GENOMICS
11,573-576
(1991)
Linkage Mapping of Highly Informative DNA Polymorphisms within the Human Interferon-a Receptor Gene on Chromosome 21 MELVIN
G. MCINNIS,*lt GEORGES LUTFALLA,$ SUSAN SLAUGENHAUPT,~ MICHAEL B. PETERSEN,* GILLES UZE,$ ARAVINDA CHAKRAVARTI,!?~ AND STYLIANOS E. ANTONARAKIS**’
*Center for Medical Genetics and Departments of Pediatrics and tfsychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; SCNRS Laboratory of Viral Oncology, Villejuif, France; and §Departments of Human Genetics and IPsychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15267 Received
Apri122,
1991;revised
1989) and variable poly(A) tracts of Alu repetitive elements (AluVpA) (Economou et aZ., 1990; Zuliani and Hobbs, 1990) are particularly useful in mapping. In this study we have identified a highly informative AluVpA DNA marker within intron 5 of the IFNAR locus and used it along with the previously described multiallelic RFLP (Viehl et al., 1990) to localize the IFNAR locus on the human genetic linkage map of human chromosome 21 (Petersen et aZ., 1991) constructed using the CEPH (Centre d’Etude Polymorphisme Humain) families (Dausset et aZ., 1990).
Two polymorphic loci within the interferon-a receptor (IFNAR) gene on human chromosome 2 1 have been identified and mapped by linkage analysis in 40 CEPH families. These markers are (1) a multiallelic RFLP with an observed heterozygosity of 0.72 and (2) a variable (AT,), short sequence repeat at the poly(A) tail of an Alu sequence (AluVpA) with an observed heterozygosity of 0.83. This locus is close to D21S58 (8 = 0.02, Z! = 36.76) and D21S17 (8 = 0.02,i = 21.76) within chromosomal band 21q22.1. Multipoint linkage analysis suggests the most likely locus order to be 21cen-D21S58-IFNAR-D2lSl7-21qter. Given its high heterozygosity, the IFNAR gene can be used as an index marker on human chromosome 21. o 1991 Academic
June 13. 1991
METHODS
Press, Inc.
The different alleles of the short sequence repeat polymorphism due to (TAAA), at the 3’ end of an Ah repeat within intron 5 (unpublished results) of the IFNAR gene (seeFig. 1) were detected using the polymerase chain reaction (PCR) as follows: The oligonucleotides bordering the Alu repeat sequence were 5’-CACACTATGTAATACTATGC-3’, termed IFNAR-IVS5-3’.2 (interferon-a receptor-intervening sequence 5); and 5’-TGCTTACTI’AACCCAGTGTG-3’, termed IFNAR-IVS5-5’. Initial experiments were performed with the 3’-oligonucleotide priming sequence5’AGCCGAGATTGAGCCACTGC-3’, which is termed IFNAR-IVS5-3’.1 and is within the repetitive Ah sequence. IFNAR-IVS5-5’ was end-labeled with [T-~“P] ATP. Amplification was carried out using 500 ng of genomic DNA in a 25-~1reaction of 10 n&f Tris-HCl, pH 8.3,50 r&f KCI, 1.5 mA4 MgCl,, 0.02% gelatin, 200 &f each dNTP, 0.8 U Z’aq polymerase, 0.4 &f IFNARIVS5-3.2 primer (0.04 pA4 IFNAI-IVS5-3.1’), and 0.08 &f IFNAR-IVS5-5’ primer. The PCR amplification was performed for 30 cycles (denaturation at 94”C, 30 s; annealing at 56”C, 30 s; extension at 72”C, 30 8). Polyacrylamide gel electrophoresis of the reaction products was performed as previously described (Petersen et al., 1990). The IFNAR-19ES polymorphism was detected
INTRODUCI’ION
The interferon-a receptor is a protein receptor molecule located on the mammalian cell surface and mediates the actions of interferon-a across the cell wall (Mogensen et al., 1989). The cDNA for the human interferon-a receptor (IFNAR) gene has been recently cloned and sequenced (Uze et al., 1990). This gene has been localized to chromosome 21q22.1 by in situ hybridization (Lutfalla et aZ., 1990) and by somatic cell hybrids (Slate et al., 1981; Langer et al., 1990). A multiallelic RFLP with Hind111 was recently reported using a cDNA probe 19ES (Viehl et al., 1990). DNA polymorphisms within human genes permit their localization on the human genetic linkage map. Highly informative multiallelic polymorphisms that include VNTRs (variable number of tandem repeats) (Jeffreys et al., 1985) or short sequence repeats (Weber and May, 1989; Litt and Luty, 1989; Tautz,
1 To whom
reprint
requests
should
be addressed. 573
All
Copyright 0 1991 rights of reproduction
OS&%7543/91 $3.00 by Academic Press, Inc. in any form reserved.
