Presence of large deletions in kindreds with autism

Abstract

Autism is caused, in part, by inheritance of multiple interacting susceptibility alleles. To identify these inherited factors, linkage analysis of multiplex families is being performed on a sample of 105 families with two or more affected sibs. Segregation patterns of short tandem repeat polymorphic markers from four chromosomes revealed null alleles at four marker sites in 12 families that were the result of deletions ranging in size from 5 to >260 kb. In one family, a deletion at marker D7S630 was complex, with two segments deleted (37 kb and 18 kb) and two retained (2,836 bp and 38 bp). Three families had deletions at D7S517, with each family having a different deletion (96 kb, 183 kb, and >69 kb). Another three families had deletions at D8S264, again with each family having a different deletion, ranging in size from <5.9 kb to >260 kb. At a fourth marker, D8S272, a 192-kb deletion was found in five families. Unrelated subjects and additional families without autism were screened for deletions at these four sites. Families screened included 40 families from Centre d'Etude du Polymorphisme Humaine and 28 families affected with learning disabilities. Unrelated samples were 299 elderly control subjects, 121 younger control subjects, and 248 subjects with Alzheimer disease. The deletion allele at D8S272 was found in all populations screened. For the other three sites, no additional deletions were identified in any of the groups without autism. Thus, these deletions appear to be specific to autism kindreds and are potential autism-susceptibility alleles. An alternative hypothesis is that autism-susceptibility alleles elsewhere cause the deletions detected here, possibly by inducing errors during meiosis.

Figure 1

Family AU018 genotypes for D7S630 and flanking polymorphic markers. Genetic markers are listed next to genotypes for subject AU01801. Markers are listed in their physical order (fig. 2). Null alleles are shown as dashed lines.

Figure 2

Physical map of D7S630 region and structure of the deletion allele. Locations of deletion boundaries are given on the top genomic contig line as positions in NT_017168.5 (accession number GI:15298273). Arrows labeled P_FB1 and P_RB1 indicate the location of primers used to amplify del-D7S630. Arrows labeled P_FA1 and P_RA1 indicate the location of primers used to amplify del-D7S630-A (table 1 and fig. 3D). Genetic markers, with locations in NT_017168.5 given in parentheses, are as follows: 1, 2127-CT (3534931); 2, 2127-CA1 (3484630); 3, 2127-GT (3466041); 4, 2127-ATCT (3457424); 5, D7S630 (3452308); 6, 4746-TET (3412376); 7, 4746-CA (3398684); 8, rs1016807 (3370218); and 9, 4746-GT (3324233). GenScan-predicted genes are shown as unblackened arrows, and an EST-predicted gene is shown as a blackened arrow.

Figure 3

Long-range PCR amplification of the D7S630 deletion allele. Genomic DNA samples are from the AU018 family (fig. 1) or unrelated unaffected control subjects (NCO samples). Amplified fragments were resolved by use of agarose gel electrophoresis and were detected with ethidium bromide staining. A, Long-range PCR performed to identify primer pairs that can amplify across the D7S630 deletion-allele junction. Primer combinations were as follows: 1, 2127-LG2-F and 4746-LG1-R; 2, 2127-LG2-F and 4746-LG2-R; 3, 2127-LG4-F and 4746-LG1-R; and 4, 2127-LG4-F and 4746-LG2-R (table 1). B, Primer pair D (2127-LG4-F and 4746-LG2-R) was used to assay for the D7S630 deletion allele in the entire AU018 family and in unaffected control subjects NCO16 and NCO25. C, Genetic markers were used as STSs in an unaffected control subject NCO087, in subject AU01803, who has the AU018 deletion allele, and in a CHO-AU01803 hybrid containing a single copy of human chromosome 7 that has the D7S630 deletion allele. D, Deleted segments del-D7S630-A and del-D7S630-B were amplified in a single PCR reaction, using primer pairs P_FA1/P_RA1 and P_FB1/P_RB1, respectively (table 1 and fig. 2). This combination of primer pairs was used to directly screen for the D7S630 complex deletion (table 4).

Figure 4

D7S517 genomic structure and deletion alleles. Deletion boundary positions listed above the top line (genomic contig) are based on numbering from contig NT_007784.5 (accession number GI:15299808). Arrows P_FA2 and P_RA2 show primer locations for amplifying del-D7S517A. Arrows P_FB2 and P_RB2 show primer locations for amplifying del-D7S517B. Genetic markers with locations in NT_007784.5 given in parentheses are as follows: 1, rs975926 (4421); 2, CE1R-AGGG (8891); 3, CE1R-CA (29444); 4, 16898-AT (53633); 5, D7S517 (74190); 6, CE1R-AGAT (93628); 7, CE1R-GT (115042); 8, D7S472 (132296); 9, rs730813 (195200); and 10, rs2005745 (219539). Genes predicted by GenScan are shown as large unblackened arrows.

Figure 5

Amplification of D7S517 deletion alleles. Deletion alleles were amplified with primer pairs P_FA2/P_RA2 and P_FB2/P_RB2 for del-D7S517A and D7S517B, respectively (table 1 and fig. 4). PCR products were resolved by agarose gel electrophoresis and were stained with ethidium bromide. Samples used and family structures are shown at the top.

Figure 6

D8S272 region genomic structure and deletion alleles. Deletion-boundary positions given beneath the top line (genomic contig) are from contig NT_008104.5 (accession number GI:15300207). Arrows mark the location of primers P_FA3 and P_RA3 that were used to directly amplify the deletion allele (fig. 7). Genetic markers with locations in NT_008104.5 given in parentheses are as follows: 1, 26261-CT (1044194); 2, 8104-CCCT (1082018); 3, 8104-TG (1084178); 4, 13646-GTT (1138446); 5, 13646-CTTT (1155366); 6, 13646-ATTT (1193790); 7, D8S272 (1214065); 8, 13646-TA (1235221); 9, 13707-ATCT (1249914); 10, 13707-AAAT (1267145); 11, 12410-GT (1373533); and 12, 12410-CA (1410210). Genes predicted by GenScan are shown as large unblackened arrows.

Figure 7

Amplification of the D7S272 deletion allele. Samples are from families with autism AU002 (left) and AU141 (right). Deletion alleles were amplified with primer pair P_FA3/P_RA3. PCR products were resolved by agarose gel electrophoresis and were stained with ethidium bromide. Samples used and family structures are shown at the top. Subjects showing a 181-bp PCR product were heterozygous for the D7S272 deletion allele and subjects showing no product were homozygous for the nondeletion long allele.

Figure 8

D8S264 region genomic and deletion-allele structures. Deletion-boundary positions given above and beneath the top line (genomic contig) are based on contig NT_008087.5 (accession number GI:15300158). The region labeled “ambiguous assembly region” potentially has contig-assembly errors due to repeat sequences (on the basis of the annotation provided for NT_008087.5). Genetic markers with locations in NT_008087.5 given in parentheses are as follows: 1, rs897445 (62264); 2, rs1824863 (79042); 3, rs922501 (87157); 4, rs899410 (87837); 5, rs1455638 (257680); 6, rs1382608 (304379); 7, rs958660 (309132); 8, 22257-ATCT (316326); 9, 8152018-CA (329355); 10, D8S264 (338805); 11, rs2119237 (344906); and 12, rs936553 (347075). Predicted genes are shown as unblackened arrows, and the known gene MYOM2 is shown as a hatched arrow.