The X Chromosome Shows Less Genetic Variation at Restriction Sites Than the Autosomes

Am J Hum Genet 39:438-451, 1986

M. H. HOFKER,l M. I. SKRAASTAD, A. A. B. BERGEN, M. C. WAPENAAR, E. BAKKER, A. MILLINGTON-WARD, G. J. B. VAN OMMEN, AND P. L. PEARSON

SUMMARY Using a standard technique, 122 single-copy probes were screened for their ability to detect restriction fragment length polymorphisms (RFLPs) in the human genome. The use of a standardized RFLP screening enables the introduction of statistical methods in the analysis of differences in RFLP content between chromosomes and enzymes. RFLPs were detected from panels containing at least 17 unrelated chromosomes, digested with Taql, MspI, BgII, HindIll, EcoRI, and PstI. Forty autosomal probes, representing a sample of 2,710 base pairs (bp) per haploid genome, were tested, and 24 RFLPs were found. With 82 X-chromosomal probes, 17 RFLPs were found in 6,228 bp per haploid genome. The frequency of X-chromosomal RFLPs is three times less than that of the autosomes; this difference is highly significant (P = < .001). The frequency of RFLPs revealed by various restriction enzymes and the possibility that the X chromosome is a "low mutation" niche in the human genome are discussed.

INTRODUCTION Restriction fragment length polymorphisms (RFLPs) have been recognized as a powerful tool in the construction of linkage maps [1] and for carrier detection and prenatal diagnosis of genetic diseases [2]. Their applicability compares favorably to other genetic markers, since they are highly abundant in the

Received February 25, 1986; revised May 9, 1986. This work was supported by grant nr. 13-23-47 from the foundation for medical research FUNGO, which is subsidized by the Netherlands Organization for the Advancement of Pure Scientific Research (ZWO), and by grant nr. 28-878 from the Netherlands Prevention Fund. ' All authors: Department of Human Genetics, Sylvius Laboratories, State University of Leiden, 2333 AL, Leiden, The Netherlands. © 1986 by the American Society of Human Genetics. All rights reserved. 0002-9297/86/3904-0002$02.00 438 GENETIC VARIATION OF THE X CHROMOSOME 439 genome and DNA can be isolated from all nucleated cells. To date, more than 300 RFLPs have been identified and assigned to specific chromosomes [3]. For some chromosomes ( lp, 21, Xp, and distal Xq), a sufficient density of markers has been reached to permit general genetic disease mapping and use in genetic counseling [3]. Early investigations of the 0-globin region suggested 1:100 bp to be polymorphic [4]. Although this figure has since been confirmed for the serum albumin locus [5], several examples have been reported (a1-antitrypsin [6], thyroglobulin [7], factor VIII [8]) in which extremely low RFLP frequencies were found. This implies that the degree of polymorphism in the human genome can vary between different gene regions. However, in order to accurately quantify the average level of DNA variation in the human genome, a random series of probes for the detection of RFLPs should be used [9, 10]. In an initial study, Cooper et al. suggested that a lower RFLP content was present in the X chromosome than in the autosomes [10]. However, because of the small number of X probes employed, the statistical significance of their observations could not be verified. Initial data from our laboratory were consistent with this finding, indicating that X-chromosomal RFLPs occur with a frequency of 1: 1,000 bp per haploid genome [ 1], substan- tially lower than proposed for random autosomal probes [12, 13]. Here, we have extended these findings by the use of many more probes. To get unbiased data, the probes were tested on the same panel of 17-38 unrelated chromosomes restricted with the same set of enzymes. Heterozygosity was estimated in two ways. The first can be defined as the probability of finding a polymorphic base pair in all sites examined and takes into account the numbers of chromosomes investigated [9]. The second can be expressed as the chance that any base pair in a haploid genome is polymorphic, according to the method of Jeffreys [4], as modified by Ewens et al. [14], and is independent of the number of chromosomes investigated. The large series of probes employed in this study can also be used to test whether significant differences exist between enzymes in their ability to reveal RFLPs. The enzyme panel includes TaqI and MspI, both containing the CpG dimer, for which a higher variability has been reported [13].

