We report here the identification of a gene associated with the hyperparathyroidism-jaw tumor (HPT-JT) syndrome. A single locus associated with HPT-JT (HRPT2) was previously mapped to chromosomal region 1q25-q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT-JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT-JT and in development of some sporadic parathyroid tumors.

Kidney stones (nephrolithiasis), which affect 12% of males and 5% of females in the western world, are familial in 45% of patients and are most commonly associated with hypercalciuria. Three disorders of hypercalciuric nephrolithiasis (Dent's disease, X-linked recessive nephrolithiasis (XRN), and X-linked recessive hypophosphataemic rickets (XLRH)) have been mapped to Xp11.22 (refs 5-7). A microdeletion in one Dent's disease kindred allowed the identification of a candidate gene, CLCN5 (refs 8,9) which encodes a putative renal chloride channel. Here we report the investigation of 11 kindreds with these renal tubular disorders for CLCN5 abnormalities; this identified three nonsense, four missense and two donor splice site mutations, together with one intragenic deletion and one microdeletion encompassing the entire gene. Heterologous expression of wild-type CLCN5 in Xenopus oocytes yielded outwardly rectifying chloride currents, which were either abolished or markedly reduced by the mutations. The common aetiology for Dent's disease, XRN and XLRH indicates that CLCN5 may be involved in other renal tubular disorders associated with kidney stones.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.

Terminal deletions of chromosome 10p result in a DiGeorge-like phenotype that includes hypoparathyroidism, heart defects, immune deficiency, deafness and renal malformations. Studies in patients with 10p deletions have defined two non-overlapping regions that contribute to this complex phenotype. These are the DiGeorge critical region II (refs 1, 2), which is located on 10p13-14, and the region for the hypoparathyroidism, sensorineural deafness, renal anomaly (HDR) syndrome (Mendelian Inheritance in Man number 146255), which is located more telomeric (10p14-10pter). We have performed deletion-mapping studies in two HDR patients, and here we define a critical 200-kilobase region which contains the GATA3 gene. This gene belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development. Investigation for GATA3 mutations in three other HDR probands identified one nonsense mutation and two intragenic deletions that predicted a loss of function, as confirmed by absence of DNA binding by the mutant GATA3 protein. These results show that GATA3 is essential in the embryonic development of the parathyroids, auditory system and kidneys, and indicate that other GATA family members may be involved in the aetiology of human malformations.

Epilepsy is resistant to drug treatment in about one-third of cases, but the mechanisms underlying this drug resistance are not understood. In cancer, drug resistance has been studied extensively. Amongst the various resistance mechanisms, overexpression of drug resistance proteins, such as multi-drug resistance gene-1 P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1), has been shown to correlate with cellular resistance to anticancer drugs. Previous studies in human epilepsy have shown that MDR1 and MRP1 may also be overexpressed in brain tissue from patients with refractory epilepsy; expression has been shown in glia and neurones, which do not normally express these proteins. We examined expression of MDR1 and MRP1 in refractory epilepsy from three common causes, dysembryoplastic neuroepithelial tumours (DNTs; eight cases), focal cortical dysplasia (FCD; 14 cases) and hippocampal sclerosis (HS; eight cases). Expression was studied immunohistochemically in lesional tissue from therapeutic resections and compared with expression in histologically normal adjacent tissue. With the most sensitive antibodies, in all eight DNT cases, reactive astrocytes within tumour nodules expressed MDR1 and MRP1. In five of eight HS cases, reactive astrocytes within the gliotic hippocampus expressed MDR1 and MRP1. Of 14 cases of FCD, MDR1 and MRP1 expression was noted in reactive astrocytes in all cases. In five FCD cases, MRP1 expression was also noted in dysplastic neurones. In FCD and DNTs, accentuation of reactivity was noted around lesional vessels. Immunoreactivity was always more frequent and intense in lesional reactive astrocytes than in glial fibrillary acidic protein-positive reactive astrocytes in adjacent histologically normal tissue. MDR1 is able to transport some antiepileptic drugs (AEDs), and MRP1 may also do so. The overexpression of these drug resistance proteins in tissue from patients with refractory epilepsy suggests one possible mechanism for drug resistance in patients with these pathologies. We propose that overexpressed resistance proteins lower the interstitial concentration of AEDs in the vicinity of the epileptogenic pathology and thereby render the epilepsy caused by these pathologies resistant to treatment with AEDs.

