However, it has been estimated that approximately 60-70% of all cases of XY GD remain unexplained at the molecular level. Attempts to identify these missing human testis determining genes have recently focused on the study of familial cases of 46,XY DSD unlinked to known genes. In one such family, 46,XY GD was transmitted as an autosomal dominant trait with highly variable expressivity, ranging from CGD to partial GD associated with normal female genitalia, sexual ambiguity or mild hypospadias in affected males. Linkage analysis in this family placed the mutated locus on the pericentric region of chromosome 5. Recently, we attributed the cause of 46,XY GD in this, and a second family, to mutations in the gene encoding the signal transduction molecule, MAP3K1. to SIN-1 chloride regulate a variety of cellular functions such as cell cycle progression, cell adherence, motility and metabolism and thereby influence a number of developmental processes. In particular, mammalian sex determination is regulated by growth factors such as insulin-like growth factors, fibroblast growth factors, prostaglandins and platelet-derived growth factors. MAP3K1 might act to regulate or integrate such signals during testis development. Analysis of MAPK signalling activity in lymphoblastoid cell lines derived from individuals with sex-reversing MAP3K1 mutations revealed enhanced phosphorylation of the MAPKs p38 and ERK after serum starvation followed by re-feeding. Moreover, RHOA, a known positive regulator of MAP3K1 kinase activity, exhibited increased binding to protein complexes containing mutant MAP3K1. These data raise the possibility that, at least in the lymphoblastoid cell line context, mutant versions of MAP3K1 behave like gain-of-function alleles, enhancing functionality of the encoded protein. This possibility is also consistent with the absence of any truncating, loss-of-function, mutations in the 46,XY GD patient cohorts examined. However, direct targets of MAP3K1 were not assayed. Moreover, crosstalk between the ERK and JNK/p38 pathways is reported to regulate apoptosis in some contexts, indicating that the distinct MAPK pathways are not insulated from each other. Thus, disruption to one element of the MAPK signalling network may conceivably cause consequential activation of other components. Along with the fact that all these functional Org 25543 hydrochloride studies were performed in heterologous lymphoblastoid cell lines, these observations indicate that no definitive explanation yet exists for how these MAP3K1 mutations disrupt human testis determination.