MATERIALS AND METHODS X-Chromosome Specific Probes The X probes were isolated from libraries from flow-sorted chromosomes [15, 16] and characterized as described [11] or obtained from a cosmid library of a hamster hybrid cell line with a single human X chromosome. In the latter case, human inserts were identified initially by their human repeats. Each cosmid was digested with EcoRI and subcloned in pAT153. Recombinant plasmids were identified as human specific single- copy probes following hybridizing to filters containing human and hamster DNA. They were further characterized on a panel consisting of cell hybrids and human cell lines with unbalanced X-chromosomal abnormalities to prove their X-chromosome specific- ity. Furthermore, DNA of 100 X-specific cosmids was pooled together, digested with EcoRI and PstI or SauIIIA, and subcloned, allowing the rapid isolation of a large series of single-copy probes (Hofker et al., submitted for publication). 440 HOFKER ET AL. Autosomal Probes EcoRI-digested genomic DNA was size selected, and the 0.5-2.0-kilobase (kb) fraction was cloned in pAT153 and transformed into E. coli. Recombinant plasmids were isolated, labeled, and hybridized to genomic filters. Single-copy probes were identified and localized to specific chromosomes with a hybrid cell panel. A fraction enriched for chromosome 21 was obtained from a mouse hybrid cell line containing chromosome 21 as the only human chromosome by using a sedimentation chamber technique [17]. This DNA was partially cut with MboI and cloned in EMBL3. DNA of phages, containing human repeats, was digested with PstI and further subcloned into pAT153. The single-copy clones were identified, their chromosome 21 origin verified, and subregional localization obtained with cell hybrids and using human cell lines with unbalanced chromosome 21 aberrations.

RFLP Investigations Single-copy probes were investigated for their ability to detect restriction site variability. A minimum of 17 chromosomes per probe per enzyme ensured a 95% chance of detecting RFLPs with a minor allele frequency greater than 10% [18]. The restriction enzymes TaqI, MspI, BglII, EcoRI, PstI, and HindIII were used throughout. To be certain that only well-characterized RFLPs are involved, potential polymorphic bands were related to a dosage change at the major allele from two to one copy in those individuals in which an RFLP was thought to occur. High-frequency RFLPs were also confirmed by their Mendelian inheritance pattern.

Calculations For ease of calculation, the number of restriction sites examined has been assumed to be equal to the number of autoradiographic bands plus one. The assumption is that the bands reflect a contiguous stretch of DNA disregarding possible small internal fragments. This will lead to a systematic underestimation both of the number of tested sites and RFLPs detected, since some fragments will either be too small or too long to be mutually detected using standard Southern blotting methods. However, such errors are likely to affect the autosomal and the X-chromosomal data equally. The number of sites, multiplied by the number of base pairs present in the restriction site, gives the total number of base pairs screened per haploid genome (X). If n-chromosomes are tested then n x X is the total number of base pairs examined. Heterozygosity may be estimated in one of two ways: (1) Cooper and Schmidtke [9] suggest the use ofthe estimator of Nei [19], which in its form suitable for RFLP analysis gives:

HN = 1 - {(alb)2 + [(b - a)Ib]2} , (1) where a is the total number of variants and b is the total number of base pairs tested (n x X). HN is the probability that two homologous DNA sequences will have different base pairs at a given site. (2) Ewens et al. [14] propose the following estimator:

HE= v/2X, (2) where HE is the fraction of sites in the haploid genome at which two or more nucleotide types appear and v is the number of polymorphic sites observed. This formula takes no account of the allele frequencies involved. GENETIC VARIATION OF THE X CHROMOSOME 441 RESULTS Heterogeneity of the X chromosome In total, 82 X-specific single-copy probes were tested for their ability to detect RFLPs (table 1A-D and table 3). From the chromosome specific A charon 21a and A gtWES libraries, 23 unique sequence X probes were isolated. Four polymorphic sites were discovered in 1,922 nonhomologous bp per haploid genome. Over all chromosomes, 11 alleles were found in 38.4 kb. From 16 X-specific cosmid clones, 16 single-copy EcoRI fragments were isolated. Five polymorphic sites were found, resulting in 10 alleles. This represents 1,484 bp of the haploid genome; 25 kb was screened over all chromosomes tested. From the pooled X-specific cosmids, 37 single-copy X probes were isolated. These detected seven polymorphic sites in 2,636 bp, yielding 25 alleles in 45 kb over all chromosomes. From the total genomic plasmid library, three X probes were detected and one polymorphism was found. There appears to be no statistical evidence that any of the isolation strategies generated probes with different ability to detect RFLPs, and the data for all X.probes were pooled accordingly. Application of formula (2) gives a value for HE of .014, that is, the probability, that a site is polymorphic, is 1:714. From formula (1), HN = .0009, that is, the probability that two homologous sites differ is 1:1,100. Heterogeneity of the Autosomes From the plasmid library, 20 random genomic single-copy probes were isolated and assigned to different autosomes (table 2A). In 1,352 different bp, 15 polymorphic sites were discovered. In total, 51 kb was screened and 90 variants observed. From the charon 21A library in parallel with the X- chromosomal probes, 10 autosomal probes were obtained (table 2B), which detected four RFLPs in 798 bp. From a chromosome 21 library, 10 unique sequence probes were isolated (table 2C), which detected five polymorphisms in a sample of 560 bp tested. Application of the X2-test shows that none of these series ofprobes have a significantly different ability to detect RFLPs, in spite of their different chromosomal origin or isolation strategy. Accordingly, the total heterogeneity of the autosomal DNA was estimated on the pooled data of all 40 probes (table 3). With formula (2), HE = .0043, or 1:230 bp of the haploid genome is polymorphic. Using formula (1), HN = .0034 states that any given base pair has a chance of 1:300 to differ from its homologue. Comparison of the Heterogeneity ofAutosomes and the X Chromosome For the autosomes, of 2,710 bp, 24 sites were found to be polymorphic. For the X chromosome, only 17 polymorphic sites were observed out of 6,228 bp. Use of the X2-test to test for homogeneity of the RFLP detection of X probes vs. autosomal probes gives a probability of 99.9% in favor of the conclusion that the X-chromosomal probes belong to a different, less polymorphic population than the autosomal probes. Since HE(autosomes)/HEX = 3.1, the frequency of polymorphic sites is approximately three times less on the X 442 HOFKER ET AL.

