMP2K, CSRNP2, MYNN, NCKAP5L, SAP30BP, SLC35B4, SP1, BAP1, GRB14, HSP90AB1, ITGA5, and TOMM5 were identified from the obesity-control study. The majority of these genes are known to be involved in T2DM and obesity. Therefore, analysis of gene expression in various tissues using GEO datasets may be an effective and feasible method to determine novel or causal genes associated with T2DM and obesity.

Characterization of the major histocompatibility complex class II DOB, DPB1, and DQB1 alleles in cynomolgus macaques of Vietnamese origin

Major histocompatibility complex (MHC) molecules play an important role in the susceptibility and/or resistance to many diseases. To gain an insight into the MHC background and to facilitate the experimental use of cynomolgus macaques, the second exon of the MhcMafa-DOB, -DPB1, and -DQB1 genes from 143 cynomolgus macaques were characterized by cloning to sequencing. A total of 16 Mafa-DOB, 16 Mafa-DPB1, and 34 Mafa-DQB1 alleles were identified, which revealed limited, moderate, and marked allelic polymorphism at DOB, DPB1, and DQB1, respectively, in a cohort of cynomolgus macaques of Vietnamese origin. In addition, 16 Mafa-DOB, 5 Mafa-DPB1, and 8 Mafa-DQB1 alleles represented novel sequences that had not been reported in earlier studies. Almost of the sequences detected at the DOB and DQB1 locus in the present study belonged to DOB*01 (100%) and DQB1*06 (62%) lineages, respectively. Interestingly, four, three, and one high-frequency alleles were detected at Mafa-DOB, -DPB1, and -DQB1, respectively, in this monkeys. The alleles with the highest frequency among these monkeys were Mafa-DOB*010102, Mafa-DPB1*13, and Mafa-DQB1*0616, and these were found in 33 (25.6%) of 129 monkeys, 32 (31.37%) of 102 monkeys, and 30 (31%) of 143 monkeys, respectively. The high-frequency alleles may represent high priority targets for additional characterization of immune function. We also carried out evolutionary and population analyses using these sequences to reveal population-specific alleles. This information will not only promote the understanding of MHC diversity and polymorphism in the cynomolgus macaque but will also increase the value of this species as a model for biomedical research.

An Effort to Use Human-Based Exome Capture Methods to Analyze Chimpanzee and Macaque Exomes

Non-human primates have emerged as an important resource for the study of human disease and evolution. The characterization of genomic variation between and within non-human primate species could advance the development of genetically defined non-human primate disease models. However, non-human primate specific reagents that would expedite such research, such as exon-capture tools, are lacking. We evaluated the efficiency of using a human exome capture design for the selective enrichment of exonic regions of non-human primates. We compared the exon sequence recovery in nine chimpanzees, two crab-eating macaques and eight Japanese macaques. Over 91% of the target regions were captured in the non-human primate samples, although the specificity of the capture decreased as evolutionary divergence from humans increased. Both intra-specific and inter-specific DNA variants were identified; Sanger-based resequencing validated 85.4% of 41 randomly selected SNPs. Among the short indels identified, a majority (54.6%–77.3%) of the variants resulted in a change of 3 base pairs, consistent with expectations for a selection against frame shift mutations. Taken together, these findings indicate that use of a human design exon-capture array can provide efficient enrichment of non-human primate gene regions. Accordingly, use of the human exon-capture methods provides an attractive, cost-effective approach for the comparative analysis of non-human primate genomes, including gene-based DNA variant discovery.

Comprehensive identification of high-frequency and co-occurring Mafa-B, Mafa-DQB1, and Mafa-DRB alleles in cynomolgus macaques of Vietnamese origin.

Abstract High-frequency alleles and/or co-occurring human leukocyte antigen (HLA) alleles across loci appear to be more important than individual alleles, because they might be markers of disease risk that have clinical value as biomarkers for targeted screening or the development of new therapies. To better elucidate the major histocompatibility complex background and to facilitate the experimental use of cynomolgus macaques, Mafa-B, Mafa-DQB1, and Mafa-DRB alleles were characterized and their combinations were investigated from 30 macaques of Vietnamese origin by cloning and sequencing. A total of 48 Mafa-B, 22 Mafa-DQB1, and 42 Mafa-DRB alleles, were detected in this study, respectively. In addition, two Mafa-DQB1 and eight Mafa-DRB alleles represented novel sequences that had not been documented in earlier studies. Our results also showed that the macaque from Vietnam might be valuable because >30% of the test animals possessed Mafa-DRB*w304 (30%) and -DQB1*0616 (30%). We report that the combination of major histocompatibility complex (MHC) class I and II alleles, including the combination of DRB3*0403-DRB*w304, DRB1*1013-DRB*w304, and Mafa-B*007:01:01-DRB*w304, which was in 17%, 13%, and 13% of the animals, respectively. Interesting, more than two Mafa-DQB1 alleles detected in one animal in this study suggest that Mafa-DQB1, like Mafa-DRB, might be a duplication in the chromosome, which have ever been documented in cynomolgus monkeys but has not yet been observed in rhesus macaques or other primates. Our results for the high frequency of commonly co-occurring MHC alleles across loci in a cohort of the Vietnamese cynomolgus macaque emphasized the value of this species as a model for biomedical research.

