Natural Selection and Functional Potentials of Human Noncoding Elements Revealed by Analysis of Next Generation Sequencing Data.

Shared genetic etiology underlying Alzheimer's disease and type 2 diabetes

ON FEBRUARY 12, 2009, WE CELEBRATE THE 200TH anniversary of the birth of Charles Darwin. He has earned the scientific reputation as being the most influential naturalist-biologist of his generation and perhaps of all time. Achievement of his esteemed status resulted initially from studying the geographical distribution of wildlife and fossils collected on his 5-year voyage on the Beagle and concluding that the “transmutation of species” was due to natural selection. In 1858, he was in the process of documenting his research when he received an essay from Alfred Russel Wallace putting forth the same concept of natural selection as the mechanism for generation of new species, resulting in a joint publication that same year. On the Origin of Species was published by Darwin in 1859, making it now the 150th anniversary of its publication. This book proposed evolution as the scientific basis of diversity and species generation produced through the effects of natural selection. Evolution has been proven to be the mechanism for species generation and the engine for change is natural selection, providing the evolved species, beginning from a common organism, adaptation and fitness to maximize reproduction and survival. Dobzhansky put it succinctly by saying that “nothing in biology makes sense except in the light of evolution.” Darwin expressed it eloquently in 1859: There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved. Two hundred years after Darwin’s birth, molecular genomic analyses of the human genome have been implemented, seeking the genetic basis for natural selection providing biological fitness and also risk of developing disease. Genome-wide association studies (GWASs) seeking gene variations, or single-nucleotide polymorphisms (SNPs), causal of several human diseases have been conducted in recent years. These diseases include autism, schizophrenia, obesity, diabetes, and heart disease. Several GWASs for risk association with neurological diseases,orneuromicstudies,havebeenreported. Increasedrisks foramyotrophic lateral sclerosis,Alzheimerdisease(AD), restless legssyndrome,andmultiplesclerosishavebeenassociated with polymorphisms in specific genes. It was hoped that these observationswouldadvanceanunderstandingof thecausation of inherited, complexpolygenetic,multifactorialneurological diseases. They have been made possible by the publication of thehumangenomeandhaplotypestudies (HapMapanalyses). Awhole-genomeanalysisofsporadicamyotrophiclateralsclerosis was published by Dunckley et al. They identified 10 genetic loci that were associated with sporadic amyotrophic lateral sclerosis in 3 independent series of patients and control subjects.Themostsignificantassociationwithdiseasewasfound foraSNPnearanuncharacterizedgeneknownasFLJ10986on chromosome1.Thus, theydescribedspecificSNPs that areassociated with onset of sporadic amyotrophic lateral sclerosis. Reiman et al conducted a genome-wide survey of 502 627 SNPs to characterize and confirm late-onset AD susceptibility genes. They found that late-onset AD was associated with 6 SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. The GAB2 gene was overexpressed in pathologically vulnerable neurons, and interference with GAB2 gene expression increased tau phosphorylation. They concluded that GAB2 modifies late-onset AD risk in apolipoprotein E (APOE) e4 allele carriers. Coon et al found a single SNP in linkage disequilibrium to the APOE e4 allele in a neurome-wide association study. Grupe et al described 3 additional SNPs on chromosome 19 in linkage disequilibrium to the APOE e4 locus also in a neurome-wide association study. Liu et al described significant linkage to 3q23 markers associated with AD in a genetically isolated Dutch population. In 2008, Li et al described SNPs associated with risk and ageatonsetofADinaGWASof469 438SNPs.They identified theAPOE linkagedisequilibriumregionasthestrongestgenetic risk factor for AD. They concluded that this event could be a consequence of the coevolution of more than 1 susceptibility allele, includingAPOC1, inthesameregion.Additionalpotential at-riskgenes forADwereof interestandrequire furtherstudies.

All vertebrate genomes are heavily methylated at CpG dinucleotide sites, and methylated CpG dinucleotides are prone to CpG→TpG mutations through spontaneous deamination. This leaves different footprints on coding and non-coding sequences. We capture these different fingerprints by five indices that can be used to discriminate between coding and non-coding (intron) sequences. We also show that a linear discriminant function derived from a training set of coding and intron sequences from human chromosome 22 can be successfully used in gene-finding of the zebrafish genome.Key wordscontent sensorDNA methylationgene findingvertebrate genomecodon structure

