Late-onset and long-lasting neutropenias in the young: A new entity anticipating immune-dysregulation disorders.

This study identifies a new chronic form of immune neutropenia in the young with or without detectable indirect anti-neutrophil antibodies, characterized by mild/moderate neutropenia low risk of severe infection (14%), tendency to develop autoimmune phenomena over the course of the disease (cumulative incidence of 58.6% after 20 years of disease duration), leukopenia, progressive reduction of absolute lymphocyte count and a T- and B-cell profile similar to autoimmune disorders like Sjogren syndrome, rheumatoid arthritis, and systemic lupus erythematosus (increased HLADR+ and CD3 + TCRγδ cells, reduced T regulatory cells, increased double-negative B and a tendency to reduced B memory cells). In a minority of patients, P/LP variants related to primary immuno-regulatory disorders were found. This new form may fit the group of "Likely acquired neutropenia," a provisional category included in the recent International Guidelines on Diagnosis and Management of Neutropenia of EHA and EUNET INNOCHRON ACTION 18233. The early recognition of this form of neutropenia would help clinicians to delineate better specific monitoring plans, genetic counseling, and potentially targeted therapies.

Novel spondyloepimetaphyseal dysplasia due to UFSP2 gene mutation.

Beukes hip dysplasia is an autosomal dominant disease which has to date been described only in a large South African family of Dutch origin. The patients presented with progressive epiphyseal dysplasia limited to femoral capital epiphysis and their height was not significantly reduced. A unique variant of the ubiquitin-fold modifier 1 (Ufm1)-specific peptidase 2 (UFSP2) gene (c.868T>C) has been reported in all individuals from Beukes family with clinical and radiological diagnosis of Beukes hip dysplasia. Three individuals, propositus, mother, and grandmother, presented with short stature, joint pain, genu vara and a novel spondyloepimetaphyseal dysplasia involving epiphyses predominantly at hips, but also at knees, ankles, wrists and hands, associated with variable degrees of metaphysis and spine involvement. Exome sequencing allowed us to identify the heterozygous variant c.1277A>C of the UFSP2 gene, leading to the missense change p.D426A, in all 3 patients. This mutation is predicted as damaging and, similarly to the mutation originally described in the Beukes family (p. Y290H), directly affects one of the catalytic residues participating in the active site of the protein. This supports the novel notion that loss of catalytic UFSP2 activity, observed in association with different mutants and already experimentally proven in vitro, may have different clinical outcomes.

Autoinflammation in pyoderma gangrenosum and its syndromic form (pyoderma gangrenosum, acne and suppurative hidradenitis).

BACKGROUND: Pyoderma gangrenosum (PG) is a rare skin disease characterized clinically by ulcers with undermined borders, and histologically by neutrophil-rich infiltrates. PG may occur alone, in syndromic forms or associated with systemic diseases, such as inflammatory bowel disease and haematological or rheumatological disorders. OBJECTIVES: To determine a specific genetic background related to autoinflammation for PG. METHODS: We assessed autoinflammation by evaluating the cytokine profile and genes involved in classic autoinflammatory diseases in 13 patients with PG and in seven patients with the syndromic form, known as PASH (pyoderma gangrenosum, acne and suppurative hidradenitis). RESULTS: In skin samples, the expression of interleukin (IL)-1ß and its receptors, IL-17 and its receptor, and tumour necrosis factor-α and its receptors were significantly higher in both PG (P = 0·001) and in PASH (P < 0·001) than in controls. The chemokines IL-8; chemokine (C-X-C motif) ligand 1/2/3; chemokine (C-X-C motif) ligand 16; and RANTES (regulated on activation, normal T-cell-expressed and secreted) were also overexpressed. Cases of PG and PASH showed mutations in the autoinflammatory genes MEFV, NLRP3, NLRP12, NOD2, LPIN2 and PSTPIP1. CONCLUSIONS: Overexpression of cytokines/chemokines, along with genetic changes, supports the hypothesis that PG and its syndromic form, PASH, are a spectrum of polygenic autoinflammatory conditions.

SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Whole genome sequencing in ROHHAD trios proved inconclusive: what's beyond?

