Simian homologues of human herpesviruses and implications for novel viral introduction to free-living mountain gorillas.

The endangered mountain gorilla (Gorilla beringei beringei) in Rwanda, Uganda, and the Democratic Republic of Congo is frequently in contact with humans through tourism, research activities, and illegal entry of people into protected gorilla habitat. Herpesviruses, which are ubiquitous in primates, have the potential to be shared in any setting where humans and gorillas share habitat. Based on serological findings and clinical observations of orofacial ulcerated lesions resembling herpetic lesions, an alpha-herpesvirus resembling human herpes simplex virus type 1 (HSV-1) has long been suspected to be present in human-habituated mountain gorillas in the wild. While the etiology of orofacial lesions in the wild has not been confirmed, HSV-1 has been suspected in captively-housed mountain gorillas and confirmed in a co-housed confiscated Grauer's gorilla (Gorilla beringei graueri). To better characterize herpesviruses infecting mountain gorillas and to determine the presence/absence of HSV-1 in the free-living population, we conducted a population-wide survey to test for the presence of orally shed herpesviruses. DNA was extracted from discarded chewed plants collected from 294 individuals from 26 groups, and samples were screened by polymerase chain reaction using pan-herpesvirus and HSV-1-specific assays. We found no evidence that human herpesviruses had infected free-ranging mountain gorillas. However, we found gorilla-specific homologs to human herpesviruses, including cytomegaloviruses (GbbCMV-1 and 2), a lymphocryptovirus (GbbLCV-1), and a new rhadinovirus (GbbRHV-1) with similar characteristics (i.e., timing of primary infection, shedding in multiple age groups, and potential modes of transmission) to their human counterparts, human cytomegalovirus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, respectively.

Fragmented mitochondrial genomes of seal lice (family Echinophthiriidae) and gorilla louse (family Pthiridae): frequent minichromosomal splits and a host switch of lice between seals.

BACKGROUND: The mitochondrial (mt) genomes of 15 species of sucking lice from seven families have been studied to date. These louse species have highly dynamic, fragmented mt genomes that differ in the number of minichromosomes, the gene content, and gene order in a minichromosome between families and even between species of the same genus. RESULTS: In the present study, we analyzed the publicly available data to understand mt genome fragmentation in seal lice (family Echinophthiriidae) and gorilla louse, Pthirus gorillae (family Pthiridae), in particular the role of minichromosome split and minichromosome merger in the evolution of fragmented mt genomes. We show that 1) at least three ancestral mt minichromosomes of sucking lice have split in the lineage leading to seal lice, 2) one minichromosome ancestral to primate lice has split in the lineage to the gorilla louse, and 3) two ancestral minichromosomes of seal lice have merged in the lineage to the northern fur seal louse. Minichromosome split occurred 15-16 times in total in the lineages leading to species in six families of sucking lice investigated. In contrast, minichromosome merger occurred only four times in the lineages leading to species in three families of sucking lice. Further, three ancestral mt minichromosomes of sucking lice have split multiple times independently in different lineages of sucking lice. Our analyses of mt karyotypes and gene sequences also indicate the possibility of a host switch of crabeater seal louse to Weddell seals. CONCLUSIONS: We conclude that: 1) minichromosome split contributes more than minichromosome merger in mt genome fragmentation of sucking lice, and 2) mt karyotype comparison helps understand the phylogenetic relationships between sucking louse species.

Adaptation to Host-Specific Bacterial Pathogens Drives Rapid Evolution of a Human Innate Immune Receptor.