574
MCINNIS
ET
AL.
TABLE
2
Two-Point Linkage Analysis between the INFAR Locus and Markers on Human Chromosome 21, Listed in the Order from 2lcen to 2lqter
FIG. 1. The sequence from the fifth intron of the IFNAR gene from which the AluVpA was identified is shown. The priming sequences are printed in lowercase letters; the Ah sequence is underlined.IFNAR-IVSB-5’: 5’-tgcttacttaacccagtgtg-3’. IFNAR-IVS53’.1: 5’-agccgagattgagccactgc-3’. IFNAR-IVS5-3’.2: B’-cacactatgtaatactatgc-3’.
by Southern blot analysis following Hi&III digestion of genomic DNA and hybridization to the 195-bp cDNA probe 19ES as described (Vi&l et al., 1990). Human genomic DNA from members of 40 CEPH families was used to genotype these two loci, to estimate the frequency of the different polymorphic alleles, and to map this polymorphic locus by linkage analysis. RESULTS Of the 40 CEPH families analyzed for the IFNAR19ES polymorphism, 7 were uninformative; 15 inforTABLE
1
Allelic Frequency and Observed Heterozygosity DNA Polymorphisms within the IFNAR Gene Allele
Size (kb)
Frequency
(%)
of
Observed heterozygosity
A. IFNAR-19ES 1 2 3 4 5 6
8.1 -.-a 11.8
2.9 8.1 30.1 18.4 25.0 15.5 B. IFNAR
1 2 3 4 5 6
lb
II’
195 199 203 207 211 215
462 466 470 474 478 480
72% (75 of 104)
AluVpA
13.1 21.7 8.2 21.7 8.7 26.6
83% (78 of 95)
’ The sizes of the intervening alleles range between 8.1 and 14.8 kb; exact lengths in kb are not given because of gel to gel variation. b Size of PCR produce (in nucleotides) between IFNAR-IVSB-5 and IFNAR-IVSB-3’.1. ’ Size of PCR produce (in nucleotides) between IFNAR-IVSS-5’ and IFNAR-IVSS-3’.2.
2lcen D21S120 D21S13 D21SllO D21Sl/Sll D21S8 APP D21Slll D21S82 SOD1 D21S58 D21S17 D21S55 ETS2 D21S3 D21S156 HMG14 D21S15 MXl D21S42 CRYAl D21S113 CD18 D21S171 PFKL D21S112 COL6Al 21qter
0.30 0.33 0.27 0.28 0.20 0.16 0.11 0.08 0.07 0.02 0.02 0.10 0.09 0.15 0.12 0.18 0.18 0.23 0.20 0.24 0.24 0.22 0.29 0.28 0.26 0.27
6.62 2.43 5.27 7.67 10.84 14.78 28.25 45.64 4.67 36.76 21.61 30.76 25.29 23.54 36.45 16.40 8.83 15.34 7.92 8.17 9.03 9.39 8.92 4.04 9.38 8.85
0.39 0.45 0.37 0.32 0.26 0.19 0.15 0.07 0.07 0.00 0.02 0.13 0.10 0.14 0.12 0.20 0.20 0.25 0.20 0.29 0.24 0.16 0.26 0.27 0.23 0.22
0.23 0.20 0.18 0.24 0.13 0.14 0.07 0.08 0.07 0.05 0.03 0.07 0.08 0.17 0.12 0.16 0.17 0.21 0.20 0.18 0.25 0.27 0.32 0.30 0.30 0.34
8.22 4.49 6.86 8.11 12.00 14.96 28.92 45.65 4.67 38.09 21.63 31.18 25.33 23.58 36.45 16.51 8.86 15.46 7.92 8.71 9.03 9.91 9.16 4.07 9.65 9.74
mative families and all CEPH parents were analyzed with IFNAR AluVpA. Genotypes were initially generated with IFNAR-IVS5-3’.1 (oligonucleotide within the Ah); however, it was found that the data were more consistent with the IFNAR-IV%-3’.2 primer, which is not part of the Ah repetitive sequence. In the 8 families on which there were data for both polymorphisms, haplotypes were constructed and used for linkage analysis. The IFNAR-19ES polymorphism showed six alleles with a heterozygosity of 0.72. The IFNAR AluVpA polymorphism also showed six alleles (see Fig. 2) with a heterozygosity of 0.83 in 95 unrelated individuals. The observed heterozygosity from unrelated individuals in the CEPH pedigrees and the frequency and size of each allele are provided in Table 1. To localize IFNAR relative to other DNA markers on the human chromosome 21 genetic map, we performed two-point linkage analyses using the computer program LINKAGE, version 4.7 (Lathrop and Lalouel, 19861, against 26 other loci on chromosome 21 as previously described (Warren et al., 1989; Petersen et al., 1991) and multipoint linkage analysis
