Is mammalian chromosomal evolution driven by regions of genome fragility?

gb-2006-7-12-r115 GBJ Research Is mammalian chromosomal evolution driven by regions of genome fragility? Ruiz-Herrera Aurora ruiz@sun.ac.za Castresana Jose jcvagr@ibmb.csic.es Robinson J Terence tjr@sun.ac.za

Evolutionary Genomics Group, Department of Botany & Zoology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa

Institut de Biologia Molecular de Barcelona, CSIC, Department of Physiology and Molecular Biodiversity, Jordi Girona 18, 08034 Barcelona, Spain

Genome Biology 1465-6906 2006 7 12 R115 http://genomebiology.com/2006/7/12/R115 17156441 10.1186/gb-2006-7-12-r115 1 8 2006 6 11 2006 8 12 2006 8 12 2006 2006 Ruiz-Herrera et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Mammalian chromosomal evolution

An analysis of the distribution of evolutionary breakpoints in eight species suggests that certain human chromosomal regions are repeatedly used during the evolutionary process, are associated with fragile sites, and show an enrichment of tandem repeats.

Abstract

Background

A fundamental question in comparative genomics concerns the identification of mechanisms that underpin chromosomal change. In an attempt to shed light on the dynamics of mammalian genome evolution, we analyzed the distribution of syntenic blocks, evolutionary breakpoint regions, and evolutionary breakpoints taken from public databases available for seven eutherian species (mouse, rat, cattle, dog, pig, cat, and horse) and the chicken, and examined these for correspondence with human fragile sites and tandem repeats.

Results

Our results confirm previous investigations that showed the presence of chromosomal regions in the human genome that have been repeatedly used as illustrated by a high breakpoint accumulation in certain chromosomes and chromosomal bands. We show, however, that there is a striking correspondence between fragile site location, the positions of evolutionary breakpoints, and the distribution of tandem repeats throughout the human genome, which similarly reflect a non-uniform pattern of occurrence.

Conclusion

These observations provide further evidence that certain chromosomal regions in the human genome have been repeatedly used in the evolutionary process. As a consequence, the genome is a composite of fragile regions prone to reorganization that have been conserved in different lineages, and genomic tracts that do not exhibit the same levels of evolutionary plasticity.

Evolution Genetics

Background

Evolutionary biologists have long sought to explain the mechanisms of chromosomal evolution in order to better understand the dynamics of mammalian genome organization. Early work in this area led Nadeau and Taylor 1 to propose the 'random breakage model' of genomic evolution, based on linkage maps of human and mouse. Their thesis relied on two assumptions: first, that many chromosomal segments are expected to be conserved among species and, second, that chromosomal rearrangements are randomly distributed within genomes. More than 20 years later, in large part due to molecular cytogenetic studies, large-scale genome sequencing efforts, and new mathematical algorithms developed for whole-genome analysis, the first assumption has been confirmed. However, the second has been questioned by the 'fragile breakage model' 2 , which considers that there are regions ('hotspots') throughout the mammalian genome that are prone to breakage and reorganization 3 4 .

Most recently, Murphy and colleagues 5 extended these analyses to include homologous synteny block (HSB) data from radiation hybrid maps of dog, cat, pig, and horse. Their findings corroborate the 'hotspot' theory and that some chromosome regions are reused 2 during mammalian chromosomal evolution. Indeed, that about 20% of the evolutionary breakpoint regions reported show reuse 5 , particularly among the more rapidly evolving genomes (cattle, dog, and rodents), led us 6 to question whether 'hotspots' identified in silico correspond to fragile sites that can be expressed in culture under specific conditions, thus mirroring findings of a correlation between the location of fragile sites and evolutionary breakpoints in primates, including human 7 8 . Our preliminary survey showed that at least 33 of the 88 cytogenetically defined common human fragile sites contain evolutionary breakpoints in at least three of the seven species analyzed by Murphy and colleagues 5 .

But what are fragile sites? These are heritable loci located in specific regions of chromosomes that are expressed as gaps or breaks when cells are exposed to specific culture conditions or certain chemical agents such as inhibitors of DNA replication or repair 9 . According to frequency of expression in the human population, and the mechanism of their induction, fragile sites have been classically divided into two groups: common and rare. Common fragile sites are considered part of the chromosome structure since they have been described in different mammalian species (Rodentia 10 , Carnivora 11 12 , Perissodactyla 13 , Cetartiodactyla 14 and Primates 7 15 16 ), whereas rare fragile sites are found expressed in a small percentage of the human population 17 . In total, 21 human fragile sites have been molecularly characterized: eight rare fragile sites (FRAXA 18 , FRAXE 19 , FRAXF 20 , FRA10A 21 , FRA10B 22 , FRA11B 23 , FRA16B 24 , and FRA16A 25 ), and 13 common human fragile sites (FRA1E 26 , FRA2G 27 , FRA3B 28 , FRA4F 29 , FRA6E 30 , FRA6F 31 , FRA7E 32 , FRA7G 33 , FRA7H 34 , FRA9E 35 , FRA13A 36 , FRA16D 37 , and FRAXB 38 ). Whereas the expression of rare fragile sites is known to be related to the amplification of specific repeat motifs (CCG repeats and AT-rich regions), no simple repeat sequences have been found to be responsible for the instability observed at common fragile sites. Rather, they appear to have a high A/T content with fragility extending over large regions (from 150 kilobases [kb] to 1 megabase [Mb]) in which the DNA can adopt structures of high flexibility and low stability 39 . Clearly, resolution differences exist between cytogenetically defined fragile sites in human chromosomes and the molecular delimitation of evolutionary breakpoints (themselves fairly gross approximations given that radiation hybrid mapping data for five of the eight species resulted in an average of 1.2 Mb for breakpoint regions 5 ). Nonetheless, the fact that fragile sites represent large 'unstable' regions of the genome 39 that in many instances span evolutionary breakpoints 7 is an observation that warrants further detailed analysis.

An intriguing aspect to emerge from comparative genomic studies performed largely on primates and rodents is the finding that breakpoint regions are rich in repetitive elements. In other words, there may be a causal link between the process of chromosome rearrangement, segmental duplications 40 41 42 43 44 , and some simple tandem repeats (for instance, the dinucleotide [TA]n 45 and [TCTG]n, [CT]n and [GTCTCT]n 46 ). In addition, microsatellites have been implicated in the mechanism underlying the chromosomal instability that characterizes some human fragile sites and constitutional human chromosomal disorders. For example, some human rare and common fragile sites have been found to be particularly rich in A/T minisatellites 39 , and certain human chromosomal aberrations have been related to palindromic AT-rich repeats 47 48 , underscoring the presence of repetitive elements in regions of chromosomal instability.

With this as the background, we analyze the distribution of 1,638 syntenic blocks, 1,152 evolutionary breakpoint regions, and 2,304 evolutionary breakpoints taken from public databases available for seven eutherian species (mouse, rat, cattle, dog, pig, cat and horse) and chicken, and examine these for correspondence with fragile sites and tandem repeat locations in the human genome. We show that evolutionary breakpoints are not uniformly distributed and that there are certain human chromosomes and chromosomal bands with high breakpoint accumulation. Additionally, there is a striking correspondence between human fragile site location, the positions of evolutionary breakpoints, and the distribution of tandem repeats throughout the human genome.

Results

Multispecies alignments

We analyzed homologous regions between the human genome and those of the rat, mouse, cattle, pig, cat, horse, dog, and chicken. By using the HSBs described by Murphy and coworkers 5 and adding data from the human/chicken and human/dog whole-genome sequence assemblies, we were able to identify 1,638 syntenic blocks in the human genome (Additional data file 4). (The dog radiation hybrid genome map data used by Murphy and coworkers 5 was replaced by the dog whole-genome assembly, which is now available.) The analysis of the human/chicken and human/dog whole-genome sequence assemblies revealed a total of 550 syntenic blocks among the three compared species (Additional data file 4). The homologous chromosomal segments of the seven mammals and the chicken were plotted against the 550 band human ideogram (Additional data file 1). We excluded the human chromosome Y from our study of evolutionary breakpoint regions (see Materials and methods, below).

In addition we identified the chromosomal position of 1,152 evolutionary breakpoint regions of 4 Mb or less in size (Additional data file 5) in the human karyotype and their corresponding evolutionary breakpoints (n = 2,304; Additional data files 1 and 5). The 2,304 evolutionary breakpoints grouped within 352 evolutionary chromosomal bands, which represents 67.77% of the human genome (2,217.46 Mb of the 3,272.19 Mb of the total human genome, NCBI35; Additional data file 5). See Figure 1 for a schematic representation of evolutionary breakpoint regions, evolutionary breakpoints and evolutionary chromosomal bands, as well as the Materials and methods section (below) for definitions of these terms. Approximately 45% (159 out of 352) of the evolutionary chromosomal bands contain evolutionary breakpoints in three or more of the eight species compared herein (Additional data file 6). These data clearly show that the distribution of the evolutionary breakpoints and breakpoint regions is concentrated in specific bands and/or chromosomes.