Rapid-onset Obesity with Hypothalamic dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD) is a rare, life-threatening, pediatric disorder of unknown etiology, whose diagnosis is made difficult by poor knowledge of clinical manifestation, and lack of any confirmatory tests. Children with ROHHAD usually present with rapid onset weight gain which may be followed, over months or years, by hypothalamic dysfunction, hypoventilation, autonomic dysfunction, including impaired bowel motility, and tumors of neural crest origin. Despite the lack of evidence of inheritance in ROHHAD, several studies have been conducted in recent years that have explored possible genetic origins, with unsuccessful results. In order to broaden the search for possible genetic risk factors, an attempt was made to analyse the non-coding variants in two trios (proband with parents), recruited in the Gaslini Children's Hospital in Genoa (Italy). Both patients were females, with a typical history of ROHHAD. Gene variants (single nucleotide variants, short insertions/deletions, splice variants or in tandem expansion of homopolymeric tracts) or altered genomic regions (copy number variations or structural variants) shared between the two probands were searched. Currently, we have not found any potentially pathogenic changes, consistent with the ROHHAD clinical phenotype, and involving genes, regions or pathways shared between the two trios. To definitively rule out the genetic etiology, third-generation sequencing technologies (e.g., long-reads sequencing, optical mapping) should be applied, as well as other pathways, including those associated with immunological and autoimmune disorders, should be explored, making use not only of genomics but also of different -omic datasets.

A unicentric cross-sectional observational study on chronic intestinal inflammation in total colonic aganglionosis: beware of an underestimated condition.

BACKGROUND: Inflammatory Bowel Diseases (IBD) are known to occur in association with Hirschsprung disease (HSCR). Most of cases are represented by Crohn Disease (CD) occurring in patients with Total Colonic Aganglionosis (TCSA) with an estimated prevalence of around 2%. Based on these considerations and on a number of provisional data belonging to our Center for Digestive Diseases, we developed a unicentric cross-sectional observational study aimed at describing phenotype, genotype, pathology and metagenomics of all patients with TCSA and Crohn-like lesions. RESULTS: Out of a series of 62 eligible TCSA patients, 48 fulfilled inclusion criteria and were enrolled in the study. Ten patients did not complete the study due to non-compliance or withdrawal of consent and were subsequently dropped out. A total of 38 patients completed the study. All patients were tested for chronic intestinal inflammation by a combination of fecal calprotectine (FC) or occult fecal blood (OFB) and underwent fecal metagenomics. Nineteen (50%) tested positive for FC, OFB, or both and subsequently underwent retrograde ileoscopy. Fourteen patients (36.8%) presented Crohn-like lesions, occurring after a median of 11.5 years after surgery (range 8 months - 21.5 years). No statistically significant differences regarding demographic, phenotype and genotype were observed comparing patients with and without lesions, except for need for blood transfusion that was more frequent in those with lesions. Faecal microbiome of patients with lesions (not that of caregivers) was less biodiverse and characterized by a reduction of Bacteroidetes, and an overabundance of Proteobacteria. FC tested negative in 3/14 patients with lesions (21%). CONCLUSIONS: Our study demonstrated an impressive 10-folds higher incidence of chronic inflammation in TCSA. Up to 50% of patients may develop IBD-like lesions postoperatively. Nonetheless, we failed in identifying specific risk factors to be used to implement prevention strategies. Based on the results of our study, we suggest screening all TCSA patients with retrograde ileoscopy regardless of FC/OFB values. The frequency of endoscopic assessments and the role of FC/OFB screening in prompting endoscopy is yet to be determined.

Guidelines for the genetic diagnosis of hereditary recurrent fevers.

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinflammatory diseases characterised by recurrent bouts of fever and serosal inflammation that are caused by pathogenic variants in genes important for the regulation of innate immunity. Discovery of the molecular defects responsible for these diseases has initiated genetic diagnostics in many countries around the world, including the Middle East, Europe, USA, Japan and Australia. However, diverse testing methods and reporting practices are employed and there is a clear need for consensus guidelines for HRF genetic testing. Draft guidelines were prepared based on current practice deduced from previous HRF external quality assurance schemes and data from the literature. The draft document was disseminated through the European Molecular Genetics Quality Network for broader consultation and amendment. A workshop was held in Bruges (Belgium) on 18 and 19 September 2011 to ratify the draft and obtain a final consensus document. An agreed set of best practice guidelines was proposed for genetic diagnostic testing of HRFs, for reporting the genetic results and for defining their clinical significance.

Long-term clinical profile of children with the low-penetrance R92Q mutation of the TNFRSF1A gene.