The selective pressure by infectious agents is a major driving force in the evolution of humans and other mammals. Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as receptors for bacterial pathogens of the genera Haemophilus, Helicobacter, Neisseria, and Moraxella, which engage CEACAMs via distinct surface adhesins. While microbial attachment to epithelial CEACAMs facilitates host colonization, recognition by CEACAM3, a phagocytic receptor expressed by granulocytes, eliminates CEACAM-binding bacteria. Sequence analysis of primate CEACAM3 orthologs reveals that this innate immune receptor is one of the most rapidly evolving human proteins. In particular, the pathogen-binding extracellular domain of CEACAM3 shows a high degree of non-synonymous versus synonymous nucleotide exchanges, indicating an exceptionally strong positive selection. Using CEACAM3 domains derived from different primates, we find that the amino acid alterations found in CEACAM3 translate into characteristic binding patterns for bacterial adhesins. One such amino acid residue is F62 in human and chimp CEACAM3, which is not present in other primates and which is critical for binding the OMP P1 adhesin of Haemophilus aegyptius. Incorporation of the F62-containing motif into gorilla CEACAM3 results in a gain-of-function phenotype with regard to phagocytosis of H. aegyptius. Moreover, CEACAM3 polymorphisms found in human subpopulations widen the spectrum of recognized bacterial adhesins, suggesting an ongoing multivariate selection acting on this innate immune receptor. The species-specific detection of diverse bacterial adhesins helps to explain the exceptionally fast evolution of CEACAM3 within the primate lineage and provides an example of Red Queen dynamics in the human genome.

Detection and analysis of ancient segmental duplications in mammalian genomes.

Although segmental duplications (SDs) represent hotbeds for genomic rearrangements and emergence of new genes, there are still no easy-to-use tools for identifying SDs. Moreover, while most previous studies focused on recently emerged SDs, detection of ancient SDs remains an open problem. We developed an SDquest algorithm for SD finding and applied it to analyzing SDs in human, gorilla, and mouse genomes. Our results demonstrate that previous studies missed many SDs in these genomes and show that SDs account for at least 6.05% of the human genome (version hg19), a 17% increase as compared to the previous estimate. Moreover, SDquest classified 6.42% of the latest GRCh38 version of the human genome as SDs, a large increase as compared to previous studies. We thus propose to re-evaluate evolution of SDs based on their accurate representation across multiple genomes. Toward this goal, we analyzed the complex mosaic structure of SDs and decomposed mosaic SDs into elementary SDs, a prerequisite for follow-up evolutionary analysis. We also introduced the concept of the breakpoint graph of mosaic SDs that revealed SD hotspots and suggested that some SDs may have originated from circular extrachromosomal DNA (ecDNA), not unlike ecDNA that contributes to accelerated evolution in cancer.

Activity analysis of LTR12C as an effective regulatory element of the RAE1 gene.

Ribonucleic acid export 1 (RAE1) plays an important role in the export of mature mRNAs from the nucleus to the cytoplasm. Long terminal repeats (LTRs) became integrated into the human genome during primate evolution. One such repeat element, LTR12C, lies within a predicted regulatory region located upstream of the RAE1 gene. We examined the transcriptional activity of LTR12C by using the luciferase assay, and showed that the tandem repeat region (TRR) located within LTR12C was required for its regulatory function. A bioinformatics analysis revealed that the LTR12C element had multiple transcription factor binding sites specific for nuclear transcription factor Y (NF-Y), and the promoter activity of LTR12C was significantly decreased after NF-Y knockdown. Additionally, we discovered novel data indicating that LTR12C was initially inserted into the gorilla genome. Taken together, our results reveal that the TRR of LTR12C has powerful regulatory activity due to its NF-Y binding sites, and the integration of the LTR12C element into the primate genome during evolution may have affected RAE1 transcription.

Functional Implications of Human-Specific Changes in Great Ape microRNAs.

microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species.

An Sp1 Modulated Regulatory Region Unique to Higher Primates Regulates Human Androgen Receptor Promoter Activity in Prostate Cancer Cells.