HIGHLY
INFORMATIVE
DNA
575
POLYMORPHISMS
12345678910
A
IFNAR AluVpA
FIG. 2. Allelic polymorphisms (B) members of the CEPH family
B
IFNAR
AluVpA
of the IFNAR AluVpA in (A) 10 Caucasians using primers 12 using primers IFNAR-IVSS-5’ and IFNAR-IVS5-3’.2.
against DNA markers APP, D21Sll1, D21S82, D21S17, D21S55, D21S58, ETSB, and D21S3. The results of two-point linkage analysis between IFNAR and 26 loci on chromosome 21 are shown in Table 2. The IFNAR locus is most closely linked to D21S58 with a lod score of 36.76 at a recombination value of 0.02 and to D21S17 with a lod score of 21.61 at a recombination value of 0.02. The most likely order is 21cen-D21S58-IFNAR-D21S17-D21S55-2lqter; the relative odds of this order compared to the second most likely order, 21cen-D21S58-D21S17-IFNARD21S55-21qter, were 103.212.
VAR (A8906303Q), Cancer. We thank
I,
The IFNAR gene maps just proximal to the minimal Down syndrome region (McCormick et al., 1989; Korenberg et aZ., 1990; RahmanietaZ., 1989). The multiple effects of IFNAR include modulation of cellular activities, inhibition of cell growth, and antitumor effects (Gresser, 1989), disturbances of which could have an effect on the full phenotype of the Down syndrome. The IFNAR AluVpA appears to be more efficacious than the IFNAR-19ES polymorphism due to the rapidity of its PCR-based detection compared to that of the Southern blot procedure. The heterozygosity value of 0.83 makes IFNAR AluVpA a good index marker in the middle of the linkage map of chromosome 2lq.
1.
2.
and
and the Association Pour la Recherche sur le Tara Cox for assistence with linkage analysis.
DAUSSET, J., CANN, H., COHEN, D., LATHROP, M., LALOIJEL, J. M., AND WHITE, R. (1990). Centre d’Etude du Polymorphisme Humain (CEPH): Collaborative genetic mapping of the human genome. Genomics 6: 575-577. ECONOMOU, E. P., BERGEN, A. W., WARREN, A. C., AND ANTONARAKIS, S. E. (1990). The polyadenylate tract of Alu repetitive elements is polymorphic in the human genome. Proc. Natl. Acad. Sci. USA 87: 2951-2954. GRESSER, I. (1989). Antitumor effects of interferon. Acta Oncol. 28: 347-353.
4.
JEFFREYS, A. J., WILSON, V., AND THEIN, S. L. (1985). Hypervariable “minisatellite” regions in human DNA. Nature 314: 67-73.
5.
KORJXNBERG, J. R., KAWASHIMA, H., I%JLST, S. M., IKEUCHI, T., OGASAWARA, N., YAMAMOTO, K., SCHONBERG, S. A., WEST, R., ALLEN, L., MAGENIS, E., IKAWA, K., TANIGUCHI, N., AND EPSTEIN, C. J. (1990). Molecular definition of a region of chromosome 21 that causes features of the Down syndrome phenotype. Am. J. Hum. Genet. 47: 236-246. LANGER, J. A., RASHIDBAIGI, A., LAI, L. W., PATTERSON, D., AND JONES, C. (1990). Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein. Somat. Cell Mol. Genet. 16: 231-240.
