Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor.

The hyperthyroidism of Graves' disease (GD) is caused by TSH-receptor (TSH-R) stimulating autoantibodies (TSAb), leading to overproduction of thyroid hormones. We present evidence for TSAb interaction with three distinct regions of the TSH-R, one in immediate vicinity of the carboxy terminal serpentine. Three murine monoclonal antibodies (MoAbs 28.1, A9 and 31.7) directed to amino acids 36-40, 147-228 and 382-415 were labelled and tested for their binding to human recombinant TSH-R on solid phase. All MoAbs bound to TSH-R with a K(d) of 8-12 nm and showed no competition among themselves. We tested 114 sera from euthyroid controls, 118 TBII positive sera from patients with GD (containing TSAb confirmed by bioassays), 16 TBII positive sera from patients with autoimmune thyroid disease (AIT), who were hypothyroid and had TSH blocking antibodies (TBAb), and 20 patients with AIT, who were hypothyroid but negative for all TRAb. Mid-regional MoAb A9 tracer achieved the highest sensitivity in the GD group (72.0%), whereas C-terminal MoAb 31.7 found most sera positive in the AIT group (87.5%). Surprisingly, the N-terminal MoAb 28.1 had the lowest sensitivity in the GD (10.4%) and AIT group (43.8%). Using a mixture of all three tracer MoAbs did not increase the sensitivity in the GD or AIT group, compared to the best single MoAb alone. Median inhibition of MoAb A9 was significantly (P < 0.001) higher than inhibition of MoAbs 28.1 or 31.7 in the group of GD patients but not in other groups. Almost all patient sera with positive reactivity in the MoAb tracer assays had TBII values in the higher range. However, there were many highly TBII positive sera, which did not show a displacement of the MoAb tracers. We conclude that, contrary to some reports, the binding of TSAb and TBAb to the TSH-R is not restricted to distinct and distant epitopes. The middle part of the TSH-R seems to be more relevant for TSAb binding than the N-terminal part, while a proportion of TSAb autoantibodies also binds to a C-terminal epitope of the TSH-R. The method described here is a TSH independent competitive assay for the detection of TSH-R autoantibodies.

Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase.

Mutations in the HNF4alpha gene are responsible for type 1 maturity-onset diabetes of the young (MODY1), which is characterized by a defect in insulin secretion. Hepatocyte nuclear factor (HNF)-4alpha is a transcription factor that plays a critical role in the transcriptional regulation of genes involved in glucose metabolism in both hepatocytes and pancreatic beta-cells. Recent evidence has implicated AMP-activated protein kinase (AMPK) in the modulation of both insulin secretion by pancreatic beta-cells and the control of glucose-dependent gene expression in both hepatocytes and beta-cells. Therefore, the question could be raised as to whether AMPK plays a role in these processes by modulating HNF-4alpha function. In this study, we show that activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) in hepatocytes greatly diminished HNF-4alpha protein levels and consequently downregulates the expression of HNF-4alpha target genes. Quantitative evaluation of HNF-4alpha target gene expression revealed diminished mRNA levels for HNF-1alpha, GLUT2, L-type pyruvate kinase, aldolase B, apolipoprotein (apo)-B, and apoCIII. Our data clearly demonstrate that the MODY1/HNF-4alpha transcription factor is a novel target of AMPK in hepatocytes. Accordingly, it can be suggested that in pancreatic beta-cells, AMPK also acts by decreasing HNF-4alpha protein level, and therefore insulin secretion. Hence, the possible role of AMPK in the physiopathology of type 2 diabetes should be considered.

Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene.

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.

Structure of the promoter and complete sequence of the gene coding for the rabbit translationally controlled tumor protein (TCTP) P23.

We have isolated genomic recombinants containing the complete gene coding for the rabbit translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF) P23. The gene is organized into five introns and six exons and its total length amounts to 3819 nucleotides. All intron/exon boundaries are in accordance with the GT/AG rule. Transcription of the gene generates two mRNAs of 843 and 1163 nucleotides differing in the length of their 3'-untranslated regions. They are formed by alternative polyadenylation. The transcription initiation site has been determined by comparison of sequences of the gene and several processed TCTP pseudogenes. The full-length 5'-untranslated region comprises 116 nucleotides and starts with an oligopyrimidine tract important for translational regulation. Additionally 1.2 kb of the 5'-flanking promoter region has been sequenced. The promoter contains a TATA box at -30 and potential binding sites for transcription factors such as stimulating protein 1 (Sp1), nuclear factor 1 (NF1), activator protein 1 (AP1), c-Ets1, cAMP-response element (CP2), myeloid-specific zinc finger protein 1 (MZF1) and others. For functional analysis 5'-flanking sequences up to -918 were fused to the chloramphenicol acetyltransferase (CAT) gene and tested using a rabbit aortic smooth-muscle cell line by cell transfection and CAT assays. The results confirm that the analyzed gene is the actively transcribed TCTP gene.

Molecular analysis of 29 pyruvate kinase-deficient patients from central Europe with hereditary hemolytic anemia.

