A Hypermutable Region in the DISP2 Gene Links to Natural Selection and Late-Onset Neurocognitive Disorders in Humans.

(CCG) short tandem repeats (STRs) are predominantly enriched in genic regions, mutation hotspots for C to T truncating substitutions, and involved in various neurological and neurodevelopmental disorders. However, intact blocks of this class of STRs are widely overlooked with respect to their link with natural selection. The human neuron-specific gene, DISP2 (dispatched RND transporter family member 2), contains a (CCG) repeat in its 5' untranslated region. Here, we sequenced this STR in a sample of 448 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N = 203) and controls (N = 245). We found that the region spanning the (CCG) repeat was highly mutated, resulting in several flanking (CCG) residues. However, an 8-repeat of the (CCG) repeat was predominantly abundant (frequency = 0.92) across the two groups. While the overall distribution of genotypes was not different between the two groups (p > 0.05), we detected four genotypes in the NCD group only (2% of the NCD genotypes, Mid-p = 0.02), consisting of extreme short alleles, 5- and 6-repeats, that were not detected in the control group. The patients harboring those genotypes received the diagnoses of probable Alzheimer's disease and vascular dementia. We also found six genotypes in the control group only (2.5% of the control genotypes, Mid-p = 0.01) that consisted of the 8-repeat and extreme long alleles, 9- and 10-repeats, of which the 10-repeat was not detected in the NCD group. The (CCG) repeat specifically expanded in primates. In conclusion, we report an indication of natural selection at a novel hypermutable region in the human genome and divergent alleles and genotypes in late-onset NhCDs and controls. These findings reinforce the hypothesis that a collection of rare alleles and genotypes in a number of genes may unambiguously contribute to the cognition impairment component of late-onset NCDs.

Dyads of GGC and GCC form hotspot colonies that coincide with the evolution of human and other great apes.

BACKGROUND: GGC and GCC short tandem repeats (STRs) are of various evolutionary, biological, and pathological implications. However, the fundamental two-repeats (dyads) of these STRs are widely unexplored. RESULTS: On a genome-wide scale, we mapped (GGC)2 and (GCC)2 dyads in human, and found monumental colonies (distance between each dyad < 500 bp) of extraordinary density, and in some instances periodicity. The largest (GCC)2 and (GGC)2 colonies were intergenic, homogeneous, and human-specific, consisting of 219 (GCC)2 on chromosome 2 (probability < 1.545E-219) and 70 (GGC)2 on chromosome 9 (probability = 1.809E-148). We also found that several colonies were shared in other great apes, and directionally increased in density and complexity in human, such as a colony of 99 (GCC)2 on chromosome 20, that specifically expanded in great apes, and reached maximum complexity in human (probability 1.545E-220). Numerous other colonies of evolutionary relevance in human were detected in other largely overlooked regions of the genome, such as chromosome Y and pseudogenes. Several of the genes containing or nearest to those colonies were divergently expressed in human. CONCLUSION: In conclusion, (GCC)2 and (GGC)2 form unprecedented genomic colonies that coincide with the evolution of human and other great apes. The extent of the genomic rearrangements leading to those colonies support overlooked recombination hotspots, shared across great apes. The identified colonies deserve to be studied in mechanistic, evolutionary, and functional platforms.

Novel islands of GGC and GCC repeats coincide with human evolution.

BACKGROUND: Because of high mutation rate, overrepresentation in genic regions, and link with various neurological, neurodegenerative, and movement disorders, GGC and GCC short tandem repeats (STRs) are prone to natural selection. Among a number of lacking data, the 3-repeats of these STRs remain widely unexplored. RESULTS: In a genome-wide search in human, here we mapped GGC and GCC STRs of ≥3-repeats, and found novel islands of up to 45 of those STRs, populating spans of 1 to 2 kb of genomic DNA. RGPD4 and NOC4L harbored the densest (GGC)3 (probability 3.09061E-71) and (GCC)3 (probability 1.72376E-61) islands, respectively, and were human-specific. We also found prime instances of directional incremented density of STRs at specific loci in human versus other species, including the FOXK2 and SKI GGC islands. The genes containing those islands significantly diverged in expression in human versus other species, and the proteins encoded by those genes interact closely in a physical interaction network, consequence of which may be human-specific characteristics such as higher order brain functions. CONCLUSION: We report novel islands of GGC and GCC STRs of evolutionary relevance to human. The density, and in some instances, periodicity of these islands support them as a novel genomic entity, which need to be further explored in evolutionary, mechanistic, and functional platforms.