OBJECTIVE: To analyze the long-term impact of the R92Q mutation of TNFRSF1A in children with periodic fever, in comparison with children with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) with TNFRSF1A structural mutations and children with periodic fever of unknown origin fulfilling the criteria for periodic fever, aphthosis, pharyngitis, and adenitis syndrome (PFAPA). METHODS: The extracellular region of TNFRSF1A was analyzed in 720 consecutive children with periodic fever, using denaturing high-performance liquid chromatography and DNA sequencing. Followup data on 11 pediatric patients with TNFRSF1A structural mutations (cysteine or T50M), 23 pediatric patients with an R92Q substitution, and 64 pediatric patients with PFAPA were collected during routine clinic visits. The 50-item Child Health Questionnaire was used to assess health-related quality of life (HRQOL). RESULTS: The frequency of typical TRAPS-related clinical manifestations was significantly lower and the impact of the disease on HRQOL was significantly reduced in patients with the R92Q mutation compared with TRAPS patients carrying structural mutations of TNFRSF1A. Followup data on 11 TRAPS patients with TNFRSF1A structural mutations (mean followup 7.9 years), 16 patients with theR92Q substitution (mean followup 7.3 years), and 64 patients with PFAPA (mean followup 5.2 years) were available. Patients with R92Q mutations and patients with PFAPA displayed a higher rate of self-resolution or amelioration of the fever episodes than did TRAPS patients with structural mutations. CONCLUSION: Although some cases may progress to a more chronic disease course, the majority of children with an R92Q mutation of the TNFRSFA1 gene show a milder disease course than that in children with TNFRSFA1 structural mutations and have a high rate of spontaneous resolution and amelioration of the recurrent fever episodes.

Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an NLRP12 mutation.

OBJECTIVE: NLRP12 mutations have been described in patients affected with peculiar autoinflammatory symptoms. This study was undertaken to characterize NLRP12 mutations in patients with autoinflammatory syndromes, particularly a novel missense mutation, p.D294E, affecting a protein sequence crucial for ATP binding, which was identified in a Caucasian family with familial cold-induced autoinflammatory syndrome in some family members. METHODS: Fifty patients were tested for NLRP12 mutations. A Caucasian family with the p.D294E missense mutation of NLRP12 in some family members was clinically characterized. In vitro analysis of the effects of the mutation on NF-κB activity was performed in HEK 293 cells after cotransfection of the cells with a luciferase NF-κB-responsive element and mutant or wild-type (WT) NLRP12 expression plasmids. NF-κB activity was also evaluated 24 hours after stimulation with tumor necrosis factor α in monocytes from individual family members carrying the mutation. Furthermore, secretion of interleukin-1ß (IL-1ß), production of reactive oxygen species (ROS), and activation of antioxidant systems in patient and healthy donor monocytes, under resting conditions and after stimulation with pathogen-associated molecular patterns (PAMPs), were also assessed. RESULTS: In the family assessed, the p.D294E mutation segregated in association with a particular sensitivity to cold exposure (especially arthralgias and myalgia), but not always with an inflammatory phenotype (e.g., urticarial rash or fever). In vitro, the mutant protein maintained the same inhibitory activity as that shown by WT NLRP12. Consistently, NLRP12-mutated monocytes showed neither increased levels of p65-induced NF-κB activity nor higher secretion of IL-1ß. However, the kinetics of PAMP-induced IL-1ß secretion were significantly accelerated, and high production of ROS and up-regulation of antioxidant systems were demonstrated. CONCLUSION: Even with a variable range of associated manifestations, the extreme sensitivity to cold represents the main clinical hallmark in an individual carrying the p.D294E mutation of the NLRP12 gene. Although regulation of NF-κB activity is not affected in patients, redox alterations and accelerated secretion of IL-1ß are associated with this mild autoinflammatory phenotype.

Megacystis, megacolon, and malrotation: a new syndromic association?

Chronic intestinal pseudo-obstruction (CIPO) can occur as a consequence of neuropathies including diffuse Intestinal Neuronal Dysplasia (IND), a relatively rare enteric nervous system (ENS) abnormality. Although various authors reported of diffuse IND associated either with intestinal malrotation or megacystis, the co-existence of these three entities in the same patient has never been described before. The aim of this paper is to report for the first time in literature a series of patient with such association, focusing on one who carries a de novo duplication of chromosome 12, suggesting a new syndromic association (megacolon, megacystis, malrotation).

Widening the Neuroimaging Features of Adenosine Deaminase 2 Deficiency.

Adenosine deaminase 2 deficiency (OMIM #615688) is an autosomal recessive disorder characterized by a wide clinical spectrum, including small- and medium-sized vessel vasculopathies, but data focusing on the associated neuroimaging features are still scarce in the literature. Here, we describe the clinical neuroimaging features of 12 patients with genetically proven adenosine deaminase 2 deficiency (6 males; median age at disease onset, 1.3 years; median age at genetic diagnosis, 15.5 years). Our findings expand the neuroimaging phenotype of this condition demonstrating, in addition to multiple, recurrent brain lacunar ischemic and/or hemorrhagic strokes, spinal infarcts, and intracranial aneurysms, also cerebral microbleeds and a peculiar, likely inflammatory, perivascular tissue in the basal and peripontine cisterns. Together with early clinical onset, positive family history, inflammatory flares and systemic abnormalities, these findings should raise the suspicion of adenosine deaminase 2 deficiency, thus prompting genetic evaluation and institution of tumor necrosis factor inhibitors, with a potential great impact on neurologic outcome.

