Evolutionary plasticity and cancer breakpoints in human chromosome 3

In this review, we focus on the evolutionary and biomedical aspects of the architecture of human chromosome 3 (HSA3) by analyzing chromosomal regions that have been conserved during the evolutionary process, compared to those that have been involved in the genomic restructuring of different placental lineages. Given that the organization of human chromosome 3 is derived when compared to the ancestral primate karyotype, and is an autosome that is commonly implicated in human tumour formation, we examined the patterns of change and the genomic consequences that have resulted from its complex evolutionary history. The data show four discrete chromosomal regions that are frequently implicated in chromosomal rearrangements (3p25, 3p22, 3p12 and 3q21). These are rich in repetitive elements and are commonly implicated in structural rearrangements that underpin human genomic disorders and neoplasias. Additional Supporting Information may be found in the online version of this article.     

An analysis of human sperm chromosome breakpoints.

Sperm chromosome analysis of 19 sperm donors with either normal or balanced karyotypes was carried out in order to explore the nature of sperm chromosome structural aberrations. A total of 2,389 cells (range 36-298/donor) were karyotyped after in vitro penetration of hamster eggs. The median percentage of sperm structural aberrations was 9.3% (SD +/- 4.7; range 0%-17.8%), with a total of 247 breakpoints, of which 220 could be characterized fully. Two sets of donors were studied in two different centers: center 1 (United States) and center 2 (Spain). The frequencies of nonrejoined and rejoined chromosome-type aberrations were very similar between center 1 and center 2: 83.6% and 10.0%, and 75.0% and 10.3%, respectively. Chromatid-type aberrations were more frequent in center 2 (14.7%) than in center 1 (6.4%) (P = .037). Chromosome 4 had less than the expected number of breakpoints (P < .001). A positive significant correlation was found between sperm breakpoints reported in this study and sites of balanced chromosome de novo rearrangements detected at prenatal diagnosis and reported in the literature (P = .0001).     

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.     

A compositional map of human chromosome 21.

GC-poor and GC-rich isochores, the long (greater than 300 kb) compositionally homogeneous DNA segments that form the genome of warm-blooded vertebrates, are located in G- and R-bands respectively of metaphase chromosomes. The precise correspondence between GC-rich isochores and R-band structure is still, however, an open problem, because GC-rich isochores are compositionally heterogeneous and only represent one-third of the genome, with the GC-richest family (which is by far the highest in gene concentration) corresponding to less than 5% of the genome. In order to clarify this issue and, more generally, to correlate DNA composition and chromosomal structure in an unequivocal way, we have developed a new approach, compositional mapping. This consists of assessing the base composition over 0.2-0.3 Mb (megabase) regions surrounding landmarks that were previously localized on the physical map. Compositional mapping was applied here to the long arm of human chromosome 21, using 53 probes that had already been used in physical mapping. The results obtained provide a direct demonstration that the DNA stretches of G-bands essentially correspond to GC-poor isochores, and that R-band DNA is characterized by a compositional heterogeneity that is much more striking than expected, in that it comprises isochores covering the full spectrum of GC levels. GC-poor isochores of R-bands may, however, correspond to 'thin' G-bands, as visualized at high resolution, leaving GC-rich and very GC-rich isochores as the real components of (high-resolution) R-band DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary history of the third chromosome gene arrangements of Drosophila pseudoobscura inferred from inversion breakpoints

The third chromosome of Drosophila pseudoobscura is polymorphic for numerous gene arrangements that form classical clines in North America. The polytene salivary chromosomes isolated from natural populations revealed changes in gene order that allowed the different gene arrangements to be linked together by paracentric inversions representing one of the first cases where genetic data were used to construct a phylogeny. Although the inversion phylogeny can be used to determine the relationships among the gene arrangements, the cytogenetic data are unable to infer the ancestral arrangement or the age of the different chromosome types. These are both important properties if one is to infer the evolutionary forces responsible for the spread and maintenance of the chromosomes. Here, we employ the nucleotide sequences of 18 regions distributed across the third chromosome in 80-100 D. pseudoobscura strains to test whether five gene arrangements are of unique or multiple origin, what the ancestral arrangement was, and what are the ages of the different arrangements. Each strain carried one of six commonly found gene arrangements and the sequences were used to infer their evolutionary relationships. Breakpoint regions in the center of the chromosome supported monophyly of the gene arrangements, whereas regions at the ends of the chromosome gave phylogenies that provided less support for monophyly of the chromosomes either because the individual markers did not have enough phylogenetically informative sites or genetic exchange scrambled information among the gene arrangements. A data set where the genetic markers were concatenated strongly supported a unique origin of the different gene arrangements. The inversion polymorphism of D. pseudoobscura is estimated to be about a million years old. We have also shown that the generated phylogeny is consistent with the cytological phylogeny of this species. In addition, the data presented here support hypothetical as the ancestral arrangement. One of the youngest arrangements, Arrowhead, has one of the highest population frequencies suggesting that selection has been responsible for its rapid increase.     