Figure 1

Schematic representation of evolutionary breakpoint regions, evolutionary breakpoints, and evolutionary chromosomal bands

Schematic representation of evolutionary breakpoint regions, evolutionary breakpoints, and evolutionary chromosomal bands. An evolutionary breakpoint region is defined as the interval between two syntenic blocks 4 megabases (Mb) or less in size. This is done in order to avoid problems of low comparative coverage. Evolutionary breakpoints are defined by sequences coordinates in any of the seven mammalian species compared with human plus the chicken, and serve to delimit the start and end of each breakpoint region. Evolutionary chromosomal bands correspond to any band in the human ideogram that contains at least one evolutionary breakpoint in any of the eight species compared with the human genome.

An analysis of the distribution of evolutionary breakpoints among the evolutionary chromosomal bands using JMP software (see Materials and methods, below) revealed a mean of six evolutionary breakpoints per evolutionary chromosomal band. Out of the 352 evolutionary chromosomal bands that were identified, 296 contain between one and ten evolutionary breakpoints, whereas 16 human chromosomal bands contain 20 or more evolutionary breakpoints each (10p11.2, 10q11.2, 15q13, 15q24, 15q25, 17p13, 17q24, 1q42.1, 22q11.2, 2p13, 2q14.3, 3p25, 3q21, 4p16, 7q22 and 8p23.1; Additional data file 6). Otherwise stated, 4.21% of the human genome (137.9 Mb of 3,272.19 Mb) accumulates 17.79% of all evolutionary breakpoints (410 of the 2,304 identified). Similarly, not all human chromosomes have been equally affected by the evolutionary process. Human chromosomes 1, 2, 3, 4, 7, 8, 10, 15, 17, and 22 carry most of the evolutionary breakpoints, whereas human chromosomes 14 and 21 are the least frequently involved.

Distribution of evolutionary breakpoints regions, breakpoints, and fragile sites

Given the distribution of evolutionary breakpoints outlined above, we proceeded to determine whether there is a significant correlation between the position of evolutionary breakpoints and the known location of fragile sites. We mapped all fragile sites (both rare and common) and evolutionary breakpoint regions (regions ≤ 4 Mb; Table 1 and Additional data file 1) to their location on the human ideogram at the 550 band resolution. Our examination reveals that 147 chromosomal bands express fragile sites (both common and rare). A contingency analysis shows that those bands that express fragility (they contain either rare or common fragile sites) have a tendency, although not significantly so (P = 0.09), to concentrate evolutionary breakpoints as compared with bands that do not express fragile sites. In fact, we observed 104 bands that contain fragile sites (rare and common) and evolutionary breakpoints, in contrast to the 95.4 bands expected if the distribution were random. A more refined analysis was subsequently conducted in which four categories of chromosomal bands (those that contain common fragile sites, those with rare fragile sites, bands with both common and rare fragile sites, and finally bands with no fragile sites) were examined using contingency analysis. There is a significant tendency (P = 0.01) for bands with rare fragile sites to accumulate evolutionary breakpoints (22 of the 24 bands known to express rare fragile sites contain evolutionary breakpoints versus the 15.6 bands expected if the distribution were random). The same tendency does not hold in the case of common fragile sites, where 73 of 111 bands that express common fragile sites contain evolutionary breakpoints (72.2 expected), or bands that contain evolutionary breakpoints but no fragile sites (248 observed versus 256.3 expected).

Table 1

The human ideogram at the 550 band resolution showing the location of fragile sites and evolutionary breakpoints