Androgen receptor (AR) mediated signalling is necessary for normal development of the prostate gland and also drives prostate cancer (PCa) cell growth and survival, with many studies showing a correlation between increased receptor levels and therapy resistance with progression to fatal castrate recurrent PCa (CRPC). Although it has been held for some time that the transcription factor Sp1 is the main stimulator of AR gene transcription, comprehensive knowledge of the regulation of the AR gene remains incomplete. Here we describe and characterise in detail two novel active regulatory elements in the 5'UTR of the human AR gene. Both of these elements contain overlapping binding sites for the positive transcription factor Sp1 and the repressor protein pur-α. Aberrant cell signalling is characteristic of PCa and the transcriptional activity of the AR promoter in PCa cells is dependent upon the relative amounts of the two transcription factors. Together with our corroboration of the dominant role of Sp1, the findings support the rationale of targeting this transcription factor to inhibit tumour progression. This should be of particular therapeutic relevance in CRPC where the levels of the repressor pur-α are reduced.

Comprehensive assembly of novel transcripts from unmapped human RNA-Seq data and their association with cancer.

Crucial parts of the genome including genes encoding microRNAs and noncoding RNAs went unnoticed for years, and even now, despite extensive annotation and assembly of the human genome, RNA-sequencing continues to yield millions of unmappable and thus uncharacterized reads. Here, we examined > 300 billion reads from 536 normal donors and 1,873 patients encompassing 21 cancer types, identified ~300 million such uncharacterized reads, and using a distinctive approach de novo assembled 2,550 novel human transcripts, which mainly represent long noncoding RNAs. Of these, 230 exhibited relatively specific expression or non-expression in certain cancer types, making them potential markers for those cancers, whereas 183 exhibited tissue specificity. Moreover, we used lentiviral-mediated expression of three selected transcripts that had higher expression in normal than in cancer patients and found that each inhibited the growth of HepG2 cells. Our analysis provides a comprehensive and unbiased resource of unmapped human transcripts and reveals their associations with specific cancers, providing potentially important new genes for therapeutic targeting.

Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2.

Humans and primates are long-lived animals with long reproductive phases. One factor that appears to contribute to longevity and fertility in humans, as well as to cancer-free survival, is the transcription factor and tumor suppressor p53, controlled by its main negative regulator MDM2. However, p53 and MDM2 homologs are found throughout the metazoan kingdom from Trichoplacidae to Hominidae. Therefore the question arises, if p53/MDM2 contributes to the shaping of primate features, then through which mechanisms. Previous findings have indicated that the appearances of novel p53-regulated genes and wild-type p53 variants during primate evolution are important in this context. Here, we report on another mechanism of potential relevance. Human endogenous retrovirus K subgroup HML-2 (HERV-K(HML-2)) type 1 proviral sequences were formed in the genomes of the predecessors of contemporary Hominoidea and can be identified in the genomes of Nomascus leucogenys (gibbon) up to Homo sapiens. We previously reported on an alternative splicing event in HERV-K(HML-2) type 1 proviruses that can give rise to nuclear protein of 9 kDa (Np9). We document here the evolution of Np9-coding capacity in human, chimpanzee and gorilla, and show that the C-terminal half of Np9 binds directly to MDM2, through a domain of MDM2 that is known to be contacted by various cellular proteins in response to stress. Np9 can inhibit the MDM2 ubiquitin ligase activity toward p53 in the cell nucleus, and can support the transactivation of genes by p53. Our findings point to the possibility that endogenous retrovirus protein Np9 contributes to the regulation of the p53-MDM2 pathway specifically in humans, chimpanzees and gorillas.

Replication profile of PCDH11X and PCDH11Y, a gene pair located in the non-pseudoautosomal homologous region Xq21.3/Yp11.2.

In order to investigate the replication timing properties of PCDH11X and PCDH11Y, a pair of protocadherin genes located in the hominid-specific non-pseudoautosomal homologous region Xq21.3/Yp11.2, we conducted a FISH-based comparative study in different human and non-human primate (Gorilla gorilla) cell types. The replication profiles of three genes from different regions of chromosome X (ZFX, XIST and ATRX) were used as terms of reference. Particular emphasis was given to the evaluation of allelic replication asynchrony in relation to the inactivation status of each gene. The human cell types analysed include neuronal cells and ICF syndrome cells, considered to be a model system for the study of X inactivation. PCDH11 appeared to be generally characterized by replication asynchrony in both male and female cells, and no significant differences were observed between human and gorilla, in which this gene lacks X-Y homologous status. However, in differentiated human neuroblastoma and cerebral cortical cells PCDH11X replication profile showed a significant shift towards allelic synchrony. Our data are relevant to the complex relationship between X-inactivation, as a chromosome-wide phenomenon, and asynchrony of replication and expression status of single genes on chromosome X.