IL28B single nucleotide polymorphisms in the treatment of hepatitis C

In the human genome, it has been estimated that considerably more sequence is under natural selection in non-coding regions [such as transcription-factor binding sites (TF-binding sites) and non-coding RNAs (ncRNAs)] compared to protein-coding ones. However, less attention has been paid to them. To study selective pressure on non-coding elements, we use next-generation sequencing data from the recently completed pilot phase of the 1000 Genomes Project, which, compared to traditional methods, allows for the characterization of a full spectrum of genomic variations, including single-nucleotide polymorphisms (SNPs), short insertions and deletions (indels) and structural variations (SVs). We develop a framework for combining these variation data with non-coding elements, calculating various population-based metrics to compare classes and subclasses of elements, and developing element-aware aggregation procedures to probe the internal structure of an element. Overall, we find that TF-binding sites and ncRNAs are less selectively constrained for SNPs than coding sequences (CDSs), but more constrained than a neutral reference. We also determine that the relative amounts of constraint for the three types of variations are, in general, correlated, but there are some differences: counter-intuitively, TF-binding sites and ncRNAs are more selectively constrained for indels than for SNPs, compared to CDSs. After inspecting the overall properties of a class of elements, we analyze selective pressure on subclasses within an element class, and show that the extent of selection is associated with the genomic properties of each subclass. We find, for instance, that ncRNAs with higher expression levels tend to be under stronger purifying selection, and the actual regions of TF-binding motifs are under stronger selective pressure than the corresponding peak regions. Further, we develop element-aware aggregation plots to analyze selective pressure across the linear structure of an element, with the confidence intervals evaluated using both simple bootstrapping and block bootstrapping techniques. We find, for example, that both micro-RNAs (particularly the seed regions) and their binding targets are under stronger selective pressure for SNPs than their immediate genomic surroundings. In addition, we demonstrate that substitutions in TF-binding motifs inversely correlate with site conservation, and SNPs unfavorable for motifs are under more selective constraints than favorable SNPs. Finally, to further investigate intra-element differences, we show that SVs have the tendency to use distinctive modes and mechanisms when they interact with genomic elements, such as enveloping whole gene(s) rather than disrupting them partially, as well as duplicating TF motifs in tandem.

To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203). Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

The author is American Cancer Society Professor of Developmental Genetics in the Department of Embryology at the Carnegie Institution of Washington in Baltimore, MD 21210. Biological organisms, unlike complex inanimate systems, contain information that regulates their day-to-day activities and, more remarkably, lets them produce new organisms from single cells. That this information resides in the chromosomes was established over 75 years ago when it was shown that the cytologically visible chromosomes were distributed at cell division, meiosis, and fertilization in the same patterns as were the genes detected by breeding experiments. Missing from the classical account, however, was any understanding of the nature of the information carried by the chromosomes. That gap was, of course, filled by later spectacular advances in molecular genetics, which showed that a gene is a segment of DNA whose linear sequence of nucleotides specifies the linear sequence of amino acids in a protein. Given the triplet code that predicts amino acids from nucleotides, anyone can read the book of instructions contained in the chromosomes. But just as a book is more than a random assortment of words, chromosomes are more than simple repositories of gene sequences. They must contain regulatory information for turning genes on and off and they must control their own replication, repair, and packaging, as well as the complex movements they carry out during mitosis and meiosis. Today's issue of Science contains five review Articles and four Reports that focus on some of these global properties of chromosomes that cannot be predicted from a knowledge of gene structure and coding alone. Central to any genetic system is the mechanism for moving the chromosomes from place to place. In higher organisms, this involves interaction between the mitotic spindle and a specific part of the chromosome, the centromere. Pluta et al . ([page 1591][1]) summarize recent information on the (noncoding) DNA sequences at the centromere as well as the special proteins that bind to this part of the chromosome. Although centromeres have often been thought of as passive structures, McKim and Hawley ([page 1595][2]) review new evidence that suggests that centromeres take an active role in establishing the spindle and in controlling the molecular “motors” that move the chromosomes to the poles. Zakian ([page 1601][3]) reviews recent studies on telomeres, the specialized ends of chromosomes. Most chromosomes have a repeated 6-base pair sequence at their ends, which is replicated by an enzyme known as telomerase. Telomerase is remarkable in that it is a ribonucleoprotein whose RNA moiety contains the template for the 6-base repeat. Like centromeres, telomeres contain specific proteins not found elsewhere on the chromosome. In a related Report, Chong et al . ([page 1663][4]) describe a human telomere-binding protein. The review by Kelley and Kuroda ([page 1607][5]) deals with dosage compensation—the raising or lowering of overall gene activity on the X chromosome to compensate for the different number of X's in females versus males. In the fly Drosophila , the single X of the male works harder, whereas in the nematode worm Caenorhabditis the two X's in the female slow down. In both cases, special proteins coat the affected X chromosomes. In mammals, dosage compensation is accomplished in still another way, by overall inactivation of one of the two X chromosomes in each cell of the female. In her review, Barlow ([page 1610][6]) describes a similar phenomenon—gametic imprinting in mammals. In this case, single genes are inactivated instead of a whole chromosome, but only when inherited from a specific parent, sometimes the father, sometimes the mother. This issue does not contain a review on chromosome replication, but the interested reader can find here three Reports dealing with the important first step in DNA replication, the formation of an initiation complex between DNA and a set of specific proteins [Gavin et al . ([page 1667][7]), Ehrenhofer-Murray et al . ([page 1671][8]), and Gossen et al . ([page 1674][9])]. What are the common themes of these papers? First, they emphasize the importance and diversity of chromosomal proteins that serve both structural and regulatory roles, most often by interacting with specific but noncoding DNA sequences. Second, they underscore the extraordinary power of genetics to identify the genes that encode these chromosomal proteins. Finally, they provide novel molecular insights into some of the oldest and most baffling problems of chromosome structure and behavior. Chromosome research is alive and well! [1]: /lookup/doi/10.1126/science.270.5242.1591 [2]: /lookup/doi/10.1126/science.270.5242.1595 [3]: /lookup/doi/10.1126/science.270.5242.1601 [4]: /lookup/doi/10.1126/science.270.5242.1663 [5]: /lookup/doi/10.1126/science.270.5242.1607 [6]: /lookup/doi/10.1126/science.270.5242.1610 [7]: /lookup/doi/10.1126/science.270.5242.1667 [8]: /lookup/doi/10.1126/science.270.5242.1671 [9]: /lookup/doi/10.1126/science.270.5242.1674

