Screening for Fragile X Syndrome (Murray et al.) - NIHR Journals ...

Health Technology Assessment 1997; Vol. 1: No. 4Chapter 4GeneticsThe genetics of fragile X syndrome was firstinvestigated in the 1940s when Martin andBell (1943) reported on a family whose pedigreeshowed a specific form of X-linked mental retardation.Hence the eponymous description of thedisorder as the Martin–Bell syndrome. However,it was a further 25 years before genetic linkage toa fragile site on the long arm of chromosome Xwas established (Lubs, 1969). Subsequently, thediscovery of the cytogenetic media conditionsneeded to demonstrate expression of the fragilesite reproducibly in vitro (Sutherland, 1977)enabled segregation studies to be performed.However, these early techniques were crude and,as a result, only limited information regardingthe nature of inheritance could be gained. Withthe recent cloning of the affected gene itself(Verkerk et al, 1991) and the development ofDNA-testing techniques, many of the previouslyinexplicable features of the syndrome can nowbe understood.Population geneticsFormally, fragile X syndrome is an X-linkeddominantly-inherited disorder with reducedpenetrance but it does not have a simple Mendelianpattern of inheritance. Females as well asmales can be affected, albeit to a lesser extent.In addition, both males and females can beunaffected carriers. Although the children ofunaffected female carriers are at increased risk ofthe disorder, those of unaffected male carriers arenot. These individuals, known as normal transmittingmales (NTMs), have sons who are neitheraffected nor carriers and daughters all of whomare unaffected carriers but whose children are atincreased risk of fragile X syndrome. This is the‘Sherman paradox’, a particular case of the generalgenetic phenomenon of ‘anticipation’ (Shermanet al, 1984). Thus, in affected families the numberof individuals with fragile X syndrome increaseswith each generation.CytogeneticsFragile sites on human chromosomes are characterisedcytologically as specific regions that exhibitconstrictions, gaps or breaks when cells arecultured and karyotyped (Sutherland & Hecht,1985). They are areas of late replication (Webb,1992; Hansen et al, 1993) and their expressioncan be induced in vitro by blocking the normalreplication pattern of DNA. This is generallyachieved by altering the media conditions of thecultured cells. Over 100 fragile sites have beenfound on the human genome (Sutherland &Ledbetter, 1989). Some are common but mostare rare, and only two are of clinical significance.Designated FRAXA and FRAXE, they are locatedon the long arm of the X chromosome, at Xq27.3and Xq28, respectively (Sutherland & Baker,1992), and both occur in families affected bymental retardation. However, whilst FRAXAexpression is specific to fragile X syndrome,FRAXE is inconsistently associated with nonspecific,mild mental retardation (Sutherland& Baker, 1992; Knight et al, 1993; 1994; Hamelet al, 1994; Mulley et al, 1995; Bullock et al,1995; Murray et al, 1996). Two other fragile sitessituated close by on the X chromosome, thecommon FRAXD at Xq27.2 (Sutherland & Baker,1990) and the rare FRAXF at Xq28 (Hirst et al,1993b; Parrish et al, 1994), are not related tomental retardation.Until recently the diagnosis of fragile X syndromewas based on the cytogenetic expression ofFRAXA in a proportion of cultured cells. However,there were a number of technical problems withthe method. First, cytogenetics cannot reliablydistinguish FRAXA from the other three neighbouringfragile sites, requiring fluorescence in situhybridisation with DNA probes to separate them(Sutherland & Baker, 1992; Hirst et al, 1993b).Second, although it was initially thought that thefrequency of cytogenetic expression was mainlycontrolled by genetic factors (Soudek et al, 1984;Hecht et al, 1986), between-laboratory variationhas been shown to contribute more to the variance(Fisch et al, 1991b). There are differences in theproportion of affected cells regarded as diagnostic;although guidelines have been published recommending4% as the lower limit (Jacky et al, 1991),some laboratories use a cut-off as low as 2%. Thereis also variability because of differences in the tissueculture medium (Sutherland, 1977), levels of folicacid and thymidylate synthetase activity (Glover,9