Understanding the recent evolution of the human genome: insights from human-chimpanzee genome comparisons

The sequencing of the chimpanzee genome and the comparison with its human counterpart have begun to reveal the spectrum of genetic changes that has accompanied human evolution. In addition to gross karyotypic rearrangements such as the fusion that formed human chromosome 2 and the human-specific pericentric inversions of chromosomes 1 and 18, there is considerable submicroscopic structural variation involving deletions, duplications, and inversions. Lineage-specific segmental duplications, detected by array comparative genomic hybridization and direct sequence comparison, have made a very significant contribution to this structural divergence, which is at least three-fold greater than that due to nucleotide substitutions. Since structural genomic changes may have given rise to irreversible functional differences between the diverging species, their detailed analysis could help to identify the biological processes that have accompanied speciation. To this end, interspecies comparisons have revealed numerous human-specific gains and losses of genes as well as changes in gene expression. The very considerable structural diversity (polymorphism) evident within both lineages has, however, hampered the analysis of the structural divergence between the human and chimpanzee genomes. The concomitant evaluation of genetic divergence and diversity at the nucleotide level has nevertheless served to identify many genes that have evolved under positive selection and may thus have been involved in the development of human lineage-specific traits. Genes that display signs of weak negative selection have also been identified and could represent candidate loci for complex genomic disorders. Here, we review recent progress in comparing the human and chimpanzee genomes and discuss how the differences detected have improved our understanding of the evolution of the human genome.     

Complete nucleotide sequences and genome organization of four chimpanzee adenoviruses

The complete nucleotide sequences for four adenoviruses-Simian Adenoviruses 21, 22, 23, and 24, originally isolated from chimpanzees, were determined. The genome organization of the chimpanzee adenoviruses was found to be similar to that of other adenoviruses. The viral gene products of the adenoviruses Simian Adenoviruses 22, 23, and 24 are very closely related to those of the (previously sequenced) chimpanzee adenovirus Simian Adenovirus 25. Simian Adenovirus 21 is most similar to human subgroup B adenoviruses HAdV-3, HAdV-7, and HAdV-35. Analysis of the capsid proteins hexon and fiber of the chimpanzee adenoviruses also supports the placement of Simian Adenovirus 21 in subgroup B and Simian Adenoviruses 22, 23, and 24 in subgroup E.     

Organization of the X and Y chromosomes in human, chimpanzee and mouse pachytene nuclei using molecular cytogenetics and three-dimensional confocal analyses

We used multicolour fluorescence in-situ hybridization on air-dried pachytene nuclei to analyse the structural and functional domains of the sex vesicle (SV) in human, chimpanzee and mouse. The same technology associated with 3-dimensional analysis was then performed on human and mouse pachytene nuclei from cytospin preparations and tissue cryosections. The human and the chimpanzee SVs were very similar, with a consistently small size and a high degree of condensation. The mouse SV was most often seen to be large and poorly condensed, although it did undergo progressive condensation during pachynema. These results suggest that the condensation of the sex chromosomes is not a prerequisite for the formation of the mouse SV, and that a different specific mechanism could be responsible for its formation. We also found that the X and Y chromosomes are organized into two separate and non-entangled chromatin domains in the SV of the three species. In each species, telomeres of the X and Y chromosomes remain clustered in a small area of the SV, even those without a pseudoautosomal region. The possible mechanisms involved in the organization of the sex chromosomes and in SV formation are discussed.     

On the sequence-directed nature of human gene mutation: the role of genomic architecture and the local DNA sequence environment in mediating gene mutations underlying human inherited disease

Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher order features of the genomic architecture. The human genome is now recognized to contain "pervasive architectural flaws" in that certain DNA sequences are inherently mutation prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here, we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of noncanonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair and may serve to increase mutation frequencies in generalized fashion (i.e., both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease.     