A GCC repeat in RAB26 undergoes natural selection in human and harbors divergent genotypes in late-onset Alzheimer's disease.

Although mainly located in genic regions and being mutation hotspots, intact blocks of CG-rich trinucleotide short tandem repeats (STRs) are largely overlooked with respect to their link with natural selection. The human RAB26 (member RAS oncogene family) directs synaptic and secretory vesicles into preautophagosomal structures, inhibition of which specifically disrupts axonal transport of degradative organelles and leads to an axonal dystrophy, resembling Alzheimer's disease (AD). Human RAB26 contains a GCC repeat in the top 1st percent in respect of length. Here we sequenced this STR in 441 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N = 216) and controls (N = 225). In both groups, the 12-repeat allele and the 12/12 genotype were predominantly abundant. We found excess of homozygosity for non-12 alleles in the NCD group (Mid-P exact = 0.027). Furthermore, divergent genotypes were detected that were specific to the NCD group (2.8% of genotypes) (Mid-P exact = 0.006) or controls (3.1% of genotypes) (Mid-P exact = 0.004). The patients harboring divergent genotypes received the diagnosis of AD. Based on the predominant abundance of the 12-repeat and 12/12 genotype in both groups, excess of non-12 homozygosity in the NCD group, and divergent genotypes across the NCD and control groups, we propose natural selection at this locus and link with late-onset AD. Our findings strengthen the hypothesis that a collection of rare genotypes unambiguously contribute to the pathogenesis of late-onset NCDs, such as AD.

Predominant monomorphism of the RIT2 and GPM6B exceptionally long GA blocks in human and enriched divergent alleles in the disease compartment.

Across human protein-coding genes, the human neuron-specific genes, RIT2 and GPM6B, contain the two longest GA short tandem repeats (STRs) of 11 and 9-repeats, respectively, the length ranges of which are functional, and result in gene expression alteration. Here we sequenced the RIT2 and GPM6B STRs in 600 human subjects, consisting of late-onset neurocognitive disorder (n = 200), multiple sclerosis (n = 200), and controls (n = 200). Furthermore, we selected two large human databases, including the general-population-based gnomAD ( https://gnomad.broadinstitute.org ) and a mainly disease-phenotype-archiving database, TOPMed ( https://www.nhlbiwgs.org ), to compare allele frequencies in the general populations vs. the disease compartment. The RIT2 and GPM6B GA-repeats were monomorphic in the human subjects studied, at lengths of 11 and 9-repeats, respectively, and were predominantly human-specific in formula. Exception included a 9/11 genotype of the RIT2 GA-STR in an isolate case of female multiple sclerosis. Exceedingly rare alleles of the two GA repeats were significantly enriched in TOPMed vs. the gnomAD. We report prime instances of predominant monomorphism for specific lengths of STRs in human, and possible enrichment of rare divergent alleles in the disease phenotype compartment. While STRs are most attended because of their high polymorphic nature, STR monomorphism is an underappreciated feature, which may have a link with natural selection and disease.

Evaluation of antioxidant potential of Heliotropium bacciferum Forssk extract and wound healing activity of its topical formulation in rat.