Identification of Mamu-DPA1, Mamu-DQA1, and Mamu-DRA alleles in a cohort of Chinese rhesus macaques

Rhesus macaques have long been used as animal models for various human diseases; the susceptibility and/or resistance to some of these diseases are related to the major histocompatibility complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, Mamu-DPA1, Mamu-DQA1, and Mamu-DRA alleles were investigated in 30 Chinese rhesus macaques by gene cloning and sequencing. A total of 14 Mamu-DPA1, 17 Mamu-DQA1, and 9 Mamu-DRA alleles were identified in this study. Of these alleles, 22 novel sequences have not been documented in earlier studies, including nine Mamu-DPA1, ten Mamu-DQA1, and three Mamu-DRA alleles. Interestingly, like Mafa-DQA1 and Mafa-DPA1, more than two Mamu-DQA1 and Mamu-DPA1 alleles were detected in one animal in this study, which suggested that they might represent gene duplication. If our findings can be validated by other studies, it will further increase the number of known Mamu-DPA1 and Mamu-DQA1 polymorphisms. Our data also indicated significant differences in MHC class II allele distribution among the Chinese rhesus macaques, Vietnamese cynomolgus macaques, and the previously reported rhesus macaques, which were mostly of Indian origin. This information will not only promote the understanding of Chinese rhesus macaque MHC diversity and polymorphism but will also facilitate the use of Chinese rhesus macaques in studies of human disease.

Twenty‐three novel major histocompatibility complex class I B alleles identified in cynomolgus macaques of Vietnamese origin

We report herein the identification of 23 novel Mafa-B alleles in cynomolgus macaques of Vietnamese origin.

Eighteen novel MHC class I A alleles identified in Vietnamese-origin cynomolgus macaques.

We report herein the identification of 18 novel Mafa-A alleles in cynomolgus macaques of Vietnamese origin.

Genome-Wide Analysis of Positively Selected Genes in Seasonal and Non-Seasonal Breeding Species

Some mammals breed throughout the year, while others breed only at certain times of year. These differences in reproductive behavior can be explained by evolution. We identified positively-selected genes in two sets of species with different degrees of relatedness including seasonal and non-seasonal breeding species, using branch-site models. After stringent filtering by sum of pairs scoring, we revealed that more genes underwent positive selection in seasonal compared with non-seasonal breeding species. Positively-selected genes were verified by cDNA mapping of the positive sites with the corresponding cDNA sequences. The design of the evolutionary analysis can effectively lower the false-positive rate and thus identify valid positive genes. Validated, positively-selected genes, including CGA, DNAH1, INVS, and CD151, were related to reproductive behaviors such as spermatogenesis and cell proliferation in non-seasonal breeding species. Genes in seasonal breeding species, including THRAP3, TH1L, and CMTM6, may be related to the evolution of sperm and the circadian rhythm system. Identification of these positively-selected genes might help to identify the molecular mechanisms underlying seasonal and non-seasonal reproductive behaviors.

Characterization of the major histocompatibility complex class I A alleles in cynomolgus macaques of Vietnamese origin.

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as an important animal model for infectious disease and transplantation research. Extensive characterization of their major histocompatibility complex (MHC) polymorphism regions therefore becomes urgently required. In this study, we identified 41 MHC class I A nucleotide sequences in 34 unrelated cynomolgus macaques of Vietnamese origin farmed in Southern China, including eight novel Mafa-A sequences. We found two sequences with perfect identity and six sequences with close similarity to previously defined MHC class I alleles from other populations, especially from Indonesian-origin macaques. We also found three Vietnamese-origin cynomolgus macaque MHC class I sequences for which the predicted protein sequences identical throughout their B and F binding pockets to Mamu-A1*001:01 and Mamu-A3*13:03, respectively. This is important because Mamu-A1*001:01 and Mamu-A3*13:03 are associated with longer survival and lower set-point viral load in simian immunodeficiency virus (SIV)-infected rhesus monkeys. These findings have implications for the evolutionary history of Vietnamese-origin cynomolgus macaque as well as for the use of this model in SIV/SHIV (a virus combining parts of the HIV and SIV genomes) research.