Chromosomal band

Type fs

Chromosomal band

Type fs

10p11.1

No fs

No EB

22q13.3^b

r-fs

10p11.2

No fs

2p11.1

No fs

No EB

10p12.1

No fs

2p11.2^b

r-fs

10p12.2

No fs

No EB

2p12

No fs

10p12.3

No fs

2p13^b

c-fs

10p13

No fs

2p14

No fs

10p14

No fs

2p15

No fs

10p15

No fs

2p16

c-fs

10q11.1

No fs

No EB

2p21

No fs

10q11.2^a

c-fs

2p22

No fs

10q21.1^a

c-fs

2p23

No fs

10q21.2^a

c-fs

2p24

c-fs

No EB

10q21.3^a

c-fs

2p25.1

No fs

No EB

10q22.1^a

c-fs

2p25.2

No fs

No EB

10q22.2

No fs

2p25.3

No fs

10q22.3

No fs

2q11.1

No fs

No EB

10q23.1

No fs

2q11.2^a

r-fs

10q23.2

No fs

2q12

No fs

10q23.3^a

r-fs

2q13^a

r-fs

10q24.1

No fs

2q14.1

No fs

10q24.2

No fs

2q14.2

No fs

10q24.3

No fs

2q14.3

No fs

10q25.1

No fs

2q21.1

No fs

10q25.2

c-fs and r-fs

No EB

2q21.2

No fs

10q25.3

No fs

No EB

2q21.3

c-fs

10q26.1^a

c-fs

2q22

r-fs

10q26.2

No fs

2q23

No fs

10q26.3

No fs

No EB

2q24.1

No fs

No EB

11p11.1

No fs

2q24.2

No fs

11p11.21

No fs

No EB

2q24.3

No fs

No EB

11p11.22

No fs

2q31^a

c-fs

No EB

11p12

No fs

No EB

2q32.1^a

c-fs

11p13

c-fs

2q32.2

No fs

11p14

c-fs

2q32.3

No fs

11p15.1^b

c-fs and r-fs

2q33^a

c-fs

11p15.2

No fs

2q34

No fs

11p15.3

No fs

No EB

2q35

No fs

11p15.4

No fs

2q36

No fs

11p15.5

No fs

2q37.1

No fs

11q11

No fs

No EB

2q37.2

No fs

11q12

No fs

2q37.3^b

c-fs

11q13.1^b

c-fs

3p11

No fs

No EB

11q13.2^b

c-fs

No EB

3p12

No fs

11q13.3^b

c-fs and r-fs

3p13

No fs

No EB

11q13.4^b

c-fs

3p14.1

No fs

11q13.5^b

c-fs

3p14.2

c-fs

No EB

11q14.1

No fs

3p14.3

No fs

11q14.2^a

c-fs

No EB

3p21.1

No fs

No EB

11q14.3

No fs

3p21.2

No fs

11q21

No fs

No EB

3p21.3

No fs

11q22.1

No fs

3p22

No fs

11q22.2

No fs

3p23

No fs

11q22.3

No fs

3p24

c-fs

11q23.3^a

c-fs and r-fs

No EB

3p25

No fs

11q23.2

No fs

No EB

3p26

No fs

No EB

11q23.1

No fs

3q11.1

No fs

No EB

11q24

No fs

3q11.2

No fs

11q25

No fs

No EB

3q12

No fs

12p11.1

No fs

No EB

3q13.1

No fs

No EB

12p11.2

No fs

3q13.2

No fs

12p12.1

No fs

No EB

3q13.3

No fs

12p12.2

No fs

No EB

3q21

No fs

12p12.3

No fs

No EB

3q22

No fs

12p13.3

No fs

3q23

No fs

12p13.2

No fs

No EB

3q24

No fs

12p13.1

No fs

No EB

3q25.1

c-fs

No EB

12q11

No fs

No EB

3q25.2

c-fs

No EB

12q12

No fs

No EB

3q25.3

c-fs

12q13.1^a

r-fs

3q26.1

No fs

12q13.2

No fs

3q26.2

No fs

12q13.3

No fs

3q26.3

No fs

12q14

No fs

3q27^a

c-fs

12q15

No fs

No EB

3q28

No fs

No EB

12q21.1

No fs

3q29

No fs

12q21.2

No fs

4p11

No fs

No EB

12q21.3^a

c-fs

No EB

4p12

No fs

12q22

No fs

4p13

No fs

12q23

No fs

4p14

No fs

No EB

12q24.1^b

c-fs and r-fs

4p15.1^b

c-fs

No EB

12q24.2^b

c-fs and r-fs

No EB

4p15.2^b

c-fs

No EB

12q24.31^b

c-fs

4p15.3^b

c-fs

12q24.32^b

c-fs

No EB

4p16^a

c-fs

12q24.33^b

c-fs

No EB

4q11

No fs

No EB

13p13

No fs

No EB

4q12

c-fs

13p12

No fs

No EB

4q13.1

No fs

No EB

13p11.2

No fs

No EB

4q13.2

No fs

No EB

13p11.1

No fs

No EB

4q13.3

No fs

13q11