Wound healing is a dynamic process that occurs in the tissue under the skin. During this process, oxidative stress biomarkers are excessively produced, which finally lead to inflammation and cellular damage. In this study, efforts have been made to evaluate the antioxidant effect and wound healing activity topical formulation containing Heliotropium bacciferum Forssk extract. The in vitro antioxidant properties were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities and the ferric reducing antioxidant power (FRAP) assay. The herbal ointments (2.5% w/w and 5% w/w) were prepared from the hydroalcoholic extract of H. bacciferum Forssk and administrated on the induced wounds in Wistar rats. The chromatic assay, percentage of wound contraction, and histopathological studies were used for evaluating the wound healing activity. For the evaluation of reactive oxygen species (ROS), catalase (CAT) activity, superoxide dismutase (SOD), and glutathione (GSH) levels were examined. The DPPH method showed tremendous radical scavenging activities at the corresponding concentrations with EC50 value of 80µg/mL. Topical application of the ointment (5% w/w) showed the highest wound contraction in comparison to the positive control (treated with CICALFATE™) and the control group (treated with normal saline). Similarly, the histological study of the group treated with the extract ointment (5% w/w) showed full collagen tissue deposition with a complete epidermal regeneration. The results of the assessment of GSH levels as well as CAT and SOD activities in the treated group (5% w/w) confirmed the scavenging property of the extract ointment. Our findings indicated the proper wound healing impact of the topical formulation of H. bacciferum Forssk due to its notable antioxidant capacity.

Natural selection at the RASGEF1C (GGC) repeat in human and divergent genotypes in late-onset neurocognitive disorder.

Expression dysregulation of the neuron-specific gene, RASGEF1C (RasGEF Domain Family Member 1C), occurs in late-onset neurocognitive disorders (NCDs), such as Alzheimer's disease. This gene contains a (GGC)13, spanning its core promoter and 5' untranslated region (RASGEF1C-201 ENST00000361132.9). Here we sequenced the (GGC)-repeat in a sample of human subjects (N = 269), consisting of late-onset NCDs (N = 115) and controls (N = 154). We also studied the status of this STR across various primate and non-primate species based on Ensembl 103. The 6-repeat allele was the predominant allele in the controls (frequency = 0.85) and NCD patients (frequency = 0.78). The NCD genotype compartment consisted of an excess of genotypes that lacked the 6-repeat (divergent genotypes) (Mid-P exact = 0.004). A number of those genotypes were not detected in the control group (Mid-P exact = 0.007). The RASGEF1C (GGC)-repeat expanded beyond 2-repeats specifically in primates, and was at maximum length in human. We conclude that there is natural selection for the 6-repeat allele of the RASGEF1C (GGC)-repeat in human, and significant divergence from that allele in late-onset NCDs. STR alleles that are predominantly abundant and genotypes that deviate from those alleles are underappreciated features, which may have deep evolutionary and pathological consequences.

Evolving evidence on a link between the ZMYM3 exceptionally long GA-STR and human cognition.

The human X-linked zinc finger MYM-type protein 3 (ZMYM3) contains the longest GA-STR identified across protein-coding gene 5' UTR sequences, at 32-repeats. This exceptionally long GA-STR is located at a complex string of GA-STRs with a human-specific formula across the complex as follows: (GA)8-(GA)4-(GA)6-(GA)32 (ZMYM3-207 ENST00000373998.5). ZMYM3 was previously reported among the top three genes involved in the progression of late-onset Alzheimer's disease. Here we sequenced the ZMYM3 GA-STR complex in 750 human male subjects, consisting of late-onset neurocognitive disorder (NCD) as a clinical entity (n = 268) and matched controls (n = 482). We detected strict monomorphism of the GA-STR complex, except of the exceptionally long STR, which was architecturally skewed in respect of allele distribution between the NCD cases and controls [F (1, 50) = 12.283; p = 0.001]. Moreover, extreme alleles of this STR at 17, 20, 42, and 43 repeats were detected in seven NCD patients and not in the control group (Mid-P exact = 0.0003). A number of these alleles overlapped with alleles previously found in schizophrenia and bipolar disorder patients. In conclusion, we propose selective advantage for the exceptional length of the ZMYM3 GA-STR in human, and its link to a spectrum of diseases in which major cognition impairment is a predominant phenotype.

Genome-scale portrait and evolutionary significance of human-specific core promoter tri- and tetranucleotide short tandem repeats.