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DISCUSSION RFLP Frequencies ofDifferent Enzymes Because of the extent of our RFLP screening, it was possible to examine the relative potency of different restriction enzymes to detect RFLPs and interpret differences in terms of the different base pair compositions in their recognition sites. In particular, CpG dimers are likely to undergo C to T transition [20]. In our study, a large fraction (48%) of the polymorphisms are revealed by either TaqI or MspI. The difference between these enzymes and the others appears to be less pronounced than that observed by Barker et al. [13], who detected 90% of the RFLPs with these two enzymes. From our data, BglII appears to be a good enzyme for the detection of RFLPs, while very few polymorphisms were detected with HindIII. In a study of Schumm et al. [21], 600 RFLPs were detected. Consistent with the above observations, these authors detected twice as many RFLPs with MspI relative to the average of the other enzymes and an elevated number for TaqI. Their finding that RsaI (GTAC) also reveals a high frequency of polymorphisms indicates that systematic studies with other enzymes may show other sequences to be highly polymorphic as well. In a thorough analysis of the relative abundance of the different restriction sites in the human genome, Wijsman [22] has questioned the significance of the increased polymorphism found at TaqI and MspI sites. Besides providing further support for the usefulness of these two enzymes in searches for RFLPs, our data do not provide much further insight in this ongoing discussion. Estimation ofHeterogeneity When studying overall heterozygosity at the DNA level, it is not possible to extrapolate from data based on studies of single gene sequences because of regional differences. Highly polymorphic regions, such as the serum albumin gene region [6], exist in parallel with very low polymorphic regions, as found in the thyroglobulin gene [7]. Cooper et al. [10] estimated the base pair variation 446 HOFKER ET AL.

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_____eq 448 HOFKER ET AL. TABLE 3 SUMMARY OF THE RESULTS

Sites (bp) screened bp screened No. No. per haploid over all variant No. Source probes genome chromosomes sites variants HE HN

Plasmid autosome ... 20 254 (1,352) 51,376 15 90 .0054 .0035 Phage autosome . 10 152 (798) 15,960 4 18 .0028 .0025 21-chromosome 10 98 (560) 11,120 5 30 .0036 .0042 Total .40 504 (2,710) 78,456 24 138 .0043 .0034 Plasmid X.3 35 (186) 4,650 1 8 .0024 .0033 Phage X.23 357 (1,922) 38,440 4 11 .0012 .0006 Cosmid X .19 277 (1,484) 25,228 5 10 .0017 .0009 Pool X.37 494 (2,636) 44,812 7 25 .0013 .0010 Total X .82 1,163 (6,228) 113,130 17 54 .0014 0.0009 with data obtained from randomly cloned probes. Fourteen autosomal probes were examined, and their heterozygosity determined with an estimator giving an overall chance of finding a variant allele. This resulted in a heterozygosity of HN = .0039. This is in good agreement with our estimate of HN = .0034, based on results with 40 autosomal probes, processed and calculated in a comparable fashion. However, one should be aware that there are some limitations on the use of these values. All of the calculations assume single base pair changes as the major cause of RFLPs. Furthermore, our investigations are performed only in the flanking sequences of single-copy probes and limited to the variation present in the recognition sites of six restriction enzymes selected for their known capability to detect RFLPs. If our estimation of the X-chromosomal heterogeneity HN (= .0009) is compared to the value obtained when the data of Aldridge et al. [23] are calculated in an identical manner (HN = .0007), the latter value is smaller. The difference may be due to the selection of the enzymes tested in our study, whereas Aldridge used a much larger panel of enzymes and a small panel of chromosomes. Many of the enzymes employed by these authors may well be less suitable for the detection of RFLPs because