Primate TNF promoters reveal markers of phylogeny and evolution of innate immunity.

BACKGROUND: Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. CONCLUSIONS/SIGNIFICANCE: Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF.

Haplotype structure of FSHB, the beta-subunit gene for fertility-associated follicle-stimulating hormone: possible influence of balancing selection.

Follicle-stimulating hormone (FSH) is essential for human reproduction. The unique functions of this hormone are provided by the FSH receptor-binding beta-subunit encoded by the FSHB gene. Resequencing and genotyping of FSHB in three European, two Asian and one African population, as well as in the great apes (chimpanzee, gorilla, orangutan), revealed low diversity and significant excess of polymorphisms with intermediate frequency alleles. Statistical tests for FSHB showed deviations from neutrality in all populations suggesting a possible effect of balancing selection. Two core haplotypes were identified (carried by 76-96.6% of each population's sample), the sequences of which are clearly separated from each other. As fertility most directly affects an organism's fitness, the carriers of these haplotypes have apparently had more success in human history to contribute to the next generation. There is a preliminary observation suggesting that the second most frequent FSHB haplotype may be associated with rapid conception success in females. Interestingly, the same haplotype is related to an ancestral FSHB variant shared with the ancestor of the great apes. The determination of the functional consequence of the two core FSHB variants may have implications for understanding and regulating human fertility, as well as in assisting infertility treatments.

Adaptive selection of mitochondrial complex I subunits during primate radiation.

Mammalian oxidative phosphorylation (OXPHOS) complexes I, III, IV and V are assembled from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) encoded subunits, with complex I encompassing 39 nDNA and seven mtDNA subunits. Yet the sequence variation of the mtDNA genes is more than ten fold greater than that of the nDNA encoded genes of the OXPHOS complexes and the mtDNA proteins have been found to be influenced by positive (adaptive) selection. To maintain a functional complex I, nDNA and mtDNA subunits must interact, implying that certain nDNA complex I genes may also have been influenced by positive selection. To determine if positive selection has influenced nDNA complex I genes, we analyzed the DNA sequences of all of the nDNA and mtDNA encoded complex I subunits from orangutan, gorilla, chimpanzee, human and all available vertebrate sequences. This revealed that three nDNA complex I genes (NDUFC2, NDUFA1, and NDUFA4) had significantly increased amino acid substitution rates by both PAML and Z-test, suggesting that they have been subjected to adaptive selection during primate radiation. Since all three of these subunits reside in the membrane domain of complex I along with the mtDNA subunits, we compared amino acid changes in these three nDNA genes with those of the mtDNA genes across species. Changes in the nDNA NDUFC2 cysteine 39 were found to correlate with those in the mtDNA ND5 cysteine 330. Therefore, adaptive selection has influenced some nDNA complex I genes and nDNA and mtDNA complex I genes may have co-evolved.

The SPANX gene family of cancer/testis-specific antigens: rapid evolution and amplification in African great apes and hominids.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes comprise a gene family with five known members (SPANX-A1, -A2, -B, -C, and -D), encoding cancer/testis-specific antigens that are potential targets for cancer immunotherapy. These highly similar paralogous genes cluster on the X chromosome at Xq27. We isolated and sequenced primate genomic clones homologous to human SPANX. Analysis of these clones and search of the human genome sequence revealed an uncharacterized group of genes, SPANX-N, which are present in all primates as well as in mouse and rat. In humans, four SPANX-N genes comprise a series of tandem duplicates at Xq27; a fifth member of this subfamily is located at Xp11. Similarly to SPANX-A/D, human SPANX-N genes are expressed in normal testis and some melanoma cell lines; testis-specific expression of SPANX is also conserved in mouse. Analysis of the taxonomic distribution of the long and short forms of the intron indicates that SPANX-N is the ancestral form, from which the SPANX-A/D subfamily evolved in the common ancestor of the hominoid lineage. Strikingly, the coding sequences of the SPANX genes evolved much faster than the intron and the 5' untranslated region. There is a strong correlation between the rates of evolution of synonymous and nonsynonymous codon positions, both of which are accelerated 2-fold or more compared to the noncoding sequences. Thus, evolution of the SPANX family appears to have involved positive selection that affected not only the protein sequence but also the synonymous sites in the coding sequence.