BACKGROUND: While there is an ongoing trend to identify single nucleotide substitutions (SNSs) that are linked to inter/intra-species differences and disease phenotypes, short tandem repeats (STRs)/microsatellites may be of equal (if not more) importance in the above processes. Genes that contain STRs in their promoters have higher expression divergence compared to genes with fixed or no STRs in the gene promoters. In line with the above, recent reports indicate a role of repetitive sequences in the rise of young transcription start sites (TSSs) in human evolution. RESULTS: Following a comparative genomics study of all human protein-coding genes annotated in the GeneCards database, here we provide a genome-scale portrait of human-specific short- and medium-size (≥ 3-repeats) tri- and tetranucleotide STRs and STR motifs in the critical core promoter region between - 120 and + 1 to the TSS and evidence of skewing of this compartment in reference to the STRs that are not human-specific (Levene's test p < 0.001). Twenty-five percent and 26% enrichment of human-specific transcripts was detected in the tri and tetra human-specific compartments (mid-p < 0.00002 and mid-p < 0.002, respectively). CONCLUSION: Our findings provide the first evidence of genome-scale skewing of STRs at a specific region of the human genome and a link between a number of these STRs and TSS selection/transcript specificity. The STRs and genes listed here may have a role in the evolution and development of characteristics and phenotypes that are unique to the human species.

Skewing of the genetic architecture at the ZMYM3 human-specific 5' UTR short tandem repeat in schizophrenia.

Differential expansion of a number of human short tandem repeats (STRs) at the critical core promoter and 5' untranslated region (UTR) support the hypothesis that at least some of these STRs may provide a selective advantage in human evolution. Following a genome-wide screen of all human protein-coding gene 5' UTRs based on the Ensembl database ( http://www.ensembl.org ), we previously reported that the longest STR in this interval is a (GA)32, which belongs to the X-linked zinc finger MYM-type containing 3 (ZMYM3) gene. In the present study, we analyzed the evolutionary implication of this region across evolution and examined the allele and genotype distribution of the "exceptionally long" STR by direct sequencing of 486 Iranian unrelated male subjects consisting of 196 cases of schizophrenia (SCZ) and 290 controls. We found that the ZMYM3 transcript containing the STR is human-specific (ENST00000373998.5). A significant allele variance difference was observed between the cases and controls (Levene's test for equality of variances F = 4.00, p < 0.03). In addition, six alleles were observed in the SCZ patients that were not detected in the control group ("disease-only" alleles) (mid p exact < 0.0003). Those alleles were at the extreme short and long ends of the allele distribution curve and composed 4% of the genotypes in the SCZ group. In conclusion, we found skewing of the genetic architecture at the ZMYM3 STR in SCZ. Further, we found a bell-shaped distribution of alleles and selection against alleles at the extreme ends of this STR. The ZMYM3 STR sets a prototype, the evolutionary course of which determines the range of alleles in a particular species. Extreme "disease-only" alleles and genotypes may change our perspective of adaptive evolution and complex disorders. The ZMYM3 gene "exceptionally long" STR should be sequenced in SCZ and other human-specific phenotypes/characteristics.

Decreased gene expression activity as a result of a mutation in the calreticulin gene promoter in a family case of schizoaffective disorder.

Accumulating evidence of population association studies support the hypothesis that the high heritability of major psychiatric disorders is a combination of relatively common alleles of modest effect, and rare alleles some with relatively larger effects. We have previously reported low frequency mutations in the proximal promoter of the human calreticulin (CALR) gene that co-occur with the spectrum of major psychiatric disorders. One of those mutations at -205C>T (rs556992558) was detected in an isolate case of schizoaffective disorder. In the current study, the functional implication of mutation -205T is studied in the human neuronal cell lines LAN-5, BE(2)-C and HEK-293. In contrast with other mutations in the promoter region which increase gene expression activity, the -205T mutation significantly decreased gene expression in those cell lines in comparison with the wild-type -205C nucleotide (p < 0.000001, p < 0.0005, and p < 0.017, respectively). Treatment of the cell lines with the mood-stabilizing drug, valproic acid (VPA) resulted in differential gene expression activity in the mutant -205T versus the wild-type -205C construct. VPA increased gene expression activity in both constructs, while a significantly higher expression activity was observed in the mutant construct (p < 0.01), indicative of the creation of a positive effector binding site for VPA as a result of the -205T mutation. We conclude that deviation from normalcy in the level of CALR in either direction is associated with major psychiatric disorders.