TABLE 4 RFLP FREQUENCIES OF DIFFERENT ENZYMES

Enzyme Site No. polymorphic sites (%) MspI ...... CCGG 10 (24%) TaqI ...... TCGA 11 (24%) PstI ...... CTGCAC 5 (12%) HindIII ...... AAGCTT 2 (5%) BgIII ...... AGATCT 9 (21%) EcoRI ...... GAATTC 5 (12%) GENETIC VARIATION OF THE X CHROMOSOME 449 either their sites are less mutable or the fragments they produce are not in the range that can be conveniently separated on agarose gels. The uniformity of the RFLP screens in our study enables the determination of interchromosomal heterogeneity. The only possible bias, the source of our probes, disclosed no significant differences. On the basis of the estimator HN, the fraction of X-chromosomal variants is three times smaller than that of the autosomes. To investigate whether this is due to a larger proportion of low- frequency polymorphic sites, or fewer polymorphic sites, the data were com- puted by the estimator for heterozygosity proposed by Ewens et al. [14]. It gives an estimation of the number of polymorphic base pairs per haploid genome. The HE value is 3.8 times lower for the X chromosome. The two ratios do not differ significantly. Thus, the lower heterogeneity of this chromosome is due to the presence of fewer polymorphic sites rather than to lower minor allele frequencies. Although Cooper and Schmidtke [9] argued that the estimator HN will be more appropriate than HE, in practice, there appears to be a fixed relationship between the two. The values of HN and HE can be expected to be approximately equal when the frequency of variant chromosomes detected with a given probe and set of enzymes is approximately l/6 the frequency of variant sites. In many situations this will be true considering that the minor allele frequencies of the majority of detected RFLPs lie between .15 and .45 and the proportion of variant sites encountered. We must be circumspect in estimating the total frequency of variation within the human genome of this type of data, since only high-frequency (> .15) RFLPs will be consistently detected and the enzymes used may be more efficient than many others available. The former will lead to an underestimate of heterozygosity and the latter to an overestimate. Considering that - CpG - transitions represent such a large proportion of all RFLPs detected, the overestimate may be very large indeed when the data are extended to all recognition sites and not just extrapolated as at present. Potential Origins of the Difference between X and Autosomes A higher stability of the X chromosome was noted by Ohno [24], who postu- lated the conservation of the X chromosome in toto in the evolution of mam- mals, because of similarity in size, banding patterns, and the finding that the same genes are X-linked in more than one species. This linkage group conservation was mainly thought to occur as a result of the mechanism of dosage compensation, unique for X-linked genes, which prevents the conservation of X-autosomal translocations in the population. Because of hemizygosity of the X chromosome in the male, a substantial loss of new X-chromosomal mutations will occur through selection. However, we assume that since RFLPs occur predominantly in noncoding regions they origi- nate from presumptively neutral mutations. Neutral mutations could only be lost when they are linked to newly mutated coding or regulatory sequences, themselves eliminated by selection. Under conditions in which many X chromosomes are eliminated by hemizygous selection of structural gene muta- 450 HOFKER ET AL. tions, an increased homozygosity of linked noncoding sequences will be achieved. Further, the hemizygosity of the X chromosome in males may lead to a stronger conservation of sequences because of two reasons: (1) The absence of X-chromosome crossing over and/or gene conversion in males reduces one of the possible sources of mutations [25] relative to autosomes. (2) Evidence has been presented that the new mutations of the X-linked diseases hemophilia A and HPRT deficiency are mainly of paternal origin [26, 27]. It has been proposed that spermatocytes acquire more mutations than oocytes because of the greater number of cell divisions of the gametic maturation cycles in males than in females. The fact that males have only one and females two X