Genes and chromosomal breakpoints in the Langer-Giedion syndrome region on human chromosome 8

The tricho-rhino-phalangeal syndrome type II (TRPS II, or Langer-Giedion syndrome) is an example of contiguous gene syndromes, as it comprises the clinical features of two autosomal dominant diseases, TRPS I and a form of multiple cartilaginous exostoses caused by mutations in the EXT1 gene. We have constructed a contig of cosmid, lambda-phage, PAC, and YAC clones, which covers the entire TRPS I critical region. Using these clones we identified a novel submicroscopic deletion in a TRPS I patient and refined the proximal border of the minimal TRPS1 gene region by precisely mapping the inversion breakpoint of another patient. As a first step towards a complete inventory of genes in the Langer-Giedion syndrome chromosome region (LGCR) with the ultimate aim to identify the TRPS1 gene, we analyzed 23 human expressed sequence tags (ESTs) and four genes (EIF3S3, RAD21, OPG, CXIV) which had been assigned to human 8q24.1. Our analyses indicate that the LGCR is gene-poor, because none of the ESTs and genes map to the minimal TRPS1 gene region and only two of these genes, RAD21 and EIF3S3, are located within the shortest region of deletion overlap of TRPS II patients. Two genes, OPG and CXIV, which are deleted only in some patients with TRPS II may contribute to the clinical variability of this syndrome.     

Molecular cytogenetic characterization of breakpoints involving pericentric inversions of human chromosome 9

Pericentric inversions involving the secondary constriction (qh) region of chromosome 9 are considered to be normal variants. The evolutionary mechanisms and conservation of these inversions via Mendelian fashion have been investigated since the advent of banding techniques. Routine cytogenetic techniques cannot provide the fine characterization necessary to determine the type of genetic material involved in these rearrangements. Therefore, the fluorescence in situ hybridization technique with the human centromere-specific alpha satellite and the beta satellite (D9Z5) and classical satellite (D9Z1) human DNA probes were used to identify the breakpoints of chromosome 9 pericentric inversions. Four unique types of pericentric inversions involving the 9qh region were observed, and the mechanism may be due to breakage and reunion at the proposed breakpoints. They are: type A inversions consist of breakpoints within the alpha and beta satellite DNA regions; type B consist of breakpoints within the beta satellite DNA region and band 9q13; type C involve breakage within the beta and classical satellite DNA regions, and type D have breakpoints within the alpha and classical satellite DNA regions. Obviously, reshuffling of satellite DNA sequences has occurred, which has given rise to a variety of heteromorphisms whose clinical significance remains obscure. Received: 21 December 1995 / Revised: 30 May 1996     

A genetic linkage map of 27 markers on human chromosome 21.

We have constructed a genetic linkage map of the long arm of human chromosome 21 comprising 27 DNA markers. This map is an updated version of that reported earlier by group (1989, Genomics 4: 579-591), which contained 17 DNA markers. The current markers consist of 10 genes and 17 anonymous sequences. Traditional methods (restriction fragment length polymorphisms) were used to map 25 of these markers, whereas 2 markers were studied by polymerase chain reaction amplification of (GT)n dinucleotide repeats. Linkage analysis was performed on 40 CEPH families using the computer program packages LINKAGE, CRI-MAP, and MAPMAKER. Recombination rates were significantly different between the sexes, with the male map being 132 cM and the female map being 161 cM, assuming Kosambi interference and a variable ratio of sex difference in recombination. Approximately one-half of the crossovers in either sex occur distally, in terminal band 21q22.3, which also contains 16 of the markers studied. The average distance between adjacent markers was 6 cM.     

Isolation of repetitive DNA sequences from human chromosome 21.

We have developed a method for the isolation of phage from the human genomic library that carry repetitive DNA sequences highly represented on specific human chromosomes. We have used this technique to select recombinants carrying inserts concentrated on chromosome 21. Five clones, representing two families of sequences, have been characterized. Members of each family show cross-homology, but the two families show no homology with each other. In all but one case, the clones do not contain members of the human Alu repeat family. Single chromosome-concentrated repetitive sequences should prove to be useful in studies of the origin, evolution, and function of repetitive DNA and in regional chromosome mapping.     

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.     