No fs

No EB

4q21.1

No fs

13q12.1

No fs

4q21.2

No fs

13q12.2

No fs

No EB

4q21.3

No fs

No EB

13q12.3

No fs

No EB

4q22

c-fs

13q13

c-fs

4q23

No fs

No EB

13q14.1

No fs

4q24

No fs

No EB

13q14.2

No fs

4q25

No fs

No EB

13q14.3

No fs

4q26

No fs

13q21.1^b

c-fs

No EB

4q27

No fs

13q21.2^b

c-fs

No EB

4q28

No fs

13q21.3^b

c-fs

No EB

4q31.1

c-fs

13q22

No fs

No EB

4q31.2

No fs

13q31

No fs

4q31.3

No fs

13q32

c-fs

No EB

4q32

No fs

13q33

No fs

4q33

No fs

13q34

No fs

No EB

4q34

No fs

14p13

No fs

No EB

4q35

No fs

14p12

No fs

No EB

5p11

No fs

No EB

14p11.2

No fs

No EB

5p12

No fs

14p11.1

No fs

No EB

5p13.1^a

c-fs

No EB

14q11.1

No fs

No EB

5p13.2^a

c-fs

No EB

14q11.2

No fs

5p13.3^a

c-fs

No EB

14q12

No fs

No EB

5p14^b

c-fs

No EB

14q13

No fs

5p15.1

No fs

14q21

No fs

5p15.2

No fs

14q22

No fs

5p15.3

No fs

14q23^a

c-fs

5q11.1

No fs

No EB

14q24.1

c-fs

No EB

5q11.2

No fs

14q24.2

No fs

No EB

5q12

No fs

14q24.3

No fs

No EB

5q13.1

No fs

14q31

No fs

No EB

5q13.2

No fs

14q32.1

No fs

No EB

5q13.3

No fs

14q32.2

No fs

No EB

5q14

No fs

14q32.3

No fs

5q15

c-fs

15p13

No fs

No EB

5q21^b

c-fs

15p12

No fs

No EB

5q22

No fs

15p11.2

No fs

No EB

5q23.1

No fs

No EB

15p11.1

No fs

No EB

5q23.2

No fs

No EB

15q11.1

No fs

No EB

5q23.3

No fs

15q11.2

No fs

5q31.1^b

c-fs

15q12

No fs

5q31.2

No fs

No EB

15q13

No fs

5q31.3

No fs

No EB

15q14

No fs

5q32

No fs

15q15

No fs

5q33.1

No fs

15q21.1

No fs

5q33.2

No fs

15q21.2

No fs

No EB

5q33.3

No fs

15q21.3

No fs

No EB

5q34

No fs

15q22.1^a

c-fs

No EB

5q35.1^b

r-fs

15q22.2^a

c-fs

No EB

5q35.2^b

r-fs

15q22.3^a

c-fs

5q35.3^b

r-fs

15q23

No fs

6p11.1

No fs

No EB

15q24

No fs

6p11.2

No fs

15q25

No fs

6p12

No fs

15q26.1

No fs

6p21.1

No fs

15q26.2

No fs

No EB

6p21.2

No fs

15q26.3

No fs

6p21.3

No fs

16p11.1

No fs

No EB

6p22.1

No fs

16p11.2

No fs

6p22.2

c-fs

16p12^a

r-fs

6p22.3

No fs

16p13.1^b

r-fs

6p23^a

r-fs

No EB

16p13.2

No fs

No EB

6p24

No fs

No EB

16p13.3

No fs

6p25^a

c-fs

No EB

16q11.1

No fs

No EB

6q11

No fs

No EB

16q11.2

No fs

No EB

6q12

No fs

16q12.1

No fs

6q13^a

c-fs

16q12.2

No fs

6q14

No fs

16q13

No fs

6q15

c-fs

16q21

No fs

6q16.1

No fs

16q22^a

c-fs and r-fs

6q16.2

No fs

No EB

16q23^a

c-fs

6q16.3

No fs

16q24

No fs

No EB

6q21^a

c-fs

No EB

17p11.1

No fs

No EB

6q22.1

No fs

17p11.2

No fs

6q22.2

No fs

No EB

17p12

r-fs

6q22.3

No fs

17p13

No fs

6q23.1

No fs

No EB

17q11.1

No fs

No EB

6q23.2

No fs

17q11.2

No fs

6q23.3

No fs

No EB

17q12

No fs

6q24

No fs

17q21.1

No fs

6q25.1

No fs

17q21.2

No fs

6q25.2

No fs

17q21.3

No fs

6q25.3

No fs

17q22

No fs

No EB

6q26^b

c-fs

No EB

17q23

c-fs

6q27

No fs

17q24

No fs

7p11.1

No fs

No EB

17q25

No fs

7p11.2^a

r-fs

18p11.1

No fs

No EB

7p12

No fs

18p11.2

No fs

7p13^a

c-fs

18p11.32

No fs

7p14

c-fs

18p11.31

No fs

No EB

7p15.1

No fs

No EB

18q11.1

No fs

No EB

