Letter to the Editor Recent Insertion of an

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85 Mol. Biol. Evol. 18(1):85–88. 2001

q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

Letter to the Editor

Recent Insertion of an Alu Element Within a Polymorphic Human-Specific Alu Insertion

David Comas,*† Ste´phanie Plaza,* Francesc Calafell,* Antti Sajantila,† and Jaume Bertranpetit*

*Unitat de Biologia Evolutiva, Facultat de Cie`ncies de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona, Catalonia, Spain; and

†Department of Forensic Medicine, University of Helsinki, Helsinki, Finland

Alu elements are a family of short interspersed re- peats that have mobilized throughout primate genomes by retrotransposition over the past 65 Myr of primate evolution (for a review, see Deininger and Batzer 1993).

In the human genome, Alu elements exist in copy num- bers of approximately 500,000 per haploid genome, rep- resenting approximately 5% of the genome, and they may be classified into groups of related subfamily members that share common diagnostic substitutions (Batzer et al. 1996b). The major subfamily branches (J, S, and Y) seem to have appeared at different evolutionary times, with J being older than S, and S being older than Y. Not only have the Alu elements contributed to the evolution of the primate genomes, but they also contrib- ute up to 0.4% of human genetic disease according to Deininger and Batzer (1999). Two main mechanisms may produce human diseases: direct insertions of Alu elements within genes (0.1% of human genetic disease), and unequal homologous recombination events between Alu repeats (0.3% of human genetic disease).

Some of the human Alu elements have retroposed so recently that their insertion at a specific location within the human genome remains polymorphic. These polymorphic insertions have been used as genetic markers in human evolution studies due to their particular prop- erties: they are rapid and easy to type, are apparently selectively neutral, and have known ancestral states. The insertion of an Alu element into the human genome is almost certainly a unique event, making any pair of Alu insertion alleles identical by descent and free of homo- plasy. The use of these polymorphisms in a worldwide survey of human populations has confirmed the African origin of modern humans (Batzer et al. 1994; Batzer et al. 1996a; Stoneking et al. 1997). One of these poly- morphic Alu insertions is the PV92 Alu site that is lo- cated in chromosome 16, and it has been proved to be human-specific (Batzer et al. 1994). The PV92 Alu in- sertion element belongs to the youngest subfamily of Alu sequences, the Alu Y subfamily, and, within that, to the Ya5 subfamily, which is defined by five diagnostic changes relative to the Y consensus (Batzer et al.

1996b). The PV92 Alu insertion is most frequent in Am- erindians (Novick et al. 1998) and East Asians (Stonek- ing et al. 1997), while it has lower frequencies elsewhere.

Key words: Alu, PV92, insertion event.

Address for correspondence and reprints: Jaume Bertranpetit, Un- itat de Biologia Evolutiva, Facultat de Cie`ncies de la Salut i de la Vida, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barce- lona, Catalonia, Spain. E-mail: jaume.bertranpetit@cexs.upf.es.

In order to understand the evolutionary context of the PV92 Alu insertion in Western Mediterranean pop- ulations, a total of 676 autochthonous individuals com- ing from the Iberian Peninsula (Andalusians, Basques, and Catalans) and North Africa (Morocco, Western Sa- hara, Algeria, and Tunisia) were typed for the human- specific PV92 Alu insertion polymorphism. The sample comprised unrelated healthy blood donors, and informed consent was obtained from all individuals participating in the study. Genomic DNA was extracted from whole blood using a phenol-chloroform extraction method after digestion with proteinase K. The PCR amplification of the PV92 Alu insertion locus was performed in 30 cycles of 958C for 1 min, 548C for 1 min, and 728C for 1 min, with a final elongation step of 728C for 7 min. The primers used were PV92A (59-AACTGGGAAAATTTGAA- GAGAAAGT-39) and PV92B (59-TGAGTTCT- CAACTCCTGTGTGTTAG-39). In order to visualize the results, the PCR product was run in a 2% agarose gel.