Cruciform-forming inverted repeats appear to have mediated many of the microinversions that distinguish the human and chimpanzee genomes

Submicroscopic inversions have contributed significantly to the genomic divergence between humans and chimpanzees over evolutionary time. Those microinversions which are flanked by segmental duplications (SDs) are presumed to have originated via non-allelic homologous recombination between SDs arranged in inverted orientation. However, the nature of the mechanisms underlying those inversions which are not flanked by SDs remains unclear. We have investigated 35 such inversions, ranging in size from 51-nt to 22056-nt, with the goal of characterizing the DNA sequences in the breakpoint-flanking regions. Using the macaque genome as an outgroup, we determined the lineage specificity of these inversions and noted that the majority (N = 31; 89%) were associated with deletions (of length beween 1-nt and 6754-nt) immediately adjacent to one or both inversion breakpoints. Overrepresentations of both direct and inverted repeats, ≥ 6-nt in length and capable of non-B DNA structure formation, were noted in the vicinity of breakpoint junctions suggesting that these repeats could have contributed to double strand breakage. Inverted repeats capable of cruciform structure formation were also found to be a common feature of the inversion breakpoint-flanking regions, consistent with these inversions having originated through the resolution of Holliday junction-like cruciforms. Sequences capable of non-B DNA structure formation have previously been implicated in promoting gross deletions and translocations causing human genetic disease. We conclude that non-B DNA forming sequences may also have promoted the occurrence of mutations in an evolutionary context, giving rise to at least some of the inversion/deletions which now serve to distinguish the human and chimpanzee genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genes,mutations, and human inherited disease at the dawn of the age of personalized genomics

The number of reported germline mutations in human nuclear genes, either underlying or associated with inherited disease, has now exceeded 100,000 in more than 3,700 different genes. The availability of these data has both revolutionized the study of the morbid anatomy of the human genome and facilitated “personalized genomics.” With ～300 new “inherited disease genes” (and ～10,000 new mutations) being identified annually, it is pertinent to ask how many “inherited disease genes” there are in the human genome, how many mutations reside within them, and where such lesions are likely to be located? To address these questions, it is necessary not only to reconsider how we define human genes but also to explore notions of gene “essentiality” and “dispensability.” Answers to these questions are now emerging from recent novel insights into genome structure and function and through complete genome sequence information derived from multiple individual human genomes. However, a change in focus toward screening functional genomic elements as opposed to genes sensu stricto will be required if we are to capitalize fully on recent technical and conceptual advances and identify new types of disease‐associated mutation within noncoding regions remote from the genes whose function they disrupt. Hum Mutat 31:631–655, 2010. © 2010 Wiley‐Liss, Inc.     

Breakpoint analysis of the pericentric inversion distinguishing human chromosome 4 from the homologous chromosome in the chimpanzee (Pan troglodytes)

The study of breakpoints that occurred during primate evolution promises to yield valuable insights into the mechanisms underlying chromosome rearrangements in both evolution and pathology. Karyotypic differences between humans and chimpanzees include nine pericentric inversions, which may have potentiated the parapatric speciation of hominids and chimpanzees 5-6 million years ago. Detailed analysis of the respective chromosomal breakpoints is a prerequisite for any assessment of the genetic consequences of these inversions. The breakpoints of the inversion that distinguishes human chromosome 4 (HSA4) from its chimpanzee counterpart were identified by fluorescence in situ hybridization (FISH) and comparative sequence analysis. These breakpoints, at HSA4p14 and 4q21.3, do not disrupt the protein coding region of a gene, although they occur in regions with an abundance of LINE and LTR-elements. At 30 kb proximal to the breakpoint in 4q21.3, we identified an as yet unannotated gene, C4orf12, that lacks an homologous counterpart in rodents and is expressed at a 33-fold higher level in human fibroblasts as compared to chimpanzee. Seven out of 11 genes that mapped to the breakpoint regions have been previously analyzed using oligonucleotide-microarrays. One of these genes, WDFY3, exhibits a three-fold difference in expression between human and chimpanzee. To investigate whether the genomic architecture might have facilitated the inversion, comparative sequence analysis was used to identify an approximately 5-kb inverted repeat in the breakpoint regions. This inverted repeat is inexact and comprises six subrepeats with 78 to 98% complementarity. (TA)-rich repeats were also noted at the breakpoints. These findings imply that genomic architecture, and specifically high-copy repetitive elements, may have made a significant contribution to hominoid karyotype evolution, predisposing specific genomic regions to rearrangements.     