7p15.2

No fs

18q11.2

No fs

7p15.3

No fs

18q12.1

No fs

No EB

7p21

No fs

18q12.2

c-fs

7p22^b

c-fs

18q12.3

No fs

7q11.1^b

c-fs

No EB

18q21.1

No fs

7q11.21^b

c-fs

No EB

18q21.2

No fs

No EB

7q11.22^b

c-fs

18q21.3^a

c-fs

7q11.23^b

c-fs

18q22

r-fs

7q21.1

No fs

18q23

No fs

7q21.2

c-fs

19p11

No fs

No EB

7q21.3

No fs

19p12

No fs

7q22^b

c-fs

19p13.1^b

r-fs

7q31.1

No fs

19p13.2^b

r-fs

7q31.2

c-fs

No EB

19p13.3^b

r-fs

7q31.3

No fs

19q11

No fs

No EB

7q32

c-fs

19q12

No fs

7q33

No fs

19q13.1

c-fs

7q34

No fs

19q13.2

c-fs

7q35

No fs

19q13.3

c-fs

7q36^b

c-fs

19q13.4

c-fs

8p11.1

No fs

No EB

1p11

No fs

No EB

8p11.2

No fs

1p12

No fs

No EB

8p12

No fs

1p13.1

No fs

8p21.1

No fs

1p13.2

No fs

8p21.2

No fs

1p13.3

No fs

8p21.3

No fs

1p21

c-fs and r-fs

8p22

No fs

1p22.1

c-fs

8p23.1

No fs

1p22.2

c-fs

8p23.2

No fs

No EB

1p22.3

c-fs

8p23.3

No fs

1p31.1

c-fs

8q11.1

No fs

No EB

1p31.2

c-fs

8q11.21

No fs

1p31.3

c-fs

No EB

8q11.23

No fs

1p32.3^b

c-fs

8q12

No fs

1p32.1^b

c-fs

No EB

8q13

No fs

1p32.2^b

c-fs

8q21.1

No fs

1p33

No fs

No EB

8q21.2

No fs

1p34.1

No fs

No EB

8q21.3

No fs

No EB

1p34.2

No fs

8q22.1^a

c-fs

1p34.3

No fs

8q22.2

No fs

No EB

1p35

No fs

8q22.3

r-fs

1p36.1^b

c-fs

8q23

No fs

1p36.2^b

c-fs

8q24.1

c-fs and r-fs

1p36.3^b

c-fs

8q24.2

No fs

No EB

1q11

No fs

No EB

8q24.3^b

c-fs

No EB

1q12

c-fs

No EB

9p11

No fs

No EB

1q21.1^b

c-fs

No EB

9p12

No fs

No EB

1q21.2^b

c-fs

No EB

9p13

No fs

1q21.3^b

c-fs

9p21

c-fs and r-fs

1q22

No fs

No EB

9p22

No fs

No EB

1q23

No fs

9p23

No fs

1q24

No fs

9p24

No fs

1q25

c-fs

9q11

No fs

No EB

1q31

c-fs

9q12^b

c-fs

No EB

1q32.1

No fs

9q13

No fs

No EB

1q32.2

No fs

9q21.1

No fs

No EB

1q32.3

No fs

9q21.2

No fs

1q41

No fs

9q21.3

No fs

1q42.1^b

c-fs

9q22.1

c-fs

1q42.2^b

c-fs

No EB

9q22.2

No fs

1q42.3^b

c-fs

9q22.3

No fs

1q43

No fs

9q31

No fs

1q44^b

c-fs

9q32^a

c-fs and r-fs

20p11.1

No fs

No EB

9q33

No fs

20p11.2

r-fs

9q34.1

No fs

20p12

c-fs

9q34.2

No fs

No EB

20p13

No fs

9q34.3

No fs

20q11.1

No fs

No EB

Xp11.1

No fs

No EB

20q11.2

No fs

Xp11.21

No fs

No EB

20q12

No fs

No EB

Xp11.22

No fs

20q13.1

No fs

Xp11.23

No fs

20q13.2

No fs

No EB

Xp11.3

No fs

20q13.3

No fs

No EB

Xp11.4

No fs

No EB

21p13

No fs

No EB

Xp21.3

No fs

No EB

21p12

No fs

No EB

Xp21.2

No fs

No EB

21p11.2

No fs

No EB

Xp21.1

No fs

21p11.1

No fs

No EB

Xp22.1

No fs

21q11

No fs

Xp22.2

No fs

No EB

21q21.1

No fs

No EB

Xp22.3

c-fs

No EB

21q21.2

No fs

No EB

Xq11

No fs

21q22.1

No fs

Xq12.1

No fs

No EB

21q22.2

No fs

No EB

Xq12.2

No fs

No EB

21q22.3

No fs

Xq13

No fs

22p13

No fs

No EB

Xq21.1

No fs

22p12

No fs

No EB

Xq21.2

No fs

No EB

22p11.2

No fs

No EB

Xq21.3

No fs

22p11.1

No fs

No EB

Xq22.1

c-fs

22q11.1

No fs

No EB

Xq22.2

No fs

22q11.2

No fs

Xq22.3

No fs

22q12.1

No fs

Xq23

No fs

22q12.2^a

c-fs

Xq24

No fs

22q12.3

