Hum Genet (1985) 69:44-46 © Springer-Verlag 1985 Linkage and recombination between fragile X-linked mental retardation and the factor IX gene Stephen T. Warren 1, Thomas W. Glover 2, Richard L. Davidson 1, and Pudur Jagadeeswaran a 1Center for Genetics, University of Illinois College of Medicine, Chicago, IL 60612, USA 2The Genetics Center of the Southwest Biomedical Research Institute, Tempe, AZ 85281, USA Summary. Linkage analysis on a family with fragile X-linked mental retardation was performed using a Taq 1 restriction fragment length polymorphism detected by a cloned human coagulation factor IX eDNA. Two affected brothers in this sibship were found to have different factor IX RFLP alleles, indicating a recombinational event occurred between the two genes. Our data therefore indicate that the gene responsible for fragile X-linked mental retardation is not as tightly linked to the factor IX gene as the previously published data may suggest. Introduction A major cause of genetically determined mental retardation is fragile X-linked mental retardation [fra(X)-linked MR]. This condition results in moderate to profound mental retardation in hemizygous males and is associated with a fragile site near the vicinity of band Xq28 (Jacobs et al. 1980; Turner and Jacobs 1983). Since carrier detection and prenatal diagnosis of this condition are associated with technical and penetrance problems (Glover 1983; Sherman et al. 1984), close linkage of a marker gene to fra(X)-linked MR would be of considerable benefit. The human coagulation factor IX gene recently has been cloned and mapped to the region from Xq26 to Xq28 by several laboratories, including our own, and a common Taq I restriction fragment length polymorphism (RFLP) has been identified (Kurachi and Davie 1982; Choo et al. 1982; Jaye et al. 1983; Camerino et al. 1984; Jagadeeswaran et al. 1984). Camerino et al. (1983) recently reported linkage between the fra(X)-linked MR gene and the factor IX Taq I RFLP. Out of 17 informative meioses in two families, recombination be- tween these genes was not observed, resulting in an estimate of the genetic distance as less than 12 centimorgans (cM). However, in the absence of recombination, it was not clear how tightly linked these genes are. We have carried out linkage analyses with a factor IX eDNA probe in families with fra(X)-linked MR and have identified a recombinant individual. Thus, our data indicate that the fra(X)-linked MR and factor IX genes are not as close as the data of Camerino et al. (1983) might suggest. Offprint requests to: P. Jagadeeswaran, Center for Genetics, Univer- sity of Illinois College of Medicine, Chicago, IL 60612, USA Materials and methods Cytogenetic analyses. Heparinized blood was cultured by con- ventional whole blood microculture techniques for 96h. To induce expression of the fra(X) site, cells were grown in either RPMI 1640 medium with 5% fetal bovine serum (FBS) but without folate (Irvine Scientific), or in folate containing RPMI 1640 medium with 10% FBS to which 0.1uM 5-fluorodeoxy- uridine (FUdR) was added at the beginning of the culture period (Glover 1981). Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines were established from some family members by the method of Jacobs et al. (1982). For fra(X) expression, lym- phoblastoid cells were cultured in RPMI 1640 medium with 10% FBS. FUdR (0.1uM) was added 24h prior to chromo- some harvest. For all cultures, air dried slides were stained with Giemsa or Q-banded and at least 50 cells scored for fra- gile X expression. DNA isolation and Southern blot analyses. High molecular weight DNA was isolated from either lymphocytes or lympho- blastoid cells and subjected to Southern blot analyses (South- ern 1975), using as probe a 1.0kb Hpa II fragment, containing the factor IX eDNA insert (Jagadeeswaran et al. 1984). Lod score calculations. The relative probability of linkage and lod scores were calculated at various recombination intervals from 0 to 50% (Morton 1955; Connea!ly and Rivas 1980). Results and discussion Figure 1 shows the pedigree of the family described in this report. This family, of English/Irish descent, was ascertained through individuals III-2 and III-3, who were diagnosed as having fra(X)-linked MR on the basis of profound psycho- motor retardation and expression of the fragile site at Xq28. The fragile site was expressed, with FUdR induction, in both lymphocytes and lymphoblastoid cells from both individuals (III-2, 22-25% fra(X) expression; III-3, 14-23% fra(X) expression). A phenotypically normal brother, III-4, did not express the fra(X) in 100 lymphocyte metaphases examined. Two female siblings were mentally normal. However, one sister (III-5) was determined to be a carrier of fra(X)-linked MR on the basis of expression of the fra(X) site (10-27% fra(X) expression in lymphocytes and lymphoblastoid cells).