6.
7.
LATHROP, G. M., AND LALOUEL, J. M. (1988). Efficient putations in multilocus linkage analysis. Am. J. Hum. 42: 498-505.
8.
LIP, M., AND LUTY, J. A. (1989). A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle gene. Am. J. Hum. Genet. 44: 397-401.
9.
LUTFALLA, G., ROECKEL, N., MOGENSEN, K. E., MATTEI, M. G., AND UZE, G. (1990). Assignment of the human interferon-a receptor gene to chromosome 21q22.1. J. Interferon Res. 10: 515-517. MCCORMICK, M. K., SCHINZEL, A., PETERSEN, M. B., STETTEN, G., DRISCOL, D. J., CANTU, E. S., TRANEBJAERG, L., MIKKELSEN, M., WATKINS, P. C., AND ANTONARAKIS, S. E. (1989). Molecular genetic approach to the characterization of the “Down syndrome region.” Genomics 5: 325-531. MOGENSEN, K. E., UZE, G., AND EID, P. (1989). The cellular
ACKNOWLEDGMENTS 10. This research was supported by NIH Grants HD24605 (S.E.A.) and HGO0344 and HD 00774 (A.C.). M.G.M. was supported by a fellowship from NIA Grant AG00149, a research training grant in the dementias of aging. We thank Dr. Ion Greaser for support. G.L. and G.U. were supported by grants from DRET (89.34.132), AN-
and IFNAR-IVS5’-3’.1
REFERENCES
3.
DISCUSSION
IFNAR-IVS5-5’
11.
comGenet.
576
MCINNIS
receptor of the alpha-beta interferons. Experimentia 508.
45: 500-
12. PETERSEN, M. B., ECONOMOU, E. P., SLAUGENHAUPT, S. E., CHAKRAVARTI, A., AND ANTONARAKIS, S. E. (1990). Linkage analysis of the human HMG 14 gene on chromosome 21 using a GT dinucleotide repeat as polymorphic marker. Genomics 7: 136-138. 13. PETERSEN, M. B., SLAUGENHAUPT, S. A., LEWIS, J. G., WARREN, A. C., CHAKRAVARTI, A., AND ANTONARAKIS, S. E. (1991). A genetic linkage map of 27 loci on human chromosome 21. Gerwmics 9: 407-419. 14. RAHMANI, Z., BLOUIN, J.-L., CREAU-GOLDBERG, N., WATKINS, P. C., MATTEI, J.-F., POISSONNIER, M., PFUEUR, M., CHETTOUH, Z., NICOLE, A., AURIAS, A., SINET, P.-M., AND DELABAR, J.-M. (1989). Critical role of the D21S55 region on chromosome 21 in the pathogenesis of Down syndrome. PFOC. N&l.
Acad.
Sci. USA
86: 5958-5962.
15. SLATE, D. L., RUDDLE, F. H., AND TAN, Y. H. (1981). Genetic control of the interferon system. In “Interferon 3” (I. Gresser, Ed.), pp. 65-76, Academic Press, London.
ET
AL.
16. TAUTZ, D. (1989). Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res. 17: 6463-6471. 17. UZE, G., LUTFALLA, G., AND GRESSER, I. (1990). Genetic transfer of a functional human interferon-a receptor into mouse cells: Cloning and expression of its cDNA. Cell 60: 225-234. 18. VIEHL, E., UZE, G., LUTFALLA, G., BANDU, M. T., AND MoGENSEN, K. E. (1990). HindIII RFLP at the IFNAR locus on chromosome 21. Nucleic Acids Res. 18: 5580. 19. WARREN, A. C., SLAUGENHAUPT, S. E., LEWIS, J. G., CHAKRAVARTI, A., AND ANTONARAKIS, S. E. (1989). A genetic linkage map of 17 markers on human chromosome 21. Genomics 4: 579-591. 20. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 388-396. 21. ZULIANI, G., AND HOBBS, H. H. (1990). A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences. Am. J. Hum. Genet. 46: 963-969.
