American Journal of Medical Genetics 43:217-223 (1992) Molecular Studies of the Fragile X Syndrome Samantha J.L. Knight, Mark C. Hirst, Anya Roche, Zoe Christodoulou, Susan M. Huson, Robin Winter, Margaret Fitchett, Mark J. McKinley, Richard H. Lindenbaum, Yutaka Nakahori, and Kay E. Davies zyxwvutsrq Molecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital (S.J.L.K., M.C.H., A.R., Z.C., Y.N.,K.E.D.); Department of Medical Genetics, Churchill Hospital (S.M.H., M.F., zyxwvu M.J.M., R.H.L.), Headington, Oxford; The Kennedy-Galton Centre, North-West Thames Regional Genetics Service, Northwick Park Hospital, Waford Road, Harrow, Middlesex (R.W.),England zyxwvut We have studied families segregating for the fragile zyxwvutsrqpo X syndrome for the presence of amplification of the CGG repeat sequence adjacent to the HpaII Tiny Fragment (ETF) island in the FMR-1 gene. We demonstrate that 138/143 fragile X positive, mentally retarded males show a characteristic smear of fragments corresponding to somatic variation in the amplification of the CGG sequence. In zyxwvutsrqp 1/8 normal transmitting males (NTM's), we show that there is a small amplification of sequence but no evidence for somatic variation. Defined mutated fragments in the size range found in NTM's are seen in daughters of NTM's. The daughters of these female carriers show either a defined fragment in the NTM size range, a defined larger fragment or a heterogeneous pattern of fragments. In the latter 2 cases the clinical phenotype of the females cannot easily be predicted, presumably because of variable X inactivation. In some families, the observed DNA genotype does not correlate with the phenotype; in others we demonstrate the occurrence of individuals with a mosaic DNA genotype. The implications of these data for diagnosis of the disease are discussed. KEY WORDS: Fragile X, mutation, diagnosis, carriers, fragile site, X-linked mental retardation INTRODUCTION zyxw The fragile X [fra(X)l syndrome is the most frequent familial form of mental retardation and is the second most common genetic cause of mental retardation after Down syndrome [for review see Fryns, 19891. It affects 1 in 1250 males and 1 in 2000 females [Kahkonen et al., 1987; Webb et al., 1986; Webb, 19891. The disease shows unusual X- linked inheritance exhibiting non-penetrance in males and semi-dominance in carrier females. Approximately 30% of female carriers show some degree of mental impairment while 20% of males are phenotypically normal transmitters of the disorder (so-called normal transmitting males (NTM's) zy ) [Sherman et al., 1984; 19851. The syndrome is associated with the cytogenetic expression of a fragile site at Xq27.3 when lymphocytes of patients are grown under defined culture conditions [Lubs, 1969; Sutherland, 19771. The expression of the fragile site has been used as a cytogenetic marker for diagnosis, carrier detection and prenatal detection of the fra(X) syndrome [for review see Tommerup, 19891. However, NTM's and their daughters do not generally express the fragile site making carrier status determinations impossible except by linkage analysis. Recently, investigators have shown that fra (X) positive, mentally retarded males are hypermethylated at a HpaII Tiny Fragment (HTF) island in Xq27.3 [Vincent et al., 1991; Bell et al., 19911. Analysis of the region of DNA around this HTF island led to the identification of tandemly arrayed CGG trinucleotide repeats within Received for publication September 30, 1991; revision received December 26, 1991. Address reprint requests to : Dr Kay E. Davies, Mol.ecular Genetics Group, Institute of Molecular Medicine, John Radclif fe Hospital, Headington, O x f o r d , OX3 9DU, England. zyxwvutsrq 0 1992 Wiley-Liss, Inc.