Ohtahara syndrome in a family with an ARX protein truncation mutation (c.81C>G/p.Y27X).

Aristaless-related homeobox (ARX) gene mutations cause a diverse spectrum of disorders of the human brain, including lissencephaly, various forms of epilepsy and non-syndromic mental retardation. We have identified a novel mutation, c.81C>G (p.Y27X), within the ARX gene in a family with two affected male cousins. One of the boys was diagnosed with an early infantile epileptic encephalopathy also known as Ohtahara syndrome, whereas his cousin had been diagnosed with West syndrome (WS). Both patients have normal genitalia and neither have lissencephaly. The ARX mutation identified is predicted to yield a severely truncated protein of only 26 amino acids and can be considered as a null mutation. Somewhat surprisingly, however, it does not yield the X-linked lissencephaly with ambiguous genitalia (XLAG) syndrome. We proposed that the ARX mRNA translation re-initiated at the next AUG codon at position c.121-123 (aa 41) and, thus, partly rescued these patients from XLAG. Our in vitro studies show that this N-terminally truncated ARX protein (p.M41_C562) is detected by western immunoblot in lysates from cells transiently transfected with an ARX over-expression construct containing the c.81C>G mutation. Although these findings widen the spectrum of clinical phenotypes because of mutations in the ARX gene, they also emphasize the molecular pathogenetic effect of individual mutations as well as the effect of genetic background resulting in intrafamilial clinical heterogeneity for these mutations.

Common chromosomal fragile site FRA16D mutation in cancer cells.

Neither the molecular basis for common fragile site DNA instability nor the contribution of this form of chromosomal instability to cancer is clearly understood. Fragile site FRA16D (16q23.2) is within regions of frequent loss-of-heterozygosity (LOH) in breast and prostate cancers, is associated with homozygous deletions in various adenocarcinomas and t(14;16) chromosomal translocations in multiple myeloma. The FOR (WWOX) gene spans FRA16D and encodes a partner of p53 that also has a role in apoptosis. Previously untested 53 cancer cell lines were screened for deletions within the FOR/WWOX gene. Deletions were detected in Co115, KM12C and KM12SM. Homozygous deletions in these and two previously identified tumour cell lines were intragenic on both alleles, indicating a distinct mutation mechanism from that causing LOH. Identical FRA16D deletions in two cell lines (one derived from the primary carcinoma and the other from a secondary metastasis) demonstrate that FRA16D DNA instability can be an early, transient event. Sequence analysis across one deletion locates one endpoint within a polymorphic AT-dinucleotide repeat and the other adjacent to an AT-rich mini-satellite repeat implicating AT-rich repeats in FRA16D DNA instability. Another deletion is associated with de novo repetition of the 9 bp AT-rich sequence at one of the deletion endpoints. FRA16D deleted cells retain cytogenetic fragile site expression indicating that the deletions are susceptible sites for breakage rather than regions that confer fragility. Most cell lines with FRA16D homozygous deletions also have FRA3B deletions, therefore common fragile sites represent highly susceptible genome-wide targets for a distinct form of mutation.

XLMR in MRX families 29, 32, 33 and 38 results from the dup24 mutation in the ARX (Aristaless related homeobox) gene.

BACKGROUND: X-linked mental retardation (XLMR) is the leading cause of mental retardation in males. Mutations in the ARX gene in Xp22.1 have been found in numerous families with both nonsyndromic and syndromic XLMR. The most frequent mutation in this gene is a 24 bp duplication in exon 2. Based on this fact, a panel of XLMR families linked to Xp22 was tested for this particular ARX mutation. METHODS: Genomic DNA from XLMR families linked to Xp22.1 was amplified for exon 2 in ARX using a Cy5 labeled primer pair. The resulting amplicons were sized using the ALFexpress automated sequencer. RESULTS: A panel of 11 families with X-linked mental retardation was screened for the ARX 24dup mutation. Four nonsyndromic XLMR families - MRX29, MRX32, MRX33 and MRX38 - were found to have this particular gene mutation. CONCLUSION: We have identified 4 additional XLMR families with the ARX dup24 mutation from a panel of 11 XLMR families linked to Xp22.1. This finding makes the ARX dup24 mutation the most common mutation in nonsyndromic XLMR families linked to Xp22.1. As this mutation can be readily tested for using an automated sequencer, screening should be considered for any male with nonsyndromic MR of unknown etiology.

Folate-sensitive fragile site FRA10A is due to an expansion of a CGG repeat in a novel gene, FRA10AC1, encoding a nuclear protein.

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5'UTR of a novel gene named FRA10AC1. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10AC1 gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of approximately 1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10AC1, of unknown function. Several splice variants leading to alternative 3' ends were identified (particularly in testis). These give rise to FRA10AC1 proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10AC1 protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10AC1 gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site.