Adenosine deaminase deficiency

Abstract Adenosine deaminase (ADA, E.C. 3.5.4.4) exhibits a wellknown polymorphism at the protein level. We have studied ADA and an STR polymorphism exhibiting variation of a TTTA repeat motif at intron 3 of the ADA gene in random samples from northern Portugal (N = 218) and southwestern Germany (N = 114). The ADA phenotype distribution and population data on the worldwide distribution of ADA favor recurrent mutation as an explanation for the maintenance of the ADA *2 gene product at polymorphic frequencies.

KEY WORDS: ADENOSINE DEAMINASE, LINKAGE, POPULATION GENETICS, PORTUGAL, GERMANY

Adenosine deaminase (ADA; adenosine aminohydrolase, E.C. 3.5.4.4) is a cytosolic enzyme that catalyzes the irreversible deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively (Conway 1939). ADA is of considerable interest mainly because several mutations at this gene lead to severe combined immunodeficiency disease (ADA-SCID) by impairing the function of T and B cells [reviewed by Hershfield and Mitchell (1995)]. The ADA gene has been localized in the region of band 20q 13. 11 (Petersen et al. 1987), and the structure and sequence of the entire gene have been published (Wiginton et al. 1986).

The 41-kDa product of the ADA gene can be easily studied in erythrocytes. The most common gene product in all populations studied so far is the ADA*1 allozyme, which is electrophoretically more acidic than the less abundant ADA *2 (Spencer et al. 1968). The gene frequency of the ADA *2 allozyme is estimated at 0.06 in Western populations, 0.03 in Africans, and 0. 11 in Indians [Spencer et al. 1968; reviewed by Weissmann et al. (1982)].

Hirschhorn et al. (1994) demonstrated that the difference between the 2 allozymes lies in the substitution of a neutral Asn for an anionic Asp at codon 8. By using restriction fragment length polymorphism (RFLP) analysis, Hirschhorn et al. also demonstrated that the same transition was found in ADA-SCID patients in association with other mutations. The occurrence of the same substitution in different genetic backgrounds led us to investigate whether the normal ADA *2 allozyme is also heterogeneous and to elucidate the genetic mechanism responsible for such heterogeneity. For that we have simultaneously analyzed the ADA protein polymorphism and a tetranucleotide short tandem repeat (STR) variation located in intron 3 of the ADA gene reported by Scozzari et al. (1996).

Materials and Methods

Two hundred eighteen unrelated individuals from northern Portugal were phenotyped for the 2 markers (ADA and the short tandem repeat at intron 3). Blood samples were collected by venipuncture and used for both protein and DNA analysis.

For the ADA protein electrophoretic typing, hemolysates were pretreated with a solution of 3 mM dithiothreitol in a final dilution of 1:5. Isoelectric focusing gels were prepared as described by Rocha et al. (1988). The zymogram was obtained essentially as previously described by Spencer et al. (1968) but using a cellulose acetate overlay technique.

DNA extraction was performed using the Chelex method according to the technique of Lareu et al. (1994). Polymerase chain reaction (PCR) was carried out using the primers described by Scozzari et al. (1996). Thermocycling conditions (Perkin-Elmer 480, 30 cycles) were 94 deg C for 1 min, 54 deg C for 1 min, 72 deg C for 1 min, and a final extension at 72 deg C for 6 min. Amplified DNA fragments were separated by horizontal electrophoresis in polyacrylamide gels (Luis and Caeiro 1995) and visualized by silver staining (Budowle et al. 1991).

Alleles were designated according to the number of TTTA repeats, and phenotyping was carried out by side-by-side comparison with an allelic ladder made from previously typed samples containing alleles *7, *9, and *10. Disequilibrium analysis was performed using the GDA software package (Lewis and Zaykin 1997).

Results

Figure 1 depicts the patterns of ADA intron 3 TTTA repeats after separation and staining of the DNA fragments according to the experimental conditions. The simultaneous distribution of the 2 ADA polymorphisms in our northern Portugal and southwestern Germany samples is presented in Table 1.

The combined analysis of the 2 polymorphisms in the northern Portugal population sample gave no evidence of haplotypic association at this locus (Fisher exact test, p = 0.89). We have also studied a nonrandom southwestern Germany sample (especially enriched in ADA *2 gene product) and confirmed the nonassociation evident in the Portuguese sample. Furthermore, we could demonstrate that among 35 unambiguous haplotypes, ADA *2 occurs twice with allele *7, once with allele *10, and in another case with either allele *7 or *10 but not with allele *9.

Discussion

Our results give the first direct evidence that the normal ADA *2 gene is genetically heterogeneous. Because common alleles at the STR locus appear to be randomly distributed among ADA *2 gene products, several explanations are possible: (1) The protein polymorphism is old enough to have accumulated subsequent mutations at the STR region; (2) recurrent mutation at the G -4 A transition site is relatively frequent; (3) intragenic recombination between the 2 sites is more frequent than expected, given their physical distance.