Genetic linkage map of human chromosome 21

Two of the most common disorders affecting the human nervous system, Down syndrome and Alzheimer's disease, involve genes residing on human chromosome 21. A genetic linkage map of human chromosome 21 has been constructed using 13 anonymous DNA markers and cDNAs encoding the genes for superoxide dismutase 1 (SOD1) and the precursor of Alzheimer's amyloid beta peptide (APP). Segregation of restriction fragment length polymorphisms (RFLPs) for these genes and DNA markers was traced in a large Venezuelan kindred established as a "reference" pedigree for human linkage analysis. The 15 loci form a single linkage group spanning 81 cM on the long arm of chromosome 21, with a markedly increased frequency of recombination occurring toward the telomere. Consequently, 40% of the genetic length of the long arm corresponds to less than 10% of its cytogenetic length, represented by the terminal half of 21q22.3. Females displayed greater recombination than males throughout the linkage group, with the difference being most striking for markers just below the centromere. Definition of the linkage relationships for these chromosome 21 markers will help refine the map position of the familial Alzheimer's disease gene and facilitate investigation of the role of recombination in nondisjunction associated with Down syndrome.     

人类21号染色体与疾病

21号染色体是人类染色体中最小的常染色体,由日本等13个国家的研究单位组成的国际基因组已完成了21号染色体的DNA序列分析,其99．7％的序列已被测定。21号染色体长臂（21q)DNA,由33546361bp组成,其中只剩下3个小的克隆裂隙和7个序列裂隙（约100kb)尚未确定,在21q,q的近着丝粒处有21q远,近区域存在很多重复序列。21号染色体有39个断裂点,原因是由于21号染色体与其他染色体发生相互易位、21号染色体内重排或受到辐射所致。人类21号染色体上的特异基因与小鼠16、17及10号染色体上的某些基因互为同质异构基因,显示了基因组在进化过程中的变异性和保守性,21号染色体基因密度较小,含有127个已知基因,98个预报基因和59个假基因,21号染色体与Down′s综合征,某些单基因遗传紊乱,某些复杂疾病（双相情感障碍、家族性复合高血脂症）、实体瘤及白血病的发生有关。     

Estimating the rate of gene conversion on human chromosome 21

There is a growing recognition that gene conversion can be an important factor in shaping fine-scale patterns of linkage disequilibrium in the human genome. We devised simple multilocus summary statistics for estimating gene-conversion rates from genomewide polymorphism data sets. In addition to being computationally feasible for very large data sets, these summaries were designed to yield robust estimates of gene-conversion rates in the presence of variation in crossing-over rates. Using our summaries, we analyzed 21,840 biallelic single-nucleotide polymorphisms (SNPs) on human chromosome 21. Our results indicate that models including both crossing over and gene conversion fit the overall short-range data (0-5 kb) of chromosome 21 much better than do models including crossing over alone. The estimated ratio of gene-conversion rate to crossing-over rate has a range of 1.6-9.4, depending on the assumed conversion tract length (in the range of 500-50 bp). Removal of the 5,696 SNPs that occur in known mutational hotspots (CpG sites) did not significantly change our conclusions, suggesting that recurrent mutations alone cannot explain our data.     

PALML, a novel paralemmin-related gene mapping on human chromosome 1p21.

We describe PALML, a novel gene encoding a 551 amino acid protein with similarity to paralemmin and the paralemmin-like amino terminal domain of AKAP2, a protein kinase A anchor protein. PALML mRNA is expressed in many tissues and is most abundant in cardiac and skeletal muscle, while absent from brain and blood. Exogenously expressed PALML fusion protein has a widespread cytoplasmic localization, and it is excluded from the nucleus. Human PALML maps on human chromosome 1p21 (between D1S2767 and D1S223). SSCP-HD analysis of exonic sequences in patients with VUR (familial non-syndromic vesicoureteral reflux syndrome) excluded mutations in the PALML gene from causing this disease. PALML, paralemmin and AKAP2 share the presence of a conserved coiled coil region that may mediate protein interactions with shared partners. Based on its resemblance to paralemmin and AKAP2, PALML is hypothesized to be involved in regulating intracellular signaling and membrane-cytoskeletal interactions.     

Stereospecific DNA anomalies in the human chromosome 21

Using our own original computer program, we analysed more than 10 millions b.p. of the complete nucleotide sequence in the human chromosome 21. A graphic catalogue of largest stereospecific anomalies of this sequence is presented. Clusters of different stereospecific anomalies, showing presumably areas of cooperative binding of different regulatory and structural proteins to DNA have been revealed. Most of the large stereospecific anomalies are situated in introns, being often accompanied by regions devoid of some specific dinucleotides.