SHORT COMMUNICATION Linkage Mapping of the AML1 Gene on Human Chromosome 21 Using a DNA Polymorphism in the 3' Untranslated Region DIMITRIOS A V R A M O P O U L O S , T A R A C O X , * JILLIAN E. BLASCHAK,* A R A V I N D A CHAKRAVARTI,*'Jr A N D STYLIANOS E. A N T O N A R A K I S 1

Center for Medical Genetics, Department of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; *Department of Human Genetics and t Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received July 1, 1992

detectable after single-strand c o n f o r m a t i o n analysis (SSCA) (5). T h e 3' U T regions of several genes are k n o w n to be p o l y m o r p h i c (3, 8), a n d S S C A has p r o v e n to be a sensitive m e t h o d for detecting such D N A sequence variation. F r o m the published nucleotide sequence of A M L 1 (4), we synthesized two oligonucleotides, 5'A G A A G T C A G T T C T T C T G T C C - 3 ' and 5'-AAGTGT A C C G G G A T C C A T G C - 3 ' , to use as primers for the p o l y m e r a s e chain reaction ( P C R ) amplification of a 345-bp s e g m e n t of genomic D N A from the 3' U T region. T h e conditions used for the P C R were: 94°C for 6 min a n d t h e n 30 cycles of 94°C for 30 s, 55°C for 30 s, a n d 72°C for 30 s. T h e P C R p r o d u c t was labeled by the addition of 2 ~Ci/gl of [a-82P]dCTP in the reaction. T h e P C R p r o d u c t was diluted 2.75 times, heat d e n a t u r e d in 43% formamide, 0.1% SDS, a n d 18 m M E D T A , cooled on ice, loaded on a 6% p o l y a c r y l a m i d e - 5 % glycerol gel, a n d electrophoresed in 0.5× T B E at 5 W for 17 h at r o o m temperature. T h e dried gel was a u t o r a d i o g r a p h e d for 17h. T h e S S C A analysis detected a two-allele D N A polym o r p h i s m (Fig.lA) with allele frequencies of 0.17 a n d 0.83 a n d a c o m p u t e d heterozygosity of 0.29. Individuals h o m o z y g o u s for each allele were identified a n d their genomic D N A c o r r e s p o n d i n g to the 3' U T region was sequenced. A n A to G t r a n s i t i o n was f o u n d at position 1615 of the published A M L 1 c D N A sequence (Fig.lB); this creates an F n u 4 H I restriction site.

W e h a v e d e t e c t e d a p o l y m o r p h i s m i n t h e 3' u n t r a n s l a t e d r e g i o n o f t h e A M L 1 g e n e , w h i c h is l o c a t e d at t h e breakpoint on chromosome 21 in the t(8;21)(q22; q22.3) translocation often associated with patients with acute myeloid leukemia. Informative CEPH famil i e s w e r e g e n o t y p e d f o r t h i s p o l y m o r p h i s m a n d u s e d to localize the gene on the linkage map of human chromosome 21. The AML1 gene is located between the markers D21S216 and D21S211, in chromosomal band 2 lq22.3. © 1992 AcademicPress, Inc.

T h e A M L 1 gene is located at the b r e a k p o i n t on chrom o s o m e 21 which is involved in the t(8;21)(q22;q22.3) translocation, often f o u n d in p a t i e n t s with acute myeloid leukemia type M2; the gene m a y be involved in the neoplastic t r a n s f o r m a t i o n (4). A yeast artificial c h r o m o some clone s p a n n i n g this t r a n s l o c a t i o n has recently been isolated (2). F u r t h e r m o r e , a c D N A clone corresponding to the A M L 1 gene has recently been identified a n d sequenced (4). W e have searched for D N A p o l y m o r p h i s m s at the A M L 1 gene t h a t could allow its localization on the linkage m a p of h u m a n c h r o m o s o m e 21. W e e x a m i n e d the 3' u n t r a n s l a t e d (3' U T ) region of the gene for variation 1To whom correspondence should be addressed.