No fs

Xq25

No fs

22q13.1^b

r-fs

Xq26

No fs

22q13.2^b

r-fs

Xq27

c-fs and r-fs

Xq28

r-fs

No EB

All rare fragile sites (r-fs) and common fragile sites (c-fs) described by Schwartz and coworkers [39] and the evolutionary breakpoints (EBs) were plotted on the ideogram. Note that human chromosome Y is not included in the study. ^aFragile sites situated in bands comprising ≥ 6000 base pairs (bp) of tandem repeats in windows of 0.250 megabases (Mb). ^bFragile sites in bands with ≥ 10,000 bp of tandem repeats in windows of 0.250 Mb.

As stated above, resolution differences exist between cytogenetically defined fragile sites in human chromosomes and the molecular delimitation of evolutionary breakpoints. That differences in resolution may confound the association between them is clearly of concern. However, of the 12 autosomal common fragile sites that have been characterized at the molecular level (Additional data file 8), six (FRA4F, FRA6E, FRA7E, FRA7G, FRA7H, and FRA9E) were shown to span evolutionary breakpoints in at least one of the species analyzed with an additional two fragile sites (FRA3B and FRA16D) located within 1 Mb of evolutionary breakpoints (Additional data file 8). Importantly, of the four autosomal common fragile sites with the highest expression frequencies (FRA3B 28 , FRA6E 30 , FRA7H 34 , and FRA16D 37 ), two (FRA6E and FRA7H) are localized within evolutionary breakpoints, and two (FRA3B and FRA16D) lie within 1 Mb of breakpoint boundaries. With respect to the eight cloned rare fragile sites 18 19 20 21 22 23 24 25 , three (FRA10A, FRA16A, and FRA16B) are located in bands that contain evolutionary breakpoints in at least one of the species analyzed by us.

Distribution of tandem repeats

The distribution of tandem repeats in human chromosomes was analyzed using 250,000 bp search windows in order to determine whether there is any correspondence between tandem repeats, fragile sites (both rare and common), and the location of evolutionary breakpoints (Additional data files 2 and 8). The tandem repeats range from microsatellites (unit size 1 bp to 6 bp) to different types of minisatellites (from 7 bp to 300 bp). We identified a high concentration of tandem repeats in the telomeres and the pericentromeric regions of each chromosome (Additional data file 2), mirroring earlier findings (for instance, see Näslund and coworkers 49 ). The distribution of tandem repeats (1 to 300 bp) along human chromosomes showed that on average 3,738.56 bp of the 250,000 bp of genomic sequence contained in each window comprised tandem repeats (about 1.5%). Chromosome 19 is exceptional for the high number of repeats found along its length 50 , which is almost double (8,377.27 bp) the average for the whole genome (Table 2 and Additional data file 3). Additionally, chromosome 19 has been shown to be exceptional in many other genomic features, most of which (including the high number of repeats) may be due to the extremely high GC content of this chromosome 51 52 .

Table 2

Mean repeat size in base pairs per window of 0.250 megabases in each human chromosome analyzed.