Alzheimer disease (AD) is a heterogeneous disorder with a substantial genetic component. A small number of cases (ie, early-onset familial AD) are caused by exceedingly rare but pathogenic and highly penetrant mutations, while most cases (ie, late-onset AD) are caused by an intricate—and still only partially understood—interplay of genetic and nongenetic risk factors.1 The past decade has seen unprecedented progress in deciphering the genetic underpinnings of lateonset AD. This advancement was achieved mostly by the application of high-throughput microarray genotyping in the context of genome-wide association studies (GWASs) comparing the allele status at millions of different base pairs on increasingly large samples of affected and unaffected individuals.2 Most AD GWAS findings to date were made with common (ie, frequency of the minor allele typically >5%) single-nucleotide polymorphisms (SNPs) typically exerting small genetic effect sizes (ie, odds ratios <1.3). In most cases, the pathogenic mechanisms underlying these associations have been difficult to discern owing to the fact thatmost common SNPs are located in noncoding regions of the genome. The study by Chen et al3 in this issue is different from most other GWASs in the field because it specifically focused on SNPs located in the coding—and thus potentially functional—portions of the genome (ie, the exome). They achieved this by using a microarray (the HumanExome Array, Illumina, Inc) specifically designed to capture the mostly rare (ie, frequency <5%, applicable to approximately 90% of all markers on the array) exome SNPs in a sample of 224 patients with AD and 224 healthy control subjects. Additional genotyping was completed on 168 patients with frontotemporal dementia and 48 patients with progressive supranuclear palsy. While the analyses of frontotemporal dementia and progressive supranuclear palsy did not yield any noteworthy new results, at least 2 novel putative loci, ie, DYSF and PAXIP1, were highlighted by the authors for AD, in addition to very strong signals near the well-established APOE locus on chromosome 19. Overall, Chen et al3 estimated that about 44% of the variance in case-control status in their sample could be explained by the SNPs typed on the exome array, a number substantially higher than the approximately 25% reported from an earlier and much larger GWAS metaanalysis mostly studying common variants.2 Themain strengthof the studybyChenet al3 is that it represents oneof the first publishedapplicationsof theexomearray technology to AD (and frontotemporal dementia and progressive supranuclearpalsy).This technology ismeant to serve as a cost-effective interim solution to studying the genetics of complex traits in times when more comprehensive approaches, such as whole-exome sequencing or wholegenome sequencing, are still relatively expensive. Thedesign of the exome array used by Chen et al3 resulted from a comprehensive analysis of whole-exome sequencing and wholegenome sequencing data of approximately 12 000 individuals with the aim to specifically capture all nonprivate and putatively functional (ie, nonsynonymous, nonsense, splice) base-pair changes (http://genome.sph.umich.edu/wiki/Exome _Chip_Design). It is estimated that 94% to 98% of these types of variants identified in an average genome by whole-exome sequencing are also captured by the exome array. While this coverage should suffice for most exome variant association studies, this typeofmicroarray technology—bydesignandunlike whole-exome sequencing or whole-genome sequencing—is not able to discover any novel sequence variants. Instead, theexomearray is limited togenotypingpolymorphisms previously shown to exist at sufficient frequency in the generalpopulationand,conceptually, is thereforenodifferent from a conventional (ie, focusing on commonvariants not selected based on functionality) GWAS or smaller-scale association study.Despite this limitation, almost90%of thevariantsgenotyped on the exome array used by Chen et al3 were not captured or analyzed in themost comprehensive AD GWAS published to date2 (variant overlap determined for the purpose of this editorial is basedondata releases fromLambert et al2 and http://www.illumina.com). Differently put, approximately 213 000 SNPs,most ofwhichwere predicted to be functional, genotyped in the study by Chen et al3 were not previously tested in patients with AD and controls of European descent, although similar studies are currently underway (see below). The only other exome genotyping study for AD published to date was performed on 400 patients with late-onset AD and 605 controls from Korea using a similar exome microarray (manufactured by Affymextrix, Inc).4 While the study by Chen et al3 thus uses state-of-the-art technology, its scientific implications currently remain limited. This is mostly owing to the small sample size investigated. In their discovery phase, Chen et al3 analyzed only 416 patients with AD and controls, followed by replication analyses in an independent set of 480 individuals. Thus, the overall sample size analyzed in the AD portion of this study was well below 1000 individuals. It was even smaller for the frontotemporal dementia (n = 168) and progressive supranuclear palsy (n = 48) analyses, forwhichno replicationanalyseswere Related article Opinion