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FIG. 1. (A) SSCA analysis of the AML1 3' UT region showing a two-allele polymorphism. Individuals 3,4, and 7 are heterozygotes for a DNA sequence variation. (B) Nucleotide sequencing of the PCR product from homozygotes for each allele shows an A to G substitution, indicated by arrow. ((3) Digestion of the PCR product with Fnu4HI. Lane 3 contains DNA from an individual homozygousfor the absence of the site, and lane 1 contains DNA from an individual homozygousfor the presence of the Fnu4HI site. Lane 2 contains DNA from a heterozygote. GENOMICS14, 506-507 (1992) 0888-7543/92 $5.00 Copyright© 1992by AcademicPress, Inc. All rights of reproductionin any formreserved.

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TABLE 1 Two-point Linkage Analysis between AML1 and Selected Markers on Chromosome 21 Markers

0

Z

Of

0m

Zm,f

X2

D21S213 IFNAR D21S216 D21S65 D21S211 D21S167 D21S156 HMG14 D21S231 D21S212

0.05 0.07 0.02 0.00 0.02 0.04 0.10 0.03 0.14 0.21

8.53 6.63 14.33 19.57 13.15 12.19 8.74 6.24 3.87 4.97

0.00 0.12 0.00 0.00 0.02 0.05 0.14 0.05 0.15 0.29

0.11 0.00 0.04 0.00 0.00 0.00 0.04 0.00 0.09 0.00

9.27 7.15 14.41 19.57 13.29 12.58 9.20 6.43 3.93 7.52

3.394 2.398 0.397 0.000 0.635 1.768 2.117 0.864 0.274 11.728

Note. 0, sex average recombination fraction; Z, maximum lod score;

0m, Of, recombination fraction for male and female meioses, respectively; Zm.f, maximum lod score for sex specific recombination; ~(2, chi square for the difference between female and male recombination fraction. The DNA polymorphic markers have been arranged from top to bottom, according to their location on the long arm of h u m a n chromosome 21. The first is closer to the centromere and the last is closer to the telomere. Information about the polymorphic markers used can be found in the Genome Database (GDB).

The genotypes of all individuals in 13 informative C E P H families were obtained by digesting the PCR product with Fnu4HI. When digested with Fnu4HI, the PCR product was cleaved into 255- and 90-bp fragments when the individual was homozygous for the presence of the restriction site, whereas it was left intact in individuals homozygous for the absence of the site (Fig.lC). The heterozygous individuals show all three fragments (90, 255, and 345 bp) after digestion with Fnu4HI. Two-point linkage analysis was performed using CRIMAP (1) for all markers in our chromosome 21 database (7) and unpublished results) versus AML1. Table 1 shows sex-equal and sex-specific recombination fractions and associated lod scores for the PCR markers that were also used for multipoint linkage analysis. The closest linkage was observed with D21S65 (0 = 0, Z = 19.57). Table 1 also provides the X2 values for testing whether the male and female recombination values are equal. There is a significant difference in male-female recombination values between D21S212 and AML1. We then performed multipoint linkage analysis using CRI-MAP to place AML1 on the map of PCR detectable polymorphisms: cen-D21S213-IFNAR-D21S216D21S65-D21S211-CBR-D21S167-D21S156-HMG14D21S231-D21S212-qter (this map has odds of 1000:1 against any permutation of the order of any two adjacent markers). The analysis showed that AML1 was located between D21S216 and D21S211 with odds of 1000:1 or greater and that AML1 could be located on either side

of D21S65, to which it is completely linked. The distances in centimorgans from the multipoint analysis are D21S216-1.6 c M - A M L I - I . 3 cM-D21S211. The localization of the AML1 gene on the linkage map is of importance since AML1 is a physical landmark in maps based on chromosomal breakpoints present in chromosomes 21. In fact, AML1 is the second marker after D21S3 (which is at the breakpoint of a ring 21 chromosome; (9), which serves as a link between the meiotic linkage map and the somatic cell hybrid breakpoint map. ACKNOWLEDGMENTS This work was supported by N I H Grants HD24605 and HG00373 to S.E.A. and HD00774 (RCDA) and HG00344 to A.C. REFERENCES 1.

2.

3. 4.

5.

6.

7.

8.

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