Genome-wide identification of human- and primate-specific core promoter short tandem repeats.

Recent reports of a link between human- and primate-specific genetic factors and human/primate-specific characteristics and diseases necessitate genome-wide identification of those factors. We have previously reported core promoter short tandem repeats (STRs) of extreme length (≥6-repeats) that have expanded exceptionally in primates vs. non-primates, and may have a function in adaptive evolution. In the study reported here, we extended our study to the human STRs of ≥3-repeats in the category of penta and hexaucleotide STRs, across the entire human protein coding gene core promoters, and analyzed their status in several superorders and orders of vertebrates, using the Ensembl database. The ConSite software was used to identify the transcription factor (TF) sets binding to those STRs. STR specificity was observed at different levels of human and non-human primate (NHP) evolution. 73% of the pentanucleotide STRs and 68% of the hexanucleotide STRs were found to be specific to human and NHPs. AP-2alpha, Sp1, and MZF were the predominantly selected TFs (90%) binding to the human-specific STRs. Furthermore, the number of TF sets binding to a given STR was found to be a selection factor for that STR. Our findings indicate that selected STRs, the cognate binding TFs, and the number of TF set binding to those STRs function as switch codes at different levels of human and NHP evolution and speciation.

BEND3 is involved in the human-specific repression of calreticulin: Implication for the evolution of higher brain functions in human.

Recent emerging evidence indicates that changes in gene expression levels are linked to human evolution. We have previously reported a human-specific nucleotide in the promoter sequence of the calreticulin (CALR) gene at position -220C, which is the site of action of valproic acid. Reversion of this nucleotide to the ancestral A-allele has been detected in patients with degrees of deficit in higher brain cognitive functions. This mutation has since been reported in the 1000 genomes database at an approximate frequency of <0.0004 in humans (rs138452745). In the study reported here, we present update on the status of rs138452745 across evolution, based on the Ensembl and NCBI databases. The DNA pulldown assay was also used to identify the proteins binding to the C- and A-alleles, using two cell lines, SK-N-BE and HeLa. Consistent with our previous findings, the C-allele is human-specific, and the A-allele is the rule across all other species (N=38). This nucleotide resides in a block of 12-nucleotides that is strictly conserved across evolution. The DNA pulldown experiments revealed that in both SK-N-BE and HeLa cells, the transcription repressor BEN domain containing 3 (BEND3) binds to the human-specific C-allele, whereas the nuclear factor I (NFI) family members, NF1A, B, C, and X, specifically bind to the ancestral A-allele. This binding pattern is consistent with a previously reported decreased promoter activity of the C-allele vs. the A-allele. We propose that there is a link between binding of BEND3 to the CALR rs138452745 C-allele and removal of NFI binding site from this nucleotide, and the evolution of human-specific higher brain functions. To our knowledge, CALR rs138452745 is the first instance of enormous nucleotide conservation across evolution, except in the human species.

An exceptionally long CA-repeat in the core promoter of SCGB2B2 links with the evolution of apes and Old World monkeys.

We have recently reported a genome-scale catalog of human protein-coding genes that contain "exceptionally long" STRs (≥6-repeats) in their core promoter, which may be of selective advantage in this species. At the top of that list, SCGB2B2 (also known as SCGBL), contains one of the longest CA-repeat STRs identified in a human gene core promoter, at 25-repeats. In the study reported here, we analyzed the conservation status of this CA-STR across evolution. The functional implication of this STR to alter gene expression activity was also analyzed in the HEK-293 cell line. We report that the SCGB2B2 core promoter CA-repeat reaches exceptional lengths, ranging from 9- to 25-repeats, across Apes (Hominoids) and the Old World monkeys (CA>2-repeats were not detected in any other species). The longest CA-repeats and highest identity in the SCGB2B2 protein sequence were observed between human and bonobo. A trend for increased gene expression activity was observed from the shorter to the longer CA-repeats (p<0.009), and the CA-repeat increased gene expression activity, per se (p<0.02). We propose that the SCGB2B2 gene core promoter CA-repeat functions as an expression code for the evolution of Apes and the Old World monkeys.