11,573-576
(1991)
Linkage Mapping of Highly Informative DNA Polymorphisms within the Human Interferon-a Receptor Gene on Chromosome 21 MELVIN
G. MCINNIS,*lt GEORGES LUTFALLA,$ SUSAN SLAUGENHAUPT,~ MICHAEL B. PETERSEN,* GILLES UZE,$ ARAVINDA CHAKRAVARTI,!?~ AND STYLIANOS E. ANTONARAKIS**’
*Center for Medical Genetics and Departments of Pediatrics and tfsychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; SCNRS Laboratory of Viral Oncology, Villejuif, France; and §Departments of Human Genetics and IPsychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15267 Received
Apri122,
1991;revised
1989) and variable poly(A) tracts of Alu repetitive elements (AluVpA) (Economou et aZ., 1990; Zuliani and Hobbs, 1990) are particularly useful in mapping. In this study we have identified a highly informative AluVpA DNA marker within intron 5 of the IFNAR locus and used it along with the previously described multiallelic RFLP (Viehl et al., 1990) to localize the IFNAR locus on the human genetic linkage map of human chromosome 21 (Petersen et aZ., 1991) constructed using the CEPH (Centre d’Etude Polymorphisme Humain) families (Dausset et aZ., 1990).
Two polymorphic loci within the interferon-a receptor (IFNAR) gene on human chromosome 2 1 have been identified and mapped by linkage analysis in 40 CEPH families. These markers are (1) a multiallelic RFLP with an observed heterozygosity of 0.72 and (2) a variable (AT,), short sequence repeat at the poly(A) tail of an Alu sequence (AluVpA) with an observed heterozygosity of 0.83. This locus is close to D21S58 (8 = 0.02, Z! = 36.76) and D21S17 (8 = 0.02,i = 21.76) within chromosomal band 21q22.1. Multipoint linkage analysis suggests the most likely locus order to be 21cen-D21S58-IFNAR-D2lSl7-21qter. Given its high heterozygosity, the IFNAR gene can be used as an index marker on human chromosome 21. o 1991 Academic
June 13. 1991
METHODS
Press, Inc.
The different alleles of the short sequence repeat polymorphism due to (TAAA), at the 3’ end of an Ah repeat within intron 5 (unpublished results) of the IFNAR gene (seeFig. 1) were detected using the polymerase chain reaction (PCR) as follows: The oligonucleotides bordering the Alu repeat sequence were 5’-CACACTATGTAATACTATGC-3’, termed IFNAR-IVS5-3’.2 (interferon-a receptor-intervening sequence 5); and 5’-TGCTTACTI’AACCCAGTGTG-3’, termed IFNAR-IVS5-5’. Initial experiments were performed with the 3’-oligonucleotide priming sequence5’AGCCGAGATTGAGCCACTGC-3’, which is termed IFNAR-IVS5-3’.1 and is within the repetitive Ah sequence. IFNAR-IVS5-5’ was end-labeled with [T-~“P] ATP. Amplification was carried out using 500 ng of genomic DNA in a 25-~1reaction of 10 n&f Tris-HCl, pH 8.3,50 r&f KCI, 1.5 mA4 MgCl,, 0.02% gelatin, 200 &f each dNTP, 0.8 U Z’aq polymerase, 0.4 &f IFNARIVS5-3.2 primer (0.04 pA4 IFNAI-IVS5-3.1’), and 0.08 &f IFNAR-IVS5-5’ primer. The PCR amplification was performed for 30 cycles (denaturation at 94”C, 30 s; annealing at 56”C, 30 s; extension at 72”C, 30 8). Polyacrylamide gel electrophoresis of the reaction products was performed as previously described (Petersen et al., 1990). The IFNAR-19ES polymorphism was detected
INTRODUCI’ION
The interferon-a receptor is a protein receptor molecule located on the mammalian cell surface and mediates the actions of interferon-a across the cell wall (Mogensen et al., 1989). The cDNA for the human interferon-a receptor (IFNAR) gene has been recently cloned and sequenced (Uze et al., 1990). This gene has been localized to chromosome 21q22.1 by in situ hybridization (Lutfalla et aZ., 1990) and by somatic cell hybrids (Slate et al., 1981; Langer et al., 1990). A multiallelic RFLP with Hind111 was recently reported using a cDNA probe 19ES (Viehl et al., 1990). DNA polymorphisms within human genes permit their localization on the human genetic linkage map. Highly informative multiallelic polymorphisms that include VNTRs (variable number of tandem repeats) (Jeffreys et al., 1985) or short sequence repeats (Weber and May, 1989; Litt and Luty, 1989; Tautz,
1 To whom
reprint
requests
should
be addressed. 573
All
Copyright 0 1991 rights of reproduction
OS&%7543/91 $3.00 by Academic Press, Inc. in any form reserved.