The chromosomes without the PV92 Alu insertion yielded an amplified fragment of 129 bp using the present set of primers (PV92A and PV92B), whereas the chromosomes with the insertion yielded an amplified fragment of;443 bp. In the present sample set of 1,352 chromosomes, 970 chromosomes did not bear the insertion, 382 bore the insertion, and the three remaining chromosomes bore a larger-than-expected insertion,

;789 bp in length (fig. 1A). These three chromosomes belonged to three different heterozygous individuals (two Basques and one northern Moroccan) whose other chromosome did not present the insertion.

In order to characterize the larger insertion detect- ed, the complete sequence of the region analyzed was obtained for two homozygous individuals without the insertion, two homozygous individuals with the standard insertion (belonging to the Ya5 subfamily), and the three individuals who presented the larger insertion. From these selected individuals, the agarose bands with the amplified product were excised, and the DNA was extracted and purified using GeneClean (BIO 101). The region was sequenced separately on both strands using primers PV92A and PV92B. Some of the sequenced frag- ments were difficult to read due to the long oligo(A) tail presented by the Alu insertions; therefore, internal sequenc- ing primers PV92C (59-AAAAGCCGGGCGTAG-39) and PV92D (59-TCGCCCAGGCTGGAG-39) were designed in order to overcome this problem.

The complete sequence of the individuals selected is shown in figure 1B. The chromosomes with the larger insertion presented an Alu sequence inserted within the standard PV92 Alu element, which produces an ampli- fied fragment around 346 bp larger than the standard

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86 Comas et al.

FIG. 1.—A, Agarose gel electrophoresis of the PCR products obtained for the PV92 site using the primers PV92A and PV92B. M 5 molecular weight marker (pBR322 DNA-Msp I Digest, New England Biolabs); 15 individual homozygous for the absence of the Alu insertion;

25 individual homozygous for the presence of the Alu Ya5 insertion; 3 5 individual heterozygous for the presence/absence of the Alu Ya5 insertion; 45 individual heterozygous with one chromosome with the Alu Ya5 plus Alu Yb8 insertions, and one chromosome without Alu

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Letter to the Editor 87

FIG. 2.—A, Polymorphisms found in the individuals sequenced.

Numbers indicate the polymorphic positions with respect to the inser- tion site of the Alu Ya5 element. The absence (2) and presence (1) of the Alu elements are indicated. BAS5 Basque; MOR 5 Moroccan.

B, Representation of the evolutionary events leading to the obtained sequences. Note that it is not possible to determine whether the inser- tion of the Alu Yb8 element preceded the appearance of the 1255 polymorphism.

←

insertions. B, Sequence of the PV92 genomic region comprising the two Alu elements (GenBank accession number AF302689). The Alu element belonging to the Ya5 subfamily is shown in dark gray, and the novel Alu element belonging to the Yb8 subfamily is shown in pale gray and is boxed. The diagnostic changes defining the Ya5 and Yb8 subfamilies are shown in bold. The polymorphic sites found are shown by superscript numbers:¹A-T polymorphism (position253);²A-G polymorphism (position1100);³A-G polymorphism (position1255). The approximate number of As of the oligo(A) tails are shown in the subscript.

fragment. This Alu element belongs to the Alu Y sub- family, in particular to the Alu Yb8 subfamily defined by eight diagnostic changes relative to the Y consensus (Batzer et al. 1996b).

Three positions of the sequenced region were found to be polymorphic in the individuals analyzed (fig. 2A):

one position upstream of the Alu insertions (position 53 upstream of the Alu Ya5 insertion site) and two positions within the Alu Ya5 element (positions 100 and 255 downstream of the site of the Alu insertion). The se- quence of one of the chromosomes with an absence of the Alu elements presented a T in position 253, while the rest of the chromosomes presented an A. One of the two individuals carrying only the Alu Ya5 element was homozygous for a G in position1100, whereas the oth- er Alu Ya5 homozygous individual presented a G in one chromosome and a C in the other. On the other hand, all of the chromosomes with the Alu Yb8 presented a C in this position. Finally, the chromosomes with only the Alu Ya5 element presented a C in position1255, where- as chromosomes with the Alu Yb8 element presented a G. The sequences of the three chromosomes with the novel Yb8 Alu insertion were identical. The novel Yb8 element was likely to be inserted in an original sequence carrying an A at position253 and a C at position 1100, but the present data do not allow us to elucidate whether the novel element was inserted in an original sequence with a C or a G at position1255 (fig. 2B). Two possible scenarios would yield the same result: the appearance of the C/G polymorphism in position1255 followed by the insertion of the Alu element or, alternatively, the in- sertion of the novel Alu element followed by a nucleo- tide change in position 1255. As none of the interme- diate sequences have been found in the present data, no conclusions about this sequence of events can be derived.