In 1916, von Economo first described encephalitis lethargica (EL), a CNS disorder presenting with pharyngitis followed by sleep disorder, basal ganglia signs (particularly parkinsonism) and neuropsychiatric sequelae. Since the 1916-1927 epidemic, only sporadic cases have been described. Pathological studies revealed an encephalitis of the midbrain and basal ganglia, with lymphocyte (predominantly plasma cell) infiltration. The EL epidemic occurred during the same time period as the 1918 influenza pandemic, and the two outbreaks have been linked in the medical literature. However, von Economo and other contemporary scientists thought that the 1918 influenza virus was not the cause of EL. Recent examination of archived EL brain material has failed to demonstrate influenza RNA, adding to the evidence that EL was not an invasive influenza encephalitis. By contrast, the findings of intrathecal oligoclonal bands (OCB) and beneficial effects of steroid treatments have provoked the hypothesis that EL may be immune-mediated. We have recently seen 20 patients with a similar EL phenotype, 55% of whom had a preceding pharyngitis. The patients had remarkable similarity to the historical descriptions of EL: sleep disorder (somnolence, sleep inversion or insomnia), lethargy, parkinsonism, dyskinesias and neuropsychiatric symptoms. CSF examination commonly showed elevated protein and OCB (75 and 69% respectively). Investigation found no evidence of viral encephalitis or other recognized causes of rapid-onset parkinsonism. MRI of the brain was normal in 60% but showed inflammatory changes localized to the deep grey matter in 40% of patients. We investigated the possibility that this phenotype could be a postinfectious autoimmune CNS disorder, and therefore similar to Sydenham's chorea. Anti-streptolysin-O titres were elevated in 65% of patients. Furthermore, western immunoblotting showed that 95% of EL patients had autoantibodies reactive against human basal ganglia antigens. These antibodies were also present in the CSF in four patients tested. By contrast, antibodies reactive against the basal ganglia were found in only 2-4% of child and adult controls (n = 173, P < 0.0001). Rather than showing polyspecific binding, these antibodies bound to common neural autoantigens of molecular weight 40, 45, 60 and 98 kDa. Regional tissue comparisons showed that the majority of these autoantigens were specific to or enriched in CNS tissue. Immunohistochemistry with secondary staining localized antibody binding to neurons rather than glial populations. Further investigation is required to determine whether these antibodies affect neuronal function (i.e. whether they are pathogenic anti-neuronal antibodies). Histopathology in one case demonstrated striatal encephalitis with perivenous B- and T-lymphocytic infiltration. We believe an EL-like syndrome is still prevalent, and propose that this syndrome may be secondary to autoimmunity against deep grey matter neurons.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. The C-terminal zinc finger (ZnF2) binds DNA, whereas the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG). We have investigated seven HDR probands and their families for GATA3 abnormalities and have identified two nonsense mutations (Glu-228 3 Stop and Arg-367 3 Stop); two intragenic deletions that result in frameshifts from codons 201 and 355 with premature terminations at codons 205 and 370, respectively; one acceptor splice site mutation that leads to a frameshift from codon 351 and a premature termination at codon 367; and two missense mutations (Cys-318 3 Arg and Asn-320 3 Lys). The functional effects of these mutations, together with a previously reported GATA3 ZnF1 mutation and seven other engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation, yeast twohybrid and glutathione S-transferase pull-down assays. Mutations involving GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding affinity. These findings are consistent with the proposed three-dimensional model of ZnF1, which has separate DNA and protein binding surfaces. Thus, our results, which expand the spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site mutation, help to elucidate the molecular mechanisms that alter the function of this zinc finger transcription factor and its role in causing this developmental anomaly.