Despite the probable accumulation of mutations at the STR site, subsequent to the origin of the ADA *2 allele, the hypothesis of recurrent mutation as a major cause is strongly supported by 3 independent arguments. First, the corresponding G ---> A transition occurs at a CpG dinucleotide, which is known to be a hotspot for C ---> 4 T and G ---> A mutations (Cooper and Youssoufian 1988). Second, concerning the STR allele frequency distribution, the European samples so far analyzed (Scozzari et al. 1996; this study) present lower heterozygosities than those reported for African populations (Scozzari et al. 1996). These findings indicate the occurrence of drift events that should have significantly reduced the STR heterogeneity associated with the ADA *2 allele in Europe, if recurrent mutation did not occur. Last, the ADA *2 gene is widely distributed in all present human populations with a very narrow low-frequency distribution (2-10%). Such a wide distribution of a low gene frequency is highly improbable given the demographic history of present human populations where random drift and differentiation are expected to have regularly occurred (Fuerst 1985).

Acknowledgments This study was partially supported by Praxis through grant PCNA/C/BIA/0106/96.

Literature Cited

Budowle, B., R. Chakraborty, A.M. Giusti et al. 1991. Analysis of the VNTR locus DIS80 by the PCR followed by high-resolution PAGE. Am. J. Hum. Genet. 48:138-144.

Conway, E.J. 1939. The deaminases of adenosine and adenylic acid in blood and tissues. Biochem. J. 33:479-492.

Cooper, D.N., and H. Youssoufian. 1988. The CpG dinucleotide and human genetic disease. Hum. Genet. 78:151-155.

Fuerst, P.A. 1985. Evolutionary differentiation and the sharing of alleles between populations. In Genetic Microdifferentiation in Humans and Other Animal Populations, Y.R. Ahuja and J.V. Neel, eds. Delhi, India: Indian Anthropological Association, 15-30.

Hershfield, M.S., and B.S. Mitchell. 1995. Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In The Metabolic and Molecular Bases of Inherited Disease, 7th ed., C.R. Scriver, A.L. Beaudet, W.S. Sly et al., eds. New York: McGraw-Hill, v. 2, 1725-1768.

Hirschhorn, R., D.R., Yang, and A. Israni. 1994. An Asp8Asn substitution results in the adenosine deaminase (ADA) genetic polymorphism (ADA*2 allozyme): Occurrence on different chromosomal backgrounds and apparent intragenic crossover. Ann. Hum. Genet. 58:1-9.

Laren, M.W., C.P. Phillips, A. Carracedo, et al. 1994. Investigation of the STR locus HUMTH01 using PCR and two electrophoresis formats: UK and Galician Caucasian population surveys and usefulness in paternity investigations. Forensic Sci. Int. 66:41-52.

Lewis, P.O., and D. Zaykin. 1997. Genetic Data Analysis: Computer Program for the Analysis of Allelic Data, Version 1.0. Free program distributed by the authors over the internet from the GDA Home Page at http://chee.unm.edu/gda/.

Luis, J.R., and B. Caeiro. 1995. Application of two STRs (VWF and hTPO) to human population profiling. Hum. Biol. 67:789-795.

Petersen, M.B., L. Tranebjaerd, N. Tommerup et al. 1987. New assignment of the adenosine deaminase gene locus to 20q13.11 by study of a patient with interstitial deletion 20q. J. Med. Genet. 24:93-96.

Rocha, J., A. Amorim, J. KC)mpf, et al. 1988. Demonstration of S-adenosylhomocysteine hydrolase polymorphism (E.C. 3.3.1.1.) by means of isoelectric focusing. Arzti. Lab. 34:283-284.

Scozzari, R., F. Cruciani, P. Santolamazza et al. 1996. Novel tetranucleotide repeat polymorphism in the human adenosine deaminase gene: Interethnic comparison of three major human groups. Hum. Biol. 68:315-320.

Spencer, N., D.A. Hopkinson, and H. Harris. 1968. Adenosine deaminase polymorphism in man. Ann. Hum. Genet. 32:9-14.

Weissmann, J., M. Vollmer, and 0. Pribilla. 1982. Survey on the distribution of adenosine dearninase and superoxide dismutase markers in different populations. Hum. Hered 32:344-356.

Wiginton, D.A., D.J. Kaplan, J.C. States et al. 1986. Complete sequence and structure of the gene for human adenosine deaminase. Biochemistry 25:8234-8244.

FATIMA SANTOS,1,2 ANT6NIO AMORIM,1,2 JORGE ROCHA, 1,2 AND JOST K6MPF3

1 Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200 Porto, Portugal.

2Faculdade de Ciencias da Universidade do Porto, Praca Gomes Teixeira, 4050 Porto, Portugal.

3 Institut fur Anthropologie und Humangenetik, Wilhelmstrasse 27, Eberhard-Karls Universitat, Tubingen D-72074, Germany.

Human Biology, December 1999, v. 71, no. 6, pp. 1009-1013.

Copyright (C) 1999 Wayne State University Press, Detroit, Michigan 48201-1309

Received 5 October 1998, revision received 8 February 1999.

Copyright Wayne State University Press Dec 1999
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