Exceptionally long 5' UTR short tandem repeats specifically linked to primates.

We have previously reported genome-scale short tandem repeats (STRs) in the core promoter interval (i.e. -120 to +1 to the transcription start site) of protein-coding genes that have evolved identically in primates vs. non-primates. Those STRs may function as evolutionary switch codes for primate speciation. In the current study, we used the Ensembl database to analyze the 5' untranslated region (5' UTR) between +1 and +60 of the transcription start site of the entire human protein-coding genes annotated in the GeneCards database, in order to identify "exceptionally long" STRs (≥5-repeats), which may be of selective/adaptive advantage. The importance of this critical interval is its function as core promoter, and its effect on transcription and translation. In order to minimize ascertainment bias, we analyzed the evolutionary status of the human 5' UTR STRs of ≥5-repeats in several species encompassing six major orders and superorders across mammals, including primates, rodents, Scandentia, Laurasiatheria, Afrotheria, and Xenarthra. We introduce primate-specific STRs, and STRs which have expanded from mouse to primates. Identical co-occurrence of the identified STRs of rare average frequency between 0.006 and 0.0001 in primates supports a role for those motifs in processes that diverged primates from other mammals, such as neuronal differentiation (e.g. APOD and FGF4), and craniofacial development (e.g. FILIP1L). A number of the identified STRs of ≥5-repeats may be human-specific (e.g. ZMYM3 and DAZAP1). Future work is warranted to examine the importance of the listed genes in primate/human evolution, development, and disease.

Dominant and Protective Role of the CYTH4 Primate-Specific GTTT-Repeat Longer Alleles Against Neurodegeneration.

Primate-specific genes and regulatory mechanisms could provide insight into human brain functioning and disease. In a genome-scale analysis of the entire protein-coding genes listed in the GeneCards database, we have recently reported human genes that contain "exceptionally long" short tandem repeats (STRs) in their core promoter, which may be of adaptive/selective evolutionary advantage in this species. The longest tetra-nucleotide repeat identified in a human gene core promoter belongs to the CYTH4 gene. This GTTT-repeat is specific to Hominidae and Old World monkeys, and the shortest allele of this repeat, (GTTT)6, is linked with neural dysfunction and type I bipolar disorder in human. In the present study, we sought a possibly broader role for the CYTH4 gene core promoter GTTT-repeat in neural functioning and investigated its allelic distribution in a total of 949 human subjects, consisting of two neurodegenerative disorders, multiple sclerosis (MS) (n = 272) and Alzheimer's disease (AD) (n = 257), and controls (n = 420). The range of the alleles of this GTTT-repeat in the human sample studied was between 6- and 9-repeats. The shortest allele, (GTTT)6, was significantly in excess in the MS and AD patients in comparison with the controls (p < 0.004). The 6/6, 6/7, and 7/7 genotypes were in excess in the MS and AD patients, whereas the overall frequency of all other genotypes (consisting of at least one longer allele, i.e., 8- or 9-repeat) was higher in the controls (p < 0.005), indicating a dominant and protective effect for the longer alleles against neurodegeneration. This is the first indication of the involvement of a primate-specific STR in neurodegeneration in humans. We propose an adaptive evolutionary role for the expansion of the CYTH4 gene core promoter GTTT-repeat in the human brain, which is supported by a link between the shortest allele of this repeat with neuropsychiatric disorders.

A primate-specific functional GTTT-repeat in the core promoter of CYTH4 is linked to bipolar disorder in human.