Human chromosome

Mean number of repeats in B bands

Mean number of repeats in E bands

Mean number of repeats in FS bands

Mean number of repeats in no-FS bands

3,338.28

3,467.81

3,647.44**

3,213.66**

3,166.83

3,314.78

3,343.16

3,261.81

2,523.47**

3,104.42**

2,807.56

3,042.48

2,884.52

3,101.81

3,267.70

2,966.80

3,012.61

3,114.22

3,335.30*

2,943.73*

3,286.71

3,039.27

3,220.15

3,056.08

3,799.18

3,571.16

5,060.35**

2,830.71**

3,693.84*

3,065.77*

3,552.78*

3,118.19*

4,018.38

3,375.42

2,877.28

3,604.23

3,232.03

3,437.11

3,098.81*

3,651.40*

3,440.03

3,269.92

3,971.77**

3,017.47**

3,419.76

3,715.78

4,456.85**

3,112.39**

2,994.29

2,959.70

2,876.41

3,014.15

3,252.94

3,112.29

2,689.67*

3,267.68*

2,789.40*

3,302.97*

2,960.15

3,212.32

6,114.65**

4,414.38**

4,669.36

4,553.13

2,279.38**

4,346.49**

3,546.25*

4,314.01*

2,675.72*

3,175.75*

3,074.19

3,045.68

8,377.27

8,889.31

8,050.54

3,875.58

3,548.22

2,952.96*

3,755.64*

3,199.52*

5,062.95*

3,924.18

5,533.07

6,132.57*

5,020.60*

Total

3,237.46**

3,501.78**

3,735.08**

3,333.41**

Tukey-Kramer tests were calculated to evaluate the statistical difference among means in each chromosome and in the whole genome. In chromosomes 19 and 22 all bands are E bands and so no test is performed. No fragile sites have been described in the literature for human chromosome 21. Significant differences among band types are indicated as follows: *P = 0.05; **P = 0.002 (after Bonferroni correction applied to 22 samples). B bands, non-evolutionary bands; E bands, evolutionary bands; FS bands, bands containing fragile sites; no-FS bands, bands without fragile sites.

Tandem repeats and evolutionary chromosomal bands

When analyzing the human genome in its entirety, but excluding the centromeric and telomeric regions from the analysis, evolutionary chromosomal bands (E bands) tend to contain significantly more (P < 0.05) tandem repeats than chromosomal bands not implicated in evolutionary change (B bands; Table 2). It is noteworthy that in the case of human chromosomes 3, 15, 17, 18, and 21, E bands contain significantly more tandem repeats than do the B bands (P < 0.05), whereas the converse holds for human chromosomes 8 and 16. In all other instances no statistically supported differences were noted. Elimination of chromosome 19 from the analysis, with its singularly high repeat content, reduces the difference between E bands and B bands but not significantly so. In addition, we detected 256 human chromosomal bands that contain regions with more than 6,000 bp of tandem repeats in the 250,000 bp of genomic sequence contained in each window. Of these high-density repeat loci, 76.95% (197 of 256) contain evolutionary breakpoints.

Tandem repeats and fragile sites

Overall, chromosomal bands that express fragile sites (rare and common combined) contain significantly more tandem repeats (P < 0.05) than do bands that do not (Table 2 and Additional data file 9). There are, however, differences evident among chromosomes. In the case of human chromosomes 1, 5, 7, 8, 11, 12, and 22, chromosomal bands that express fragile sites contain more tandem repeats than do bands that do not show fragility (P < 0.05). The converse holds for chromosomes 10, 14, 17, and 20, where regions of fragility are not characterized by elevated tandem repeat levels. In the remaining human chromosomes (2, 3, 4, 6, 9, 13, 15, 16, 18, and 19), there is no statistical relationship between those bands that express fragile sites and have high numbers of tandem repeats, and bands that do not (Table 2). Moreover, the statistically significant differences detailed above hold irrespective of whether chromosome 19 is omitted from the analysis or not. Interestingly, 62.6% (92 out of 147; Table 1) of the human bands that contain human fragile sites are localized in regions that contain high densities of repeats (for instance, regions containing >6,000 bp of tandem repeats in the 250,000 bp of genomic sequence contained in each window; see above). No fragile sites have been described in the literature for human chromosome 21.

We examined the repeat content of the four categories of chromosomal bands (those that express common fragile sites, bands with rare fragile sites, bands with both common and rare fragile sites, and finally bands that do not contain fragile sites; Additional data file 9). Those containing rare fragile sites were shown to have significantly (P < 0.05) greater numbers of tandem repeats (average of 4,852.53 bp per 250,000 bp of genomic sequence contained in each window) than any other category (3,714.86 bp per 250,000 bp of genomic sequence contained in each window in the case of common fragile sites, the next most frequent category).