Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR/ABCC7). Despite the extensive and enduring efforts of many CF researchers over the past 14 years, up to 30% of disease alleles still remain to be identified in some populations. It has long been suggested that gross genomic rearrangements could account for these unidentified alleles. To date, however, only a few large deletions have been found in the CFTR gene and only three have been fully characterized. Here, we report the first systematic screening of the 27 exons of the CFTR gene for large genomic rearrangements, by means of the quantitative multiplex PCR of short fluorescent fragments (QMPSF). A well-characterized cohort of 39 classical CF patients carrying at least one unidentified allele (after extensive and complete screening of the CFTR gene by both denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography) participated in this study. Using QMPSF, some 16% of the previously unidentified CF mutant alleles were identified and characterized, including five novel mutations (one large deletion and four indels). The breakpoints of these five mutations were precisely determined, enabling us to explore the underlying mechanisms of mutagenesis. Although non-homologous recombination may be invoked to explain all five complex lesions, each mutation appears to have arisen through a different mechanism. One of the indels was highly unusual in that it involved the insertion of a short 41 bp sequence with partial homology to a retrotranspositionally-competent LINE-1 element. The insertion of this ultra-short LINE-1 element (dubbed a "hyphen element") may constitute a novel type of mutation associated with human genetic disease.

Hereditary hemochromatosis (HH) is a common autosomal recessive genetic disorder of iron metabolism. The HFE candidate gene encoding an HLA class I-like protein involved in HH was identified in 1996. Two missense mutations have been described: C282Y, accounting for 80% to 90% of HH chromosomes, and H63D, which is associated with a milder form of the disease representing 40% to 70% of non-C282Y HH chromosomes. We report here on the analysis of C282Y, H63D, and the 193A→T substitution leading to the S65C missense substitution in a large series of probands and controls. The results confirm that the C282Y substitution was the main mutation involved in hemochromatosis, accounting for 85% of carrier chromosomes, whereas the H63D substitution represented 39% of the HH chromosomes that did not carry the C282Y mutation. In addition, our screening showed that the S65C substitution was significantly enriched in probands with at least one chromosome without an assigned mutation. This substitution accounted for 7.8% of HH chromosomes that were neither C282Y nor H63D. This enrichment of S65C among HH chromosomes suggests that the S65C substitution is associated with the mild form of hemochromatosis.

The aristaless-related homeobox (ARX) gene has been implicated in a wide spectrum of disorders ranging from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of X-linked mental retardation without apparent brain abnormalities. To better understand its role in corticogenesis, we used in utero electroporation to knock down or overexpress ARX. We show here that targeted inhibition of ARX causes cortical progenitor cells to exit the cell cycle prematurely and impairs their migration toward the cortical plate. In contrast, ARX overexpression increases the length of the cell cycle. In addition, we report that RNA interferencemediated inactivation of ARX prevents cells from acquiring multipolar morphology in the subventricular and intermediate zones, resulting in decreased neuronal motility. In contrast, ARX overexpression appears to promote the development of tangentially oriented processes of cells in the subventricular and intermediate zones and affects radial migration of pyramidal neurons. We also demonstrate that the level of ARX expression is important for tangential migration of GABA-containing interneurons, because both inactivation and overexpression of the gene impair their migration from the ganglionic eminence. However, our data suggest that ARX is not directly involved in GABAergic cell fate specification. Overall, these results identify multiple and distinct cell-autonomous roles for ARX in corticogenesis.

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here. This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.