Systematic analysis and functional annotation of variations in the genome of an Indian individual

Whole genome sequencing of personal genomes has revealed a large repertoire of genomic variations and has provided a rich template for identification of common and rare variants in genomes in addition to understanding the genetic basis of diseases. The widespread application of personal genome sequencing in clinical settings for predictive and preventive medicine has been limited due to the lack of comprehensive computational analysis pipelines. We have used next-generation sequencing technology to sequence the whole genome of a self-declared healthy male of Indian origin. We have generated around 28X of the reference human genome with over 99% coverage. Analysis revealed over 3 million single nucleotide variations and about 490,000 small insertion-deletion events including several novel variants. Using this dataset as a template, we designed a comprehensive computational analysis pipeline for the systematic analysis and annotation of functionally relevant variants in the genome. This study follows a systematic and intuitive data analysis workflow to annotate genome variations and its potential functional effects. Moreover, we integrate predictive analysis of pharmacogenomic traits with emphasis on drugs for which pharmacogenomic testing has been recommended. This study thus provides the template for genome-scale analysis of personal genomes for personalized medicine.     

Prevaccination genomic diversity of human papillomavirus genotype 11: a study on 63 clinical isolates and 10 full-length genome sequences

Prevaccination genomic diversity of human papillomavirus genotype 11 (HPV 11) was established by sequencing 40% of the genome of 63 clinical isolates obtained from an ethnogeographically closed Caucasian cohort, and full-length genome sequencing of the ten most divergent isolates. In the study, which included the largest number of isolates to date, by analyzing pooled L1, LCR, E6, E5a, and E5b sequences (3,217 bp) of an individual isolate, a total of 23 genomic variants were identified, of which three (5 isolates) and twenty (58 isolates) corresponded to prototypic and non-prototypic variant groups, respectively. Several novel, potentially important mutations are described. Full-length genome sequences of ten isolates revealed more than 99% similarity to the HPV 11 prototype isolate. The minimum genomic distance between the full-length sequences of genomic variants and the prototype was 3 point mutations and 2 inserts and the maximum distance 31 point mutations, one insertion and one deletion. Within the ethnogeographically closed cohort investigated in this study, HPV 11 was shown to be less polymorphic in comparison to the majority of HPV genotypes studied to date.     

Improving the rigor of mutation reports: Biologic parentage and de novo mutations

The accurate determination and dissemination of the causality or pathogenicity of human DNA sequence variants is a crucial function of genetics journals. Published reports of pathogenic mutations are a common source of information for mutation databases, which are in turn used to make recommendations to patients. One of the strongest pieces of evidence in support of causality or pathogenicity for mutation reports is the occurrence of a de novo mutation. Yet, many publications describing such changes do not demonstrate that the mutation is truly de novo, by performing biologic parentage testing. I argue here that all mutation reports that describe such mutations should include biologic parentage testing, or in the absence of such testing, the mutation should be described as “apparently de novo.” This proposed standard should improve the transparency of the evidence that underlies our literature, and ultimately improve the databases of mutations in human disease. Hum Mutat 33:1501–1502, 2012. Published 2012 Wiley Periodicals, Inc.*     

Characterization of hepatitis B virus mutations in untreated patients co‐infected with HIV and HBV based on complete genome sequencing

Co‐infection of HBV with HIV results in an accelerated course of HBV‐associated chronic liver disease. Several studies have shown that viral mutations are related to disease progression in mono‐infection with HBV. However, it is unclear whether HBV mutation patterns might differ between co‐infected and mono‐infected patients. To compare the frequencies and mutation patterns in the HBV genome between co‐infection and mono‐infection. Twenty‐four treatment‐naïve co‐infected and 31 treatment‐naïve mono‐infected Thai patients were included. HBV mutations were characterized by whole genome sequencing of virus serum samples. The clinical features and frequency of known clinically significant mutations were compared between the two groups. No significant difference between the groups was found with respect to sex, age and HBeAg. However, HBV DNA levels were significantly higher in co‐infected patients. The distribution of HBV genotypes was comparable between the two groups and restricted mostly to sub‐genotypes C1 and B2. An isolate with recombinants of genotypes G/C1 was also identified in a patient with co‐infection. There was no difference in the prevalence of mutations in the enhancer II/basal core promoter/precore region, pre‐S/S and polymerase genes between the two groups. In conclusion, dual infections tend to engender increased HBV DNA levels. There was no major difference in the frequencies of common HBV mutations between co‐infected and mono‐infected patients. Thus, HBV mutations may not contribute to disease pathogenesis in Thai patients with co‐infection. J. Med. Virol. 85:16–25, 2012. © 2012 Wiley Periodicals, Inc.     