Congenital central hypoventilation syndrome: genotype-phenotype correlation in parents of affected children carrying a PHOX2B expansion mutation.

Congenital Central Hypoventilation Syndrome (CCHS) is a rare genetic disorder. Although most CCHS associated PHOX2B mutations occur de novo, about 10% of the cases are inherited from apparently asymptomatic parents, thus confirming variable expressivity and incomplete penetrance of PHOX2B mutations. Three asymptomatic parents of children affected with CCHS, and found to carry the same PHOX2B expansion mutations as their siblings, were studied by overnight polysomnography and somatic mosaicism analysis. In one case, significant sleep breathing control anomalies were detected, while the other two resulted in normal. In tissue-specific allele studies, mosaicism with a comparatively low mutant allele proportion was showed in the two unaffected adult carriers. Accurate polysomnography and assessment of the degree of somatic mosaicism should be conducted in asymptomatic carriers of PHOX2B mutations, as they may unmask subclinical but significant anomalies.

Genomics approach to the analysis of bacterial communities dynamics in Hirschsprung's disease-associated enterocolitis: a pilot study.

INTRODUCTION: The most invalidating and life-threatening complication in Hirschsprung's disease patients (HSCR) is Hirschsprung's disease-associated enterocolitis (HAEC). The mechanisms underlying enterocolitis have not been identified. The limited knowledge of the role of intestinal microflora is in part due to the complexity of the intestinal microbiome and to the limitation of cultivation-based technologies, given that less than 25% of the intestinal bacterial species can be cultured. MATERIALS AND METHODS: We used amplified ribosomal DNA restriction analysis (ARDRA) with four different restriction enzymes to study variations of microflora composition of the stools of a selected HSCR patient in different clinical conditions (acute phase vs. remission). RESULTS: We assessed a total of 15 stool specimens belonging to the same 3-year-old male patient suffering from HSCR, which were harvested during 4 HAEC episodes and remission phases. Restriction analysis showed that HAEC episodes seem to cluster together at ARDRA analysis, thus suggesting a sort of predisposing bacterial community for HAEC development and the need for a microflora equilibrium to maintain wellness. CONCLUSIONS: This approach proved to be effective, useful and powerful in assessing microflora dynamics and indicated that the differences in microflora associated with acute HAEC or remission are likely to result from a combination of disease activity and different antibiotic therapies. ARDRA proved to be useful in discriminating disease versus remission. Our findings indicated that HAEC results from a change in the equilibrium between bacterial species or from altered discrimination of harmless from harmful microorganisms, challenging the definition of pathogenic and non-pathogenic species. Based on these results, we propose ARDRA as a rapid inexpensive tool to assess microflora dynamics during HAEC episodes.

Hirschsprung disease, associated syndromes and genetics: a review.

Hirschsprung disease (HSCR, aganglionic megacolon) represents the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births. This developmental disorder is a neurocristopathy and is characterised by the absence of the enteric ganglia along a variable length of the intestine. In the last decades, the development of surgical approaches has importantly decreased mortality and morbidity which allowed the emergence of familial cases. Isolated HSCR appears to be a non-Mendelian malformation with low, sex-dependent penetrance, and variable expression according to the length of the aganglionic segment. While all Mendelian modes of inheritance have been described in syndromic HSCR, isolated HSCR stands as a model for genetic disorders with complex patterns of inheritance. The tyrosine kinase receptor RET is the major gene with both rare coding sequence mutations and/or a frequent variant located in an enhancer element predisposing to the disease. Hitherto, 10 genes and five loci have been found to be involved in HSCR development.

Search for pathogenetic variants of the SPRY2 gene in intestinal innervation defects.

SPRY2 is an inducible inhibitor of signalling mediated by tyrosine kinases receptors, whose targeting causes intestinal hyperganglionosis in mice. In this light, we have undertaken a mutational analysis of the SPRY2 gene in patients affected with intestinal neuronal dysplasia (IND), without detecting nucleotide changes in any of the 26 DNA samples analysed, with the exception of two already known polymorphic variants. A role of the SPRY2 gene in IND pathogenesis can be thus excluded.