574
MCINNIS
ET
AL.
TABLE
2
Two-Point Linkage Analysis between the INFAR Locus and Markers on Human Chromosome 21, Listed in the Order from 2lcen to 2lqter
FIG. 1. The sequence from the fifth intron of the IFNAR gene from which the AluVpA was identified is shown. The priming sequences are printed in lowercase letters; the Ah sequence is underlined.IFNAR-IVSB-5’: 5’-tgcttacttaacccagtgtg-3’. IFNAR-IVS53’.1: 5’-agccgagattgagccactgc-3’. IFNAR-IVS5-3’.2: B’-cacactatgtaatactatgc-3’.
by Southern blot analysis following Hi&III digestion of genomic DNA and hybridization to the 195-bp cDNA probe 19ES as described (Vi&l et al., 1990). Human genomic DNA from members of 40 CEPH families was used to genotype these two loci, to estimate the frequency of the different polymorphic alleles, and to map this polymorphic locus by linkage analysis. RESULTS Of the 40 CEPH families analyzed for the IFNAR19ES polymorphism, 7 were uninformative; 15 inforTABLE
1
Allelic Frequency and Observed Heterozygosity DNA Polymorphisms within the IFNAR Gene Allele
Size (kb)
Frequency
(%)
of
Observed heterozygosity
A. IFNAR-19ES 1 2 3 4 5 6
8.1 -.-a 11.8
2.9 8.1 30.1 18.4 25.0 15.5 B. IFNAR
1 2 3 4 5 6
lb
II’
195 199 203 207 211 215
462 466 470 474 478 480
72% (75 of 104)
AluVpA
13.1 21.7 8.2 21.7 8.7 26.6
83% (78 of 95)
’ The sizes of the intervening alleles range between 8.1 and 14.8 kb; exact lengths in kb are not given because of gel to gel variation. b Size of PCR produce (in nucleotides) between IFNAR-IVSB-5 and IFNAR-IVSB-3’.1. ’ Size of PCR produce (in nucleotides) between IFNAR-IVSS-5’ and IFNAR-IVSS-3’.2.
2lcen D21S120 D21S13 D21SllO D21Sl/Sll D21S8 APP D21Slll D21S82 SOD1 D21S58 D21S17 D21S55 ETS2 D21S3 D21S156 HMG14 D21S15 MXl D21S42 CRYAl D21S113 CD18 D21S171 PFKL D21S112 COL6Al 21qter
0.30 0.33 0.27 0.28 0.20 0.16 0.11 0.08 0.07 0.02 0.02 0.10 0.09 0.15 0.12 0.18 0.18 0.23 0.20 0.24 0.24 0.22 0.29 0.28 0.26 0.27
6.62 2.43 5.27 7.67 10.84 14.78 28.25 45.64 4.67 36.76 21.61 30.76 25.29 23.54 36.45 16.40 8.83 15.34 7.92 8.17 9.03 9.39 8.92 4.04 9.38 8.85
0.39 0.45 0.37 0.32 0.26 0.19 0.15 0.07 0.07 0.00 0.02 0.13 0.10 0.14 0.12 0.20 0.20 0.25 0.20 0.29 0.24 0.16 0.26 0.27 0.23 0.22
0.23 0.20 0.18 0.24 0.13 0.14 0.07 0.08 0.07 0.05 0.03 0.07 0.08 0.17 0.12 0.16 0.17 0.21 0.20 0.18 0.25 0.27 0.32 0.30 0.30 0.34
8.22 4.49 6.86 8.11 12.00 14.96 28.92 45.65 4.67 38.09 21.63 31.18 25.33 23.58 36.45 16.51 8.86 15.46 7.92 8.71 9.03 9.91 9.16 4.07 9.65 9.74
mative families and all CEPH parents were analyzed with IFNAR AluVpA. Genotypes were initially generated with IFNAR-IVS5-3’.1 (oligonucleotide within the Ah); however, it was found that the data were more consistent with the IFNAR-IV%-3’.2 primer, which is not part of the Ah repetitive sequence. In the 8 families on which there were data for both polymorphisms, haplotypes were constructed and used for linkage analysis. The IFNAR-19ES polymorphism showed six alleles with a heterozygosity of 0.72. The IFNAR AluVpA polymorphism also showed six alleles (see Fig. 2) with a heterozygosity of 0.83 in 95 unrelated individuals. The observed heterozygosity from unrelated individuals in the CEPH pedigrees and the frequency and size of each allele are provided in Table 1. To localize IFNAR relative to other DNA markers on the human chromosome 21 genetic map, we performed two-point linkage analyses using the computer program LINKAGE, version 4.7 (Lathrop and Lalouel, 19861, against 26 other loci on chromosome 21 as previously described (Warren et al., 1989; Petersen et al., 1991) and multipoint linkage analysis