Natural selection leaves imprints on DNA, offering the opportunity to identify functionally important regions of the genome. Identifying the genomic regions affected by natural selection within pathogens can aid in the pursuit of effective strategies to control diseases. In this study, we analyzed genome-wide patterns of selection acting on different classes of sequences in a worldwide sample of eight strains of the model plant-pathogenic fungus Colletotrichum graminicola. We found evidence of selective sweeps, balancing selection, and positive selection affecting both protein-coding and noncoding DNA of pathogenicity-related sequences. Genes encoding putative effector proteins and secondary metabolite biosynthetic enzymes show evidence of positive selection acting on the coding sequence, consistent with an Arms Race model of evolution. The 5′ untranslated regions (UTRs) of genes coding for effector proteins and genes upregulated during infection show an excess of high-frequency polymorphisms likely the consequence of balancing selection and consistent with the Red Queen hypothesis of evolution acting on these putative regulatory sequences. Based on the findings of this work, we propose that even though adaptive substitutions on coding sequences are important for proteins that interact directly with the host, polymorphisms in the regulatory sequences may confer flexibility of gene expression in the virulence processes of this important plant pathogen.

Gene-guided therapy for catheter-ablation of atrial fibrillation: are we there yet?

Genome-wide association studies (GWAS) using population-based designs have identified many genetic loci, at which common variants can influence the risk of developing the sporadic colon cancers. These are single nucleotide polymorphisms (SNPs) located on different chromosomes, close to genes involved in cancer developing process, and the SNPs modify their functions, and as a consequence the cancer risk is increased. Our aim was to provide frequency distributions data of variable (risk) allele of six independent SNPs in patients with colorectal cancers and in control Hungarian population, predicting the increased risk effect of sequence variant of SNPs. We also investigated the frequency distribution of tumor localization between right or left half of large bowel as well as the RAS mutation status. 47 non-tumorous patients and 47 patients with colorectal cancer were given oral mucosa cells or blood samples for SNP analysis. After DNA isolation, an LC480 (Roche) type PCR instrument, asymmetric LATE PCR method and melting point analysis were used for detection of sequence variations, by the assistance of two SNP specific primers, unlabeled specific probe and intercalating fluorescent dye. Genomic frequency distribution of variable alleles of SNPs predisposed to tumor development have been investigated in colorectal cancer carrier patients and the results have been compared with the same allele frequency distribution data obtained from the non-tumorous control patients and from CEU population stored in SNPnexus data base. The homozygous risk alleles of SNPs showed a 1.5-2.3-time increase in colorectal cancer carrier patients then in control and CEU patients, but the heterozygous risk allele distribution was identical in tumorous and control population. The frequency distribution of homozygous risk alleles of six SNPs was also investigated in the same time and some patients. Among 47 patients with colorectal cancer, in 3 patients carrying 3 SNPs with homozygous risk alleles, in additional 5 tumor samples two and 24 samples contain only one SNP's homozygous risk alleles, and in 15 patients, SNPs with homozygous risk alleles do not occur. In 47 control patients, only 3 samples contain two SNPs with homozygous risk alleles and 17 samples contain only one SNP with homozygous risk alleles. Significant differences of the tumorous and the control population can be seen detected. NRAS mutation was not found. Our results showed a real increased risk effect of several newly recognized low-penetrance colorectal cancer susceptibility genetic variants by influence of the regulation of neighboring genes, however, the degree of cancer risk is individual, and influenced by others environmental factors, such as dietary factors. Orv Hetil. 2018; 159(40): 1614-1623.

