The Phox2b gene is necessary for autonomic nervous-system development. Phox2b-/- mice die in utero with absent autonomic nervous system circuits, since autonomic nervous system neurons either fail to form or degenerate. We first identified the Phox2b human ortholog, PHOX2B, as the gene underlying congenital central hypoventilation syndrome (CCHS, or Ondine curse), with an autosomal dominant mode of inheritance and de novo mutation at the first generation. We have subsequently shown that heterozygous mutations of PHOX2B may account for several combined or isolated disorders of autonomic nervous-system development--namely, tumors of the sympathetic nervous system (TSNS), such as neuroblastoma and late-onset central hypoventilation syndrome. Here, we report the clinical and molecular assessments of a cohort of 188 probands with CCHS, either isolated or associated with Hirschsprung disease and/or TSNS. The mutation-detection rate was 92.6% (174/188) in our series, and the most prevalent mutation was an in-frame duplication leading to an expansion of +5 to +13 alanines in the 20-alanine stretch at the carboxy terminal of the protein. Such findings suggest PHOX2B mutation screening as a simple and reliable tool for the diagnosis of CCHS, independent of the clinically variable phenotype. In addition, somatic mosaicism was detected in 4.5% of parents. Most interestingly, analysis of genotype-phenotype interactions strongly supports the contention that patients with CCHS who develop malignant TSNS will harbor either a missense or a frameshift heterozygous mutation of the PHOX2B gene. These data further highlight the link between congenital malformations and tumor predisposition when a master gene in development is mutated.

Spinal muscular atrophies (SMA, also known as hereditary motor neuropathies) and hereditary motor and sensory neuropathies (HMSN) are clinically and genetically heterogeneous disorders of the peripheral nervous system. Here we report that mutations in the TRPV4 gene cause congenital © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to M.A.-G. (michaela.auergrumbach@medunigraz.at).. METHODS: Methods and any associated references are available in the online version of the paper at http://www.nature.com/ naturegenetics/. Accession codes. GenBank: human TRPV4 cDNA, NM_021625; human TRPV4, NP_067638 IsoA. Pfam: ankyrin repeat, PF00023.Note: Supplementary information is available on the Nature Genetics website. AUTHOR CONTRIBUTIONS: M.A.-G., S.U., J.S., M.E.M., A.H.C., K.J.D., C.M.A.v.R.-A., N.E.A., H.L., B.S.-W., R.P., C.L., G.W.P., H.J.S., H.K. and T.R.P. recruited the study participants, acquired clinical data, conducted neurological and neurophysiological evaluations and performed linkage analysis. M.A.-G, C.G., L.P. and C.F. carried out the Affymetrix array linkage studies and identified the mutations. A.O., Z.B. and B.T. designed, carried out and analyzed the electrophysiological and Ca 2+ -imaging studies. E.F. conducted immunofluorescence and immunohistochemistry studies. H.S. conducted fluorescence-activated cell sorting (FACS) and biotinylation studies. A.K. performed structural biology and biocomputing analyses. A.H.C., M.E.M. and H.K. participated in the data analysis and reviewed the manuscript. M.A.-G. and C.G. analyzed the data, designed and supervised the study and wrote the manuscript. Supplementary Fig. 1) and observed linkage to three chromosomal regions with log 10 of odds (lod) scores >2 for several SNP markers, including the chromosome 12q23-24 region (data not shown). We constructed haplotypes by including additional distantly related family members (right branch of the pedigree; Supplementary Fig. 1). The genetic interval transmitted with the disease resides between SNPs rs2374688 and rs35426 (Chr. 12: 106,197,054,429 bp; Supplementary Table 1) and overlaps with the intervals reported for risk of congenital distal SMA, SPSMA and HMSN2C 2-4 . Europe PMC Funders GroupIn an affected individual from family FAM_1, we began sequencing all protein-coding exons and exon-intron boundaries of 19 genes but initially observed only known SNPs (Supplementary Table 2). However, sequencing of all protein-coding exons of TRPV4 (transient receptor potential vanilloid 4; chr. 12: 108,705,277-108,755,595; reverse strand) revealed a heterozygous C-to-T nucleotide change at position 943 in exon 6 (Supplementary Fig. 2a), which is predicted to cause the substitution of arginine with tryptophan at position 315 of TRPV4 (R315W). We then screened DNA samples from additional families showing one of the phenotypes described above, including two families previously reported 1,3,4 . All affected individuals from the chromosome 12q23-24-linked family (here called FAM_2) described by...

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes—the 22 autosomes and the X and Y sex chromosomes—can be stained or “painted” with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.

Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.