The novel Yb8 insertion reported occurred in the middle A-rich region of the preexisting Ya5 element.

Alu elements insert preferentially into A-rich regions (Daniels and Deininger 1985; Batzer et al. 1990; Matera et al. 1990); therefore, the presence of two A-rich re- gions within the Alu elements (in the middle and in the 39 oligo(A) tail) could increase the likelihood that one Alu element might insert within another Alu, as previ- ously described (Hammarstrom et al. 1984; Stoppa- Lyonnet et al. 1990). In this sense, there are a number of examples of insertions of Alu elements into the 39 oligo(A) tail of a preexisting element, but there are few examples of mobile element insertions into the middle A-rich region. Moreover, both A-rich regions have been reported to be associated with the presence of microsatellites (Batzer et al. 1990; Economou et al. 1990; Ar- cot et al. 1995) and even trinucleotide repeats that mu-

tated to form the Friedreich’s ataxia triplet repeat (Cam- puzano et al. 1996).

The Ya5 and Yb8 Alu subfamilies appear to be ac- tively undergoing concurrent amplification and mobili- zation within the human genome, as reported in a de- tailed study of these two subfamilies (Batzer et al. 1995) as well as evidenced by the de novo Alu insertions that have resulted in diseases (Deininger and Batzer 1999).

The present Yb8 Alu insertion is one of the four youn- gest repeats reported to date (after three Yb8 Alu inser- tions already reported in Deininger and Batzer [1999]), reinforcing the argument that these two Alu subfamilies are undergoing concurrent retroposition in the human genome.

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88 Comas et al.

The finding of an Alu element inserted within an- other polymorphic and human-specific Alu insertion suggests that the insertion of the novel Alu element rep- resents a very recent event. The present polymorphism shows a precise case of migration through the Straits of Gibraltar, an important barrier to human migration (Bosch et al. 1999). This fact, plus its limited geograph- ical distribution and its absence in a broad set of populations already typed in the literature, makes the novel Alu insertion described in the present study a clear case of private or population-specific polymorphism. A population-specific polymorphism is defined as a genetic variant found in a particular group that might be used as a genetic marker for the genetic affiliation of one individual (Calafell et al. 1999). Moreover, the descrip- tion of the Alu element insertions provides a first level of genetic analysis, but there is a second, more precise level of analysis that integrates nucleotidic variations within the Alu elements and even, as in the present case, new insertional events.

Acknowledgments

We thank Kirsti Hoö¨k and Marjo Leppa¨la¨ for tech- nical assistance. This research was supported by Direc- cioń General de Investigacioń Cientı´fica y Tećnica in Spain (PB98-1064) and Direccio´ General de Recerca, Generalitat de Catalunya (1998SGR00009). D.C. was fi- nancially supported by the Finnish Ministry of Educa- tion and by Centre for International Mobility (CIMO) Scholarships for foreign postgraduates and young re- searchers. S.P. is the recipient of a predoctoral fellow- ship (2000FI00696) by the Comissionat per a Univer- sitats i Recerca, Catalan Autonomous Government.

LITERATURE CITED

ARCOT, S. S., Z. WANG, J. L. WEBER, P. L. DEININGER, and M. A. BATZER. 1995. Alu repeats: a source for the genesis of primate microsatellites. Genomics 29:136–144.

BATZER, M. A., S. S. ARCOT, J. W. PHINNEY et al. (16 co- authors). 1996a. Genetic variation of recent Alu insertions in human populations. J. Mol. Evol. 42:22–29.

BATZER, M. A., P. L. DEININGER, U. HELLMANN-BLUMBERG, J.