The worldwide pattern of single nucleotide polymorphism (SNP) variation is of great interest to human geneticists, population geneticists, and evolutionists, but remains incompletely understood. We studied the pattern in noncoding regions, because they are less affected by natural selection than are coding regions. Thus, it can reflect better the history of human evolution and can serve as a baseline for understanding the maintenance of SNPs in human populations. We sequenced 50 noncoding DNA segments each approximately 500 bp long in 10 Africans, 10 Europeans, and 10 Asians. An analysis of the data suggests that the sampling scheme is adequate for our purpose. The average nucleotide diversity (pi) for the 50 segments is only 0.061% +/- 0.010% among Asians and 0.064% +/- 0.011% among Europeans but almost twice as high (0.115% +/- 0.016%) among Africans. The African diversity estimate is even higher than that between Africans and Eurasians (0.096% +/- 0.012%). From available data for noncoding autosomal regions (total length = 47,038 bp) and X-linked regions (47,421 bp), we estimated the pi-values for autosomal regions to be 0.105, 0.070, 0.069, and 0.097% for Africans, Asians, Europeans, and between Africans and Eurasians, and the corresponding values for X-linked regions to be 0.088, 0.042, 0.053, and 0.082%. Thus, Africans differ from one another slightly more than from Eurasians, and the genetic diversity in Eurasians is largely a subset of that in Africans, supporting the out of Africa model of human evolution. Clearly, one must specify the geographic origins of the individuals sampled when studying pi or SNP density.

Bovine mastitis is a common inflammatory disease caused by multiple factors in early lactation or dry period. Genome wide association studies (GWAS) can provide a convenient and effective strategy for understanding the biological basis of mastitis and better prevention. 2b-RADseq is a high-throughput sequencing technique that offers a powerful method for genome-wide genetic marker development and genotyping. In this study, single nucleotide polymorphisms (SNPs) of the immune-regulated gene correlative with mastitis were screened and identified by two stage association analysis via GWAS-2b-RADseq in Chinese Holstein cows. We have screened 10,058 high quality SNPs from 7,957,920 tags and calculated their allele frequencies. Twenty-seven significant SNPs were co-labeled in two GWAS analysis models [Bayesian (P < 0.001) and Logistic regression (P < 0.01)], and only three SNPs (rs75762330, C > T, PIC = 0.2999; rs88640083, A > G, PIC = 0.1676; rs20438858, G > A, PIC = 0.3366) were annotated to immune-regulated genes (PTK2B, SYK, and TNFRSF21). Identified three SNPs are located in non-coding regions with low or moderate genetic polymorphisms. However, independent sample population validation (Case-control study) data showed that three important SNPs (rs75762330, P < 0.025, OR > 1; rs88640083, P < 0.005, OR > 1; rs20438858, P < 0.001, OR < 1) were significantly associated with clinical mastitis trait. Importantly, PTK2B and SYK expression was down-regulated in both peripheral blood leukocytes (PBLs) of clinical mastitis cows and in vitro LPS (E. coli)–stimulated bovine mammary epithelial cells, while TNFRSF21 was up-regulated. Under the same conditions, expression of Toll-like receptor 4 (TLR4), AKT1, and pro-inflammatory factors (IL-1β and IL-8) were also up-regulated. Interestingly, network analysis indicated that PTK2B and SYK are co-expressed in innate immune signaling pathway of Chinese Holstein. Taken together, these results provided strong evidence for the study of SNPs in bovine mastitis, and revealed the role of SYK, PTK2B, and TNFRSF21 in bovine mastitis susceptibility/tolerance.

Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes.

Human leukocyte antigen–G allele polymorphisms have evolved following three different evolutionary lineages based on intron sequences