PyEvolve: a toolkit for statistical modelling of molecular evolution.

BACKGROUND: Examining the distribution of variation has proven an extremely profitable technique in the effort to identify sequences of biological significance. Most approaches in the field, however, evaluate only the conserved portions of sequences - ignoring the biological significance of sequence differences. A suite of sophisticated likelihood based statistical models from the field of molecular evolution provides the basis for extracting the information from the full distribution of sequence variation. The number of different problems to which phylogeny-based maximum likelihood calculations can be applied is extensive. Available software packages that can perform likelihood calculations suffer from a lack of flexibility and scalability, or employ error-prone approaches to model parameterisation. RESULTS: Here we describe the implementation of PyEvolve, a toolkit for the application of existing, and development of new, statistical methods for molecular evolution. We present the object architecture and design schema of PyEvolve, which includes an adaptable multi-level parallelisation schema. The approach for defining new methods is illustrated by implementing a novel dinucleotide model of substitution that includes a parameter for mutation of methylated CpG's, which required 8 lines of standard Python code to define. Benchmarking was performed using either a dinucleotide or codon substitution model applied to an alignment of BRCA1 sequences from 20 mammals, or a 10 species subset. Up to five-fold parallel performance gains over serial were recorded. Compared to leading alternative software, PyEvolve exhibited significantly better real world performance for parameter rich models with a large data set, reducing the time required for optimisation from approximately 10 days to approximately 6 hours. CONCLUSION: PyEvolve provides flexible functionality that can be used either for statistical modelling of molecular evolution, or the development of new methods in the field. The toolkit can be used interactively or by writing and executing scripts. The toolkit uses efficient processes for specifying the parameterisation of statistical models, and implements numerous optimisations that make highly parameter rich likelihood functions solvable within hours on multi-cpu hardware. PyEvolve can be readily adapted in response to changing computational demands and hardware configurations to maximise performance. PyEvolve is released under the GPL and can be downloaded from http://cbis.anu.edu.au/software.

Cataract surgery with foldable intraocular lens implants in captive lowland gorillas (Gorilla gorilla gorilla).

Two juvenile, male, captive-born lowland gorillas (Gorilla gorilla gorilla) with the same father but different mothers developed bilateral cataracts. The cataracts were surgically removed within 6 yr and 3 mo, respectively, of diagnosis, and foldable intraocular lenses were implanted. Although vision was not restored in one eye with a mature, 6-yr-old cataract in gorilla A, surgical intervention on the other eye was performed before a complete cataract developed, and vision was fully restored. Gorilla B was treated at the age of 17 mo, and normal visual development proceeded in both eyes. This animal developed bilateral after-cataract and therefore needed a second intervention in both eyes using Nd:YAG laser treatment. The genetic component of juvenile cataracts should be considered in breeding management programs.

Possible polyphyletic origin of major histocompatibility complex class I chain-related gene A (MICA) alleles.