chromosomes implies that X chromosomes are in 2/3 Of the cases replicated via oogenesis and only in /3 of cases via spermatogenesis. In contrast, the autosomes are equally distributed over female and male gametogenesis. With the proposed difference in mutation acquisition, this would ultimately lead to proportionally less polymorphisms on the X chromosome. The mechanisms proposed above clearly show the possibility of a lower mutation rate, even in absence of selection, which extends the evolutionary conservation of the X chromosome from linkage group conservation to the nucleotide level. ACKNOWLEDGMENTS We thank N. Goor and E. Klein-Breteler for the excellent technical assistance and Drs. M. H. Breuning and E. C. Klasen for critically reviewing the manuscript. REFERENCES 1. BOTSTEIN D, WHITE RL, SKOLNICK M, DAvIs RV: Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314-331, 1980 2. KAN YW, Dozy AM: Polymorphism of DNA sequence adjacent to 13-globin structural gene: relationship to sickle mutation. Proc Natl Acad Sci USA 75:5631-5635, 1978 3. WILLARD HF, SKOLNICK MH, PEARSON PL, MANDEL JL: Report of the Committee on Human Gene Mapping by Recombinant DNA Techniques. Cytogenet Cell Genet 40:360-489, 1985 4. JEFFREYS AT: DNA sequence variants in the G-y, Ay, 13, and 8 globin genes of man. Cell 18:1-10, 1979 5. MURRAY JC, MILLS KA, DEMOPULOS CM, HORNUNG S, MOTULSKY AG: Linkage disequilibrium and evolutionary relationships of DNA variants (restriction fragment length polymorphisms) at the serum albumin locus. Proc Natl Acad Sci USA 81:3486-3490, 1984 6. MATTESON KJ, OSTER H, CHAKRAVARTI A, ET AL.: A study of restriction fragment length polymorphisms at the human alpha-l-antitrypsin locus. Hum Genet 69:263- 267, 1985 7. BAAS F, BIKKER H, VAN OMMEN GJB, DE VIJLDER JJM: Unusual scarcity of restriction site polymorphism in the human thyroglobulin gene. A linkage study suggesting autosomal dominance of a defective thyroglobulin allele. Hum Genet 67:301-305, 1984 8. 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BARKER D, SCHAFER M, WHITE R: Restriction sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 36:131-138, 1984 14. EWENS WJ, SPIELMAN RS, HARRIS H: Estimation of genetic variation at the DNA level from restriction endonuclease data. Proc Natl Acad Sci USA 78:3748-3750, 1981 15. DAVIES KE, YOUNG B, ELLEs R, HILL M, WILLIAMSON R: Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry. Nature 293:374-381, 1981 16. KUNKEL LM, TANTRAVAHI U, EISENHARD M, LATT SA: Regional localization on the human X of DNA sequences cloned from flow sorted chromosomes. Nucleic Acids Res 10:1557-1561, 1982 17. COLLARD JG, PHILIPPUs E, TULP A, LEBO RV, GRAY JW: Separation and analysis of human chromosomes by combined velocity sedimentation and flow sorting applying single- and dual-laser flow cytometry. Cytometry 5:9-19, 1984 18. SKOLNICK M, WHITE R: Strategies for detecting and characterizing restriction fragment length polymorphisms (RFLPs). Cytogenet Cell Genet 32:58-67, 1982 19. NEI M: Molecular Population Genetics and Evolution. Amsterdam, North-Holland, 1975 20. COULONDRE C, MILLER JH, FARABAUGH PJ, GILBERT W: Molecular basis of base substitution hotspots in E. coli. Nature 274:775-780, 1978 21. SCHUMM J, KNOWLTON R, BRAMAN J, ET AL.: Detection of more than 500 single copy RFLPs by random screening. Cytogenet Cell Genet 40:739, 1985 22. WIJSMAN EM; Optimizing selection of restriction enzymes in the search for DNA variants. Nucleic Acids Res 12:9209-9226, 1984 23. ALDRIDGE J, KUNKEL L, BRUNS G, ET AL.: A strategy to reveal high-frequency RFLPs along the human X chromosome. Am J Hum Genet 36:546-564, 1984 24. OHNO S: Ancient linkage groups conserved in human chromosomes and the concept of frozen accidents. Nature 244:259-262, 1973 25. COWAN EP, JORDAN BR, COLIGAN JE: Molecular cloning and DNA sequence analysis of genes encoding cytotoxic T lymphocytes defined HLA-A3 subtypes: the El subtype. Proc Natl Acad Sci USA 135:2835-2841, 1985 26. HALDANE JBS: The mutation rate of the gene for hemophilia, and its segregation ratios in males and females. Ann Eugen (Lond) 13:262-271, 1947 27. FRANCKE U, FELSENSTEIN J, GARTLER SM, ET AL.: The occurrence of new mutants in the X-linked recessive Lesch-Nyhan disease. Am J Hum Genet 28:123-137, 1976