HIGHLY
INFORMATIVE
DNA
575
POLYMORPHISMS
12345678910
A
IFNAR AluVpA
FIG. 2. Allelic polymorphisms (B) members of the CEPH family
B
IFNAR
AluVpA
of the IFNAR AluVpA in (A) 10 Caucasians using primers 12 using primers IFNAR-IVSS-5’ and IFNAR-IVS5-3’.2.
against DNA markers APP, D21Sll1, D21S82, D21S17, D21S55, D21S58, ETSB, and D21S3. The results of two-point linkage analysis between IFNAR and 26 loci on chromosome 21 are shown in Table 2. The IFNAR locus is most closely linked to D21S58 with a lod score of 36.76 at a recombination value of 0.02 and to D21S17 with a lod score of 21.61 at a recombination value of 0.02. The most likely order is 21cen-D21S58-IFNAR-D21S17-D21S55-2lqter; the relative odds of this order compared to the second most likely order, 21cen-D21S58-D21S17-IFNARD21S55-21qter, were 103.212.
VAR (A8906303Q), Cancer. We thank
I,
The IFNAR gene maps just proximal to the minimal Down syndrome region (McCormick et al., 1989; Korenberg et aZ., 1990; RahmanietaZ., 1989). The multiple effects of IFNAR include modulation of cellular activities, inhibition of cell growth, and antitumor effects (Gresser, 1989), disturbances of which could have an effect on the full phenotype of the Down syndrome. The IFNAR AluVpA appears to be more efficacious than the IFNAR-19ES polymorphism due to the rapidity of its PCR-based detection compared to that of the Southern blot procedure. The heterozygosity value of 0.83 makes IFNAR AluVpA a good index marker in the middle of the linkage map of chromosome 2lq.
1.
2.
and
and the Association Pour la Recherche sur le Tara Cox for assistence with linkage analysis.
DAUSSET, J., CANN, H., COHEN, D., LATHROP, M., LALOIJEL, J. M., AND WHITE, R. (1990). Centre d’Etude du Polymorphisme Humain (CEPH): Collaborative genetic mapping of the human genome. Genomics 6: 575-577. ECONOMOU, E. P., BERGEN, A. W., WARREN, A. C., AND ANTONARAKIS, S. E. (1990). The polyadenylate tract of Alu repetitive elements is polymorphic in the human genome. Proc. Natl. Acad. Sci. USA 87: 2951-2954. GRESSER, I. (1989). Antitumor effects of interferon. Acta Oncol. 28: 347-353.
4.
JEFFREYS, A. J., WILSON, V., AND THEIN, S. L. (1985). Hypervariable “minisatellite” regions in human DNA. Nature 314: 67-73.
5.
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ACKNOWLEDGMENTS 10. This research was supported by NIH Grants HD24605 (S.E.A.) and HGO0344 and HD 00774 (A.C.). M.G.M. was supported by a fellowship from NIA Grant AG00149, a research training grant in the dementias of aging. We thank Dr. Ion Greaser for support. G.L. and G.U. were supported by grants from DRET (89.34.132), AN-
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