Multiple endocrine neoplasia type 2 (MEN 2) comprises three clinically distinct, dominantly inherited cancer syndromes. MEN 2A patients develop medullary thyroid carcinoma (MTC) and phaeochromocytoma. MEN 2B patients show in addition ganglioneuromas of the gastrointestinal tract and skeletal abnormalities. In familial MTC, only the thyroid is affected. Germ-line mutations of the RET proto-oncogene have recently been reported in association with MEN 2A and familial MTC. All mutations occurred within codons specifying cysteine residues in the transition point between the RET protein extracellular and transmembrane domains. We now show that MEN 2B is also associated with mutation of the RET proto-oncogene. A mutation in codon 664, causing the substitution of a threonine for a methionine in the tyrosine kinase domain of the protein, was found in all nine unrelated MEN 2B patients studied. The same mutation was found in six out of 18 sporadic tumours.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

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.

Nonsense-mediated mRNA decay (NMD) is of universal biological significance1-3. It has emerged as an important global RNA, DNA and translation regulatory pathway4. By systematically sequencing 737 genes (annotated in the Vertebrate Genome Annotation database) on the human X chromosome in 250 families with X-linked mental retardation, we identified mutations in the UPF3 regulator of nonsense transcripts homolog B (yeast) (UPF3B) leading to protein truncations in three families: two with the Lujan-Fryns phenotype5,6 and one with the FG phenotype7. We also identified a missense mutation in another family with nonsyndromic mental retardation. Three mutations lead to the introduction of a premature termination codon and subsequent NMD of mutant UPF3B mRNA. Protein blot analysis using lymphoblastoid cell lines from affected individuals showed an absence of the UPF3B protein in two families. The UPF3B protein is an important component of the NMD surveillance machinery8,9. Our results directly implicate abnormalities of NMD in human disease and suggest at least partial redundancy of NMD pathways.

SAP is an adaptor protein expressed in T cells and natural killer cells. It plays a critical role in immunity, as it is mutated in humans with X-linked lymphoproliferative syndrome (XLP), a fatal immunodeficiency characterized by an abnormal response to Epstein-Barr virus (EBV) infection. SAP interacts with the SLAM family receptors and promotes transduction signal events by these receptors through its capacity to recruit and activate the Src kinase FynT. Because it has been previously established that FynT is selectively required for the development of NKT cells, we examined NKT cells in SAP-deficient mice and in humans with XLP. In the absence of SAP, the development of NKT cells is severely impaired both in mice and in humans. These results imply that SAP is a potent regulator of NKT cell development. They also identify for the first time a defect in NKT cells associated with a human primary immunodeficiency, revealing a potential role of NKT cells in the immune response to EBV.

EAT-2 is an adaptor expressed in innate immune cells, including natural killer (NK) cells. It is closely related to the adaptor SAP, which regulates signaling lymphocyte activation molecule (SLAM)-related receptors by recruiting the kinase FynT to the receptors. Here we have studied the function of EAT-2 in NK cells by creating mice lacking or overexpressing EAT-2. Like SAP, EAT-2 was associated with the SLAM-related receptor 2B4 in NK cells. However, unlike SAP, EAT-2 was an inhibitor of NK cell function. EAT-2 repressed natural cytotoxicity and interferon-gamma secretion by a mechanism involving tyrosine phosphorylation of its C terminus. We have demonstrated a similar function for the adaptor ERT, a newly identified SAP family member expressed in mouse NK cells. These data identify a previously unknown mechanism of NK cell inhibition. Moreover, they indicate that EAT-2 and SAP have distinct and at times opposing functions in natural immunity.

We have made the following observations regarding self-recognition during the development and progression of murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS): 1) chronic progression of EAE is accompanied by a sequential, predictable cascade of neo-autoreactivity, commonly referred to as epitope spreading, presumably caused by endogenous self-priming during autoimmune-mediated tissue damage; 2) there is an invariant relationship between the progression of EAE and the emergence of epitope spreading; 3) progression of EAE can be inhibited by the induction of antigen-specific tolerance to spreading determinants after onset of initial neurologic symptoms; 4) CD4+ Th 1 cells responding to spreading determinants are autonomously encephalitogenic; 5) epitope spreading occurs during the development of MS and in some cases involves HLA-DP class II-restricted self-recognition; and 6) progression of both EAE and MS is accompanied by the decline of primary T-cell autoreactivity associated with disease onset and by the concurrent emergence of the epitope spreading cascade. Our studies directly challenge the traditional view that EAE and MS are initiated and maintained by autoreactivity directed against a single predominant myelin protein or determinant. Our results indicate that progression of EAE and MS involves a shifting of T-cell autoreactivity from primary initiating self-determinants to defined cascades of secondary determinants that sustain the inflammatory self-recognition process during disease progression.

SAP is an adaptor protein that is expressed in NK and T cells. It is mutated in humans who have X-linked lymphoproliferative (XLP) disease. By interacting with SLAM family receptors, SAP enables tyrosine phosphorylation signaling of these receptors by its ability to recruit the Src-related kinase, Fyn. Here, we analyzed the role of SAP in NK cell functions using the SAP-deficient mouse model. Our results showed that SAP was required for the ability of NK cells to eliminate tumor cells in vitro and in vivo. This effect strongly correlated with expression of CD48 on tumor cells, the ligand of 2B4, a SLAM-related receptor expressed in NK cells. In keeping with earlier reports that studied human NK cells, we showed that SAP was necessary for the ability of 2B4 to trigger cytotoxicity and IFN-γ secretion. In the absence of SAP, 2B4 function was shifted toward inhibition of NK cell–mediated cytotoxicity. By analyzing mice lacking Fyn, we showed that similarly to SAP, Fyn was strictly required for 2B4 function. Taken together, these results provide evidence that the 2B4-SAP-Fyn cascade defines a potent activating pathway of natural cytotoxicity. They also could help to explain the high propensity of patients who have XLP disease to develop lymphoproliferative disorders.