JURKA, D. LABUDA, C. M. RUBIN, C. W. SCHMID, E. ZIET-

KIEWICZ, and E. ZUCKERKANDL. 1996b. Standardized no- menclature for Alu repeats. J. Mol. Evol. 42:3–6.

BATZER, M. A., G. E. KILROY, P. E. RICHARD, T. H. SHAIKH, T. D. DESSELLE, C. L. HOPPENS, and P. L. DEININGER. 1990.

Structure and variability of recently inserted Alu family members. Nucleic Acids Res. 18:6793–6798.

BATZER, M. A., C. M. RUBIN, U. HELLMANN-BLUMBERG, M.

ALEGRIA-HARTMAN, E. P. LEEFLANG, J. D. STERN, H. A.

BAZAN, T. H. SHAIKH, P. L. DEININGER, and C. W. SCHMID.

1995. Dispersion and insertion polymorphism in two small subfamilies of recently amplified human Alu repeats. J. Mol.

Biol. 247:418–427.

BATZER, M. A., M. STONEKING, M. ALEGRIA-HARTMANet al.

(11 co-authors). 1994. African origin of human-specific polymorphic Alu insertions. Proc. Natl. Acad. Sci. USA 91:

12288–12292.

BOSCH, E., F. CALAFELL, F. R. SANTOS, A. PE´ REZ-LEZAUN, D.

COMAS, N. BENCHEMSI, C. TYLER-SMITH, and J. BERTRAN-

PETIT. 1999. Variation in short tandem repeats is deeply structured by genetic background on the Y chromosome.

Am. J. Hum. Genet. 65:1623–1638.

CALAFELL, F., D. COMAS, A. PE´ REZ-LEZAUN, E. BOSCH, B.

MORERA, and J. BERTRANPETIT. 1999. Evolutionary pro- cesses and micro-differentiation: emergence and detection of population-specific variation. Pp. 15–28 in C. RENFREW

ed. America past, America present: genes and languages in the Americas and beyond. Oxbow Books, Oxford, England.

CAMPUZANO, V., L. MONTERMINI, M. D. MOLTOet al. (27 co- authors). 1996. Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.

Science 271:1423–1427.

DANIELS, G. R., and P. L. DEININGER. 1985. Integration site preferences of the Alu family and similar repetitive sequenc- es. Nucleic Acids Res. 13:8939–8946.

DEININGER, P. L., and M. A. BATZER. 1993. Evolution of re- trotransposons. Evol. Biol. 27:157–196.

———. 1999. Alu repeats and human disease. Mol. Genet.

Metab. 67:183–193.

ECONOMOU, E. P., A. W. BERGEN, A. C. WARREN, and S. E.

ANTONARAKIS. 1990. The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome.

Proc. Natl. Acad. Sci. USA 87:2951–2954.

HAMMARSTROM, K., G. WESTIN, C. BARK, J. ZABIELSKI, and U. PETTERSON. 1984. Genes and pseudogenes for human U2 RNA. Implications for the mechanism of pseudogene formation. J. Mol. Biol. 179:157–169.

MATERA, A. G., U. HELLMAN, M. F. HINTZ, and C. W. SCHMID. 1990. Recently transposed Alu repeats result form multiple source genes. Nucleic Acids Res. 18:6019–6026.

NOVICK, G. E., C. C. NOVICK, J. YUNISet al. (11 co-authors).

1998. Polymorphic Alu insertions and the Asian origin of Native American populations. Hum. Biol. 70:23–39.

STONEKING, M., J. J. FONTIUS, S. L. CLIFFORD, H. SOODYALL, S. S. ARCOT, N. SAHA, T. JENKINS, M. A. TAHIR, P. L.

DEININGER, and M. A. BATZER. 1997. Alu insertion polymorphisms and human evolution: evidence for a larger pop- ulation size in Africa. Genome Res. 7:1061–1071.

STOPPA-LYONNET, D., P. E. CARTER, T. MEO, and M. TOSI. 1990. Clusters of intragenic Alu repeats predispose the hu- man C1 inhibitor locus to deleterious rearrangements. Proc.

Natl. Acad. Sci. USA 87:1551–1555.

JEFFREYC. LONG, reviewing editor Accepted September 7, 2000