Whole exome sequencing approach to childhood onset familial erythrodermic psoriasis unravels a novel mutation of CARD14 requiring unusual high doses of ustekinumab.

BACKGROUND: Autosomal dominant gain of function mutations in caspase recruitment domain family member 14 (CARD14) is a rare condition associated with plaque-type psoriasis, generalized pustular psoriasis, palmoplantar pustular psoriasis and pityriasis rubra pilaris. Recently, a new CARD14 -associated phenotype defined as CAPE (CARD14-associated papulosquamous eruption) with clinical features of both psoriasis and pityriasis rubra pilaris was reported. We describe a family carrying a novel heterozygous mutation in CARD14 gene, with childhood-onset erythrodermic psoriasis requiring an unusual extremely high dose (up to 2 mg/kg every 8 weeks) of ustekinumab to achieve disease remission. CASE PRESENTATION: We describe a large family with three pairs of twins presenting a clinical phenotype characterized by childhood-onset erythrodermic psoriasis; in some family members is also reported psoriatic arthritis. The two probands presented poor clinical response to topic and systemic therapy with antihistamine, steroid, retinoids, cyclosporine and etanercept. After exclusion of the most common genes associated to autoinflammatory diseases (IL36RN, IL1RN, MVK, TNFRSF1A, NLRP3, NLRP12, MEFV, NOD2, PSMB8, PSTPIP1, LPIN2) we approached a new gene search by subjecting to Whole Exome Sequencing (WES) analysis five members of the family. A novel heterozygous mutation (c.446 T > G, leading to the missense amino acid substitution p.L149R) in the exon 4 of the CARD14 gene was identified in all affected members. Increasing dosages (up to 2 mg/kg every 8 weeks) of ustekinumab, a human monoclonal antibody targeting interleukin-12 (IL-12) and interleukin-23 (IL-23), allowed the complete control of the clinical manifestations, with an evident reduction of circulating Th17 and Th22 CD4+ T cell subsets. CONCLUSIONS: We describe the association of mutations of the CARD14 gene with an erythrodermic psoriasis pedigree, underlying the necessity to investigate CARD14 mutations in childhood-onset psoriasis cases and confirming the presence of CARD14 causative mutations also in erythrodermic psoriasis form, as recently reported. Also in pediatric age, ustekinumab represents a powerful therapeutic option for this rare condition, that is usually refractory to other treatments. In young children, high and frequent dosages allowed a complete control of the clinical manifestations without any severe side effects, with a long-term follow-up.

PHOX2A and PHOX2B genes are highly co-expressed in human neuroblastoma.

The detection of PHOX2B mutations in a small proportion of patients affected with either familial or sporadic neuroblastoma (NB), has arisen interest on the possible pathogenic role of this gene in the disease determination. In this light, we have carried out a quantitative expression analysis of PHOX2B and its paralogue PHOX2A on a panel of NB cell lines and NB tumour samples to identify a possible differential expression between NB cells and their normal counterpart (adrenal medulla cells). Our results revealed that both PHOX2A and PHOX2B are over-expressed in tumour samples and NB cell lines. Particularly, the expression levels of the two genes in NB cell lines show a highly significant correlation, suggesting their possible synergistic role or a coordinated expression regulation. Furthermore, PHOX2 gene over-expression in NB tumours and cell lines suggests these genes may be widely involved in NB development through either a direct mechanism of up-regulation or a failure in maintaining proper transcript levels after embryonic development.

RET mutations in human disease.

The RET proto-oncogene is at the origin of one of the most interesting models of human disease caused by mutations in a receptor tyrosine kinase gene. Somatic rearrangements of RET are involved in the aetiology of a variable proportion of papillary thyroid carcinomas (PTC), the most common type of thyroid tumour whose prevalence is increasing in areas heavily exposed to radioactive fallout after the Chernobyl accident of 1986. Moreover, germline RET mutations are associated with the three variants of the inherited cancer syndrome known as multiple endocrine neoplasia type 2 (MEN2A, MEN2B and FMTC). Finally, RET mutations or heterozygous deletions of the whole gene cause the autosomal dominant form of Hirschsprung disease (HSCR), a congenital disorder of the enteric nervous system (ENS).

The sensitivity of activated Cys Ret mutants to glial cell line-derived neurotrophic factor is mandatory to rescue neuroectodermic cells from apoptosis.

Hirschsprung's disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys(609), Cys(620), and Cys(634)) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys(609)- or Cys(620)-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys(634) Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys(634) Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys(609) or Cys(620) mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.

Somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the RET proto-oncogene.

Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.