Hirschsprung's disease is a genetic disorder of neural crest development affecting 1 in 5,000 births. It is characterized by the absence of intramural ganglion cells in the hindgut, which often results in partial to complete intestinal obstruction during the first years of life. An autosomal dominant gene causing this disease was recently mapped to chromosome 10q11.2 (refs 1,2), using an interstitial deletion of this region isolated in a cell hybrid. It was subsequently localized to a 250-kilobase interval which contains the RET proto-oncogene. Using flanking intronic sequences as primers to amplify 12 of the 20 exons of RET from genomic DNA of 27 Hirschsprung's disease patients, we have now identified four mutations (one frameshift and three missense) that totally disrupt or partially change the structure of the tyrosine kinase domain of the RET protein (Ret). Mutations in the extracellular cysteine-rich domain of Ret have been identified previously in patients with multiple endocrine neoplasia type 2A, and a targeted mutation in the tyrosine kinase domain of the same gene produces intestinal aganglionosis and kidney agenesis in homozygous transgenic mice. Our results support the hypothesis that RET, in addition to its potential role in tumorigenesis, plays a critical role in the embryogenesis of the mammalian enteric nervous system.

To better understand the role of TBX5, a T-box containing transcription factor in forelimb and heart development, we have studied the clinical features of HoltOram syndrome caused by 10 different TBX5 mutations. Defects predicted to create null alleles caused substantial abnormalities both in limb and heart. In contrast, missense mutations produced distinct phenotypes: Gly80Arg caused significant cardiac malformations but only minor skeletal abnormalities; and Arg237Gln and Arg237Trp caused extensive upper limb malformations but less significant cardiac abnormalities. Amino acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is highly conserved within T-box sequences that interact with the major groove of target DNA; residue 237 is located in the T-box domain that selectively binds to the minor groove of DNA. These structural data, taken together with the predominant cardiac or skeletal phenotype produced by each missense mutation, suggest that organ-specific gene activation by TBX5 is predicated on biophysical interactions with different target DNA sequences.

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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM *240300, also called APS 1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is generally characterized by two of the three major clinical symptoms that may be present, Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have a number of other clinical symptoms including chronic gastritis, gonadal failure, and rarely, autoimmune thyroid disease and insulin-dependent diabetes mellitus. We and others have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes, and fetal liver and encodes a protein containing motifs suggestive of a transcriptional regulator, including two zinc finger motifs (PHD finger), a proline-rich region, and three LXXLL motifs. Six mutations, in cluding R257X, the predominant Finnish APECED allele, have been defined. R257X was also observed in non-Finnish APECED patients occurring on different chromosomal haplotypes suggesting different mutational origins. Here we present mutation analyses in an extended series of patients, mainly of Northern Italian origin. We have detected 12 polymorphisms, including one amino acid substitution, and two additional mutations, R203X and X546C, in addition to the previously described mutations, R257X, 1096-1097insCCTG, and a 13-bp deletion (1094-1106del). R257X was also the common mutation in the Northern Italian patients (10 of 18 alleles), and 1094-1106del accounted for 5 of 18 Northern Italian alleles. Both R257X and 1094-1106del were both observed in patients of four different geo-ethnic origins, and both were associated with multiple different haplotypes using closely flanking polymorphic markers showing likely multiple mutation events (six and four, respectively). The identification of common AIRE mutations in different APECED patient groups will facilitate its genetic diagnosis. In addition, the polymorphisms presented provide the tools for investigation of the involvement of AIRE in other autoimmune diseases, particularly those affecting the endocrine system.

Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. We report the molecular characterization of a de novo balanced t(2;7)(q37.1;q21.3) translocation in a young female with Marfanoid habitus and skeletal anomalies. The translocation was characterized by fluorescence in situ hybridization (FISH), checked for other abnormalities by array-comparative genomic hybridization (CGH), and finally, the breakpoints were cloned, sequenced, and compared. Biochemical dosage was applied to study the possible mechanisms that may cause the proposita's phenotype. The breakpoint on chromosome 2 disrupts the hypothetical gene MGC42174 (HUGO-approved symbol DIS3L2) and is located in the proximity of the NPPC gene coding for C-type natriuretic peptide (CNP), a molecule that regulates endochondral bone growth. CNP plasma concentration was doubled in the proband compared to five normal controls, while NPPC was substantially overexpressed in her fibroblasts. A transgenic mouse generated to target NPPC overexpression in bone showed a phenotype highly reminiscent of the patient's phenotype. The breakpoint on chromosome 7 is localized proximally at about 75 kb from the COL1A2 gene. The COL1A2 allele on the derivative chromosome was strongly underexpressed in fibroblasts, but total collagen was not significantly different from controls. Several evidences support the conclusion that the proband's abnormal phenotype is associated with C-type natriuretic peptide overexpression.

Hirschsprung disease (HSCR) is a frequent congenital disorder (1 in 5,000 newborns) of unknown origin characterized by the absence of parasympathetic intrinsic ganglion cells of the hindgut. Taking advantage of a proximal deletion of chromosome 10q (del 10q11.2-q21.2) in a patient with total colonic aganglionosis, and of a high-density genetic map of microsatellite DNA markers, we performed genetic linkage analysis in 15 non-syndromic long-segment and short-segment HSCR families. Multipoint linkage analysis indicated that the most likely location for a HSCR locus is between loci D10S208 and D10S196, suggesting that a dominant gene for HSCR maps to 10q11.2, a region to which other neural crest defects have been mapped.

The ectodermal dysplasias (EDs) are a large and complex nosologic group of diseases; more than 170 different pathologic clinical conditions have been identified. Despite the great number of EDs described so far, few causative genes have been identified. We review EDs in the light of the most recent molecular findings and propose a new classification of EDs integrating both molecular-genetic data and corresponding clinical findings of related diseases.

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement).