New mutations in MID1 provide support for loss of function as the cause ofX-linked Opitz syndrome

Opitz syndrome (OS) is a genetically heterogeneous malformation disorder. P atients with OS may present with a variable array of malformations that are indicative of a disturbance of the primary midline developmental field. Mu tations in the C-terminal half of MID1, an RBCC (RING, a-box and coiled-coi l) protein, have recently been shown to underlie the X-linked form of OS. H ere we show that the MID1 gene spans at least 400 kb, almost twice the dist ance originally reported and has a minimum of six mRNA isoforms as a result of the alternative use of 5' untranslated exons, In addition, our detailed mutational analysis of MID1 in a cohort of 15 patients with OS has resulte d in the identification of seven novel mutations, two of which disrupt the N-terminus of the protein, The most severe of these (E115X) is predicted to truncate the protein before the a-box motifs, In a separate patient, a mis sense change (L626P) was found that also represents the most C-terminal alt eration reported to date, As noted with other C-terminal mutations, GFP fus ion constructs demonstrated that the L626P mutant formed cytoplasmic clumps in contrast to the microtubular distribution seen with the wild-type seque nce, Notably, however, both N-terminal mutants showed no evidence of cytopl asmic aggregation, inferring that this feature is not pathognomonic for X-l inked OS, These new data and the finding of linkage to MID1 in the absence of a demonstrable open reading frame mutation in a further family support t he conclusion that X-linked OS results from loss of function of MID1.