Phylogenetic relationships among 23 nonhuman primate (NHP) major histocompatibility complex class I chain-related gene (MIC) sequences, 54 confirmed human MICA alleles, and 16 human MICE alleles were constructed with methods of sequence analysis. Topology of the phylogenetic tree showed separation between NHP MICs and human MICs. For human MICs, the topology indicated monophyly for the MICB alleles, while MICA alleles were separated into two lineages, LI and LII. Of these, LI MICA alleles shared a common ancestry with gorilla (Ggo) MIC. One conservative amino acid difference and two nonconservative amino acid differences in the alpha3 domain were found between the MICA lineages. The nonconservative amino acid differences might imply structural and functional differences. Transmembrane (TM) trinucleotide-repeat variants were found to be specific to the MICA lineages such as A4, A9, and A10 to LI and A5 to LII. Variants such as A5.1 and A6 were commonly found in both MICA lineages. Based on these analyses, we postulate a polyphyletic origin for MICA alleles and their division into two lineages, LI and LII. As such, there would be 30 alleles in LI and 24 alleles in LII, thereby reducing the current level of polymorphism that exists, based on a presumed monophyletic origin. The lower degree of polymorphism in MICA would then be in line with the rest of the human major histocompatibility complex nonclassical class I genes.

Mutations in the factor IX gene (F9) during the past 150 years have relative rates similar to ancient mutations.

Pollutants and dietary mutagens have been associated with somatic mutation and cancer, but the extent of their influence on germline mutation is not clear. Since deleterious germline mutations can be transmitted for thousands of years, any influence on germline mutation from the vast increase in man-made chemicals of the past 150 years would be an important public health issue. Observed disease causing mutations in the X-linked factor IX gene (F9) of hemophilia B patients originated predominantly in the past 150 years, since the half-life of these mutations in human populations had been about two generations before effective treatment became available about a generation ago. Recent changes in germline mutational processes may be detected by comparison of the observed hemophilia B causing mutation pattern in F9 with the pattern of neutral polymorphisms which occurred over a much longer period of time. By scanning a total of 1.5 megabases of deep intronic regions of F9 in the genomic DNA from 84 individuals, 42 neutral polymorphisms were found in 23 haplotypes that differed by at least 11 mutations from the ancestral primate haplotype. By sequencing F9 in seven non-human primates, 39 of these polymorphisms were characterized as ancient mutations relative to a unanimous ancestral primate allele. This ancient mutation pattern was compared to the recent pattern of hemophilia B causing mutations. Remarkably, no significant difference was found (P=0.5), suggesting that the vast increase in man-made chemicals during the past 150 years has not had a major impact on the pattern of human germline mutation. This result is consistent with the hypothesis that endogenous processes dominate germline mutation.

CAG repeat instability at SCA2 locus: anchoring CAA interruptions and linked single nucleotide polymorphisms.

Spinocerebellar ataxia 2 (SCA2) is an autosomal dominant neurodegenerative disorder that results from the expansion of a cryptic CAG repeat within the exon 1 of the SCA2 gene. The CAG repeat in normal individuals varies in length from 14 to 31 repeats and is frequently interrupted by one or more CAA triplets, whereas the expanded alleles contain a pure uninterrupted stretch of 34 to 59 CAG repeats. We have previously reported the presence of a limited pool of 'ancestral' or 'at risk' haplotypes for the expanded SCA2 alleles in the Indian population. We now report the identification of two novel single nucleotide polymorphisms (SNPs) in exon 1 of the SCA2 gene and their characterization in 215 normal and 64 expanded chromosomes. The two biallelic SNPs distinguished two haplotypes, GT and CC, each of which formed a predominant haplotype associated with normal and expanded SCA2 alleles. All the expanded alleles segregated with CC haplotype, which otherwise was associated with only 29.3% of the normal chromosomes. CAA interspersion analysis revealed that majority of the normal alleles with CC haplotype were either pure or lacked the most proximal 5' CAA interruption. The repeat length variation at SCA2 locus also appeared to be polar with changes occurring mostly at the 5' end of the repeat. Our results demonstrate that CAA interruptions play an important role in conferring stability to SCA2 repeat and their absence predisposes alleles towards instability and pathological expansion. Our study also provides new haplotypes associated with SCA2 that should prove useful in further understanding the mutational history and mechanism of repeat instability at the SCA2 locus.