Closely spaced multiple mutations as potential signatures of transient hypermutability in human genes

Data from diverse organisms suggests that transient hypermutability is a general mutational mechanism with the potential to generate multiple synchronous mutations, a phenomenon probably best exemplified by closely spaced multiple mutations (CSMMs). Here we have attempted to extend the concept of transient hypermutability from somatic cells to the germline, using human inherited disease‐causing multiple mutations as a model system. Employing stringent criteria for data inclusion, we have retrospectively identified numerous potential examples of pathogenic CSMMs that exhibit marked similarities to the CSMMs reported in other systems. These examples include (1) eight multiple mutations, each comprising three or more components within a sequence tract of <100 bp; (2) three possible instances of “mutation showers”; and (3) numerous highly informative “homocoordinate” mutations. Using the proportion of CpG substitution as a crude indicator of the relative likelihood of transient hypermutability, we present evidence to suggest that CSMMs comprising at least one pair of mutations separated by ≤100 bp may constitute signatures of transient hypermutability in human genes. Although this analysis extends the generality of the concept of transient hypermutability and provides new insights into what may be considered a novel mechanism of mutagenesis underlying human inherited disease, it has raised serious concerns regarding current practices in mutation screening.Hum Mutat 30:1–14, 2009. © 2009 Wiley‐Liss, Inc.     

Manipulation of the human genome in human embryonic stem cells

Human embryonic stem cells (HESCs) have a tremendous clinical and scientific importance since they may serve as a cell source for transplantation and as a system for the study of human development and disease. The genetic engineering of HESCs has become instrumental in achieving these goals. Here we discuss various methodologies to genetically manipulate HESCs and propose a variety of applications of the modified cells in basic and applied research.     

Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group

We report here the complete nucleotide sequence and predicted polyprotein sequence of HeLa cell-adapted human rhinovirus 16 (HRV16). This virus is more suitable than human rhinovirus 14 (HRV14) for clinical studies, and its growth and physical properties are favorable for biochemical and crystallographic analysis. The complete message-sense RNA genome of HRV16 is composed of 7124 bases, not including the poly(A) tail. An open reading frame, extending from base 626 to 7084 predicts a polyprotein containing 2152 amino acid residues. Comparison with other rhinovirus sequences shows HRV16 is much more representative of human rhinoviruses than HRV14. No apparent relationship was found between receptor group and amino acid sequence in VP1, the capsid protein bearing the binding site for the intercullular adhesion molecule-1 (ICAM-1) in both HRV14 and HRV16.Genbank accession number: L24917.     

从MT2、HeLa细胞DNA及人和黑猩猩PBMC DNA中扩增GBV-C核苷酸

目的　检测MT2和HeLa细胞DNA、6例人和 1例黑猩猩PBMCDNA中是否存在与GBV C同源的核苷酸序列。方法　直接以上述DNA为模板、采用GBV C 5′ NCR和NS3区引物的直接套式PCR(dPCR)、核苷酸序列分析、引物介导原位扩增 (PRINS)NDA序列特异荧光标记技术。结果　从MT2和HeLa细胞DNA、4例人PBMCDNA标本中获得 5′ NCR引物扩增片段 ,从MT2和HeLa细胞DNA、5例人和黑猩猩PBMCDNA标本中获得NS3区引物扩增片段。这些扩增产物的核苷酸序列与GBV C同源性分别为 74 2 9%～ 77 14%和 73 80 %～ 79 15 %。PRINS检测结果显示 ,dPCR阳性的PBMC及其染色体上有荧光着色。结论　MT2和HeLa细胞DNA、人和黑猩猩PBMCDNA中存在与GBV C 5′ NCR和 或NS3区同源性较高的核苷酸序列 ,这些序列可能位于dPCR阳性的PBMC染色体上。     

Molecular cloning and complete nucleotide sequence of the genome of Japanese encephalitis virus Beijing-1 strain

The genomic RNA of the Japanese encephalitis virus (JEV) Beijing-1 strain was reversely transcribed and the synthesized cDNA was molecularly cloned. Six continuous cDNA clones that cover the entire virus genome were established and sequenced to determine the complete nucleotide sequence of the JEV RNA. The precise genomic size was estimated as 10,965 bases long. With flanking 95 bases at the 5 and 583 bases at the 3 non-coding regions, one long open reading frame (ORF) was revealed encoding a virus polyprotein with 3,429 amino acid residues. Because of sequence homologies observed between JEV and other flaviviruses, the genome organization of JEV appears to be identical with other flaviviruses. Genetic variation detected among flavivirus genomes is consistent with the established serological relatedness between JEV and other members of flaviviruses. The secondary structure of the JEV genome is deduced and discussed concerning its involvement in genome replication.