Evidence of primate-specific genes and gene regulatory mechanisms linked to bipolar disorder (BD) lend support to evolutionary/adaptive processes in the pathogenesis of this disorder. Following a genome-scale analysis of the entire protein coding genes annotated in the GeneCards database, we have recently reported that cytohesin-4 (CYTH4) contains the longest tetra-nucleotide short tandem repeat (STR) identified in a human protein-coding gene core promoter, which may be of adaptive advantage to this species. In the current study, we analyzed the evolutionary trend of this STR across evolution. We also analyzed the functional implication and distribution of this STR in a group of patients with type 1 BD (n=233) and controls (n=262). We found that this STR is exceptionally expanded in primates (Fisher exact p<0.00003). Association was observed between type I BD and the 6-repeat allele of this STR, (GTTT)6 (Yates corrected Χ(2)=12.68, p<0.0001, OR: 1.68). This allele is the shortest length of the GTTT-repeat identified in the human subjects studied. Consistent with that finding, excess homozygosity was observed for the shorter alleles, (GTTT)6 and (GTTT)7, vs. the longer alleles, (GTTT)8 and (GTTT)9 in the BD group (Yates corrected Χ(2)=5.18, p<0.01, 1 df, OR: 1.96). Using Dual Glo luciferase system in HEK-293 cells, a trend for gene expression repression was observed from the 6- to the 9-repeat allele (p<0.003), and the GTTT-repeat significantly down-regulated gene expression, per se (p<0.0006). This is the first evidence of a link between a primate-specific STR and a major psychiatric disorder in human. It may be speculated that the CYTH4 GTTT-repeat in primates may have conferred selective advantage to this order, reflected in neural function and neurophenotypes. The role of the CYTH4 gene in the pathogenesis of type I BD remains to be clarified in the future studies.

Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes.

Protein complexes that bind to 'GAGA' DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p<1 × 10(-8)). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution.

Evolutionary trend of exceptionally long human core promoter short tandem repeats.

Short tandem repeats (STRs) are variable elements that play a significant role in genome evolution by creating and maintaining quantitative genetic variation. Because of their proximity to the +1 transcription start site (TSS) and polymorphic nature, core promoter STRs may be considered a novel source of variation across species. In a genome-scale analysis of the entire human protein-coding genes annotated in the GeneCards database (19,927), we analyze the prevalence and repeat numbers of different classes of core promoter STRs in the interval between -120 and +1 to the TSS. We also analyze the evolutionary trend of exceptionally long core promoter STRs of ≥6-repeats. 133 genes (~2%) had core promoter STRs of ≥6-repeats. In the majority of those genes, the STR motifs were found to be conserved across evolution. Di-nucleotide repeats had the highest representation in the human core promoter long STRs (72 genes). Tri- (52 genes), tetra-, penta-, and hexa-nucleotide STRs (9 genes) were also present in the descending prevalence. The majority of those genes (84 genes) revealed directional expansion of core promoter STRs from mouse to human. However, in a number of genes, the difference in average allele size across species was sufficiently small that there might be a constraint on the evolution of average allele size. Random drift of STRs from mouse to human was also observed in a minority of genes. Future work on the genes listed in the current study may further our knowledge into the potential importance of core promoter STRs in human evolution.

Haplotypes across the human caveolin 1 gene upstream purine complex significantly alter gene expression: implication in neurodegenerative disorders.

We have previously reported a polymorphic purine complex at the 1.5 kb upstream region of the human caveolin 1 (CAV1) gene that is conserved across several species in respect with sequence motifs and the location of the complex. The IRF and Ets transcription factors have common binding sites for this region across those species. We have also shown skew in the homozygote haplotype compartment of this complex in two neurodegenerative disorders, sporadic late-onset Alzheimer's disease (AD), and multiple sclerosis (MS), versus disease-free controls (p<0.0000001). In the current study, we analyze the functional implication of the disease homozygote haplotypes (i.e. 102-bp and 142-bp) vs. control homozygote haplotype (110-bp) in three neuronal cell lines, LAN-5, U-87 MG, and N2A, using dual luciferase reporter system. A significant increase in gene expression was observed in the cell lines with the disease haplotype constructs vs. control haplotype in the three cell lines (t-test p<4 × 10(-4), 1 × 10(-6), and 3 × 10(-4)), respectively. We conclude that the human CAV1 upstream purine complex modifies gene expression. An additive effect of the haplotypes in the homozygous status is speculated based on the skew in the homozygote haplotypes in neurodegenerative disorders.