We investigated the DNA of 29 unrelated pyruvate kinase (PK) deficiency (PKD) patients from Central Europe with hereditary nonspherocytic hemolytic anemia for mutations in the PK-L/R gene. Among 58 potentially affected alleles, 53 mutations were identified, of which 17 were different from each other. Of these 17 mutations, 13 were single-nucleotide (nt) substitutions resulting in amino acid exchanges, G787A (Gly263-Arg), G994A (Gly332-Ser), G1006T (Ala336-Ser), G1010A (Arg337-Gln), A1081G (Asn361-Asp), G1127T (Ser376-Ile), G1174A (Ala392-Thr), G1281T (Glu427-Asp), C1454T (Ser485-Phe), C1456T (Arg486-Trp), G1493A (Arg498-His), G1529A (Arg510-Gin), and C1594T (Arg532-Trp); 1 in-frame triplet deletion, 1060delAAG (delLys354); 1 in-frame triplet insertion, 1203insAGC (insSer after Cys401); 1 splicesite mutation, 101-1G-A; and 1 frameshift deletion, 628delGT. Six mutations, 628delGT, G787A, G1010A, G1127T, G1281T, and C1454T, are described for the first time. To test the hypothesis of a single origin of the most common PK mutation in the European population, G1529A, we investigated all patients at four polymorphic sites in the PK-L/R gene: C/A at nt 1705, C/T at nt 1992, the (ATT)n microsatellite in intron J, and a polymorphism (T)10/(T)19 in intron I. Nine patients homozygous for mutation G1529A were consistent in all four markers. In the group of patients homozygous for mutation G1529A, the hematologic parameters and clinical manifestations have been studied in detail. Although having an identical mutation in the PK-L/R gene, the patients are affected differently. Their appearance ranges from a very mild compensated hemolysis to a severe anemia. Possible molecular explanations are discussed.

Complete genomic sequence of the human PK-L/R-gene includes four intragenic polymorphisms defining different haplotype backgrounds of normal and mutant PK-genes.

The human pyruvate kinase L/R-gene has been completely sequenced in unrelated normal individuals and in pyruvate kinase-deficient patients by a PCR-based direct genomic sequencing approach and analyzed for polymorphisms. The total length of the gene is 8409 nucleotides. Four polymorphic sites have been detected: C/A1705 and C/T1992 in exon 12, a T-stretch in intron 1 occurring in the two polymorphic forms (T)10 and (T)19 and an (ATT)n microsatellite in intron J which has been found in the variation (ATT)11-17. Haplotype analysis using these four markers has been applied to trace the genetic background in PK-deficiencies. The results support the idea of a single origin of most of the individual PK-mutations.

A novel mutation in the coding region for neurophysin-II is associated with autosomal dominant neurohypophyseal diabetes insipidus.

OBJECTIVE: Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare cause of diabetes insipidus, in which AVP serum levels are insufficient. AVP is synthesized along with neurophysin-II (NPII) as an AVP-NPII precursor polypeptide in the hypothalamus. After proteolytic cleavage during axonal transport, AVP and NPII are reassembled and stored loosely bound to each other in the posterior pituitary until both are released into the circulation. In this study, we investigated the genetic basis of ADNDI in a German kindred with 10 affected members spanning three generations. DESIGN: Genomic DNA was isolated from peripheral blood leucocytes. The entire coding region of the AVP-NPII gene of one of the affected persons was amplified by polymerase chain reaction (PCR) and subjected to nucleotide sequence analysis. Sequencing results were confirmed by restriction enzyme analysis of PCR products. PATIENTS: Six affected and two unaffected members of a family with ADNDI and 54 unrelated healthy control subjects were studied. RESULTS: The index patient was found by direct sequencing to be heterozygous for a G to T transversion at nucleotide position 1884 (exon 2) of the AVP-NPII gene. This mutation introduced a new recognition site for the restriction enzyme Ava II, which was used to test for the presence of the mutation in other family members and in control subjects. The mutation was detected in all family members with ADNDI, but was not found in unaffected family members or in control subjects. The mutation encodes a valine in place of the normal glycine at amino acid 65 of NPII, which is known to be highly conserved during evolution. CONCLUSIONS: In this family, the autosomal dominant neurohypophyseal diabetes insipidus phenotype cosegregates with a point mutation in a region of the AVP-neurophysin-II gene which codes for the carboxy-terminal domain of neurophysin-II. Although the altered amino acid is not directly involved in AVP binding, the mutation might lead to conformational changes that impair the dimerization of neurophysin-II molecules. This could in turn affect the AVP binding affinity of neurophysin-II or might interfere with the transport of the AVP-neurophysin-II precursor in the AVP-producing cells of the hypothalamus.

Mutations in the pyruvate kinase L gene in patients with hereditary hemolytic anemia.

We have completely sequenced the introns of the human L-type pyruvate kinase (PK) gene using the published cDNA sequence. Subsequently, DNA from 12 unrelated PK deficiency (PKD) patients of Central European origin was investigated for mutations in this gene by solid-phase sequencing. We detected 10 different mutations, 9 of which result in single amino acid alterations, whereas the tenth destroys a splice site. Eight of the 10 mutations have not been described before. We found 7 missense mutations: G994-->A (Gly-332-->Ser), G1006-->T (Ala-336-->Ser), A1081-->G (Asn-361-->Asp), G1174-->A (Ala-392-->Thr), G1493-->A (Arg-498-->His), G1529-->A (Arg-510-->Gln), C1594-->T (Arg-532-->Trp), one in-frame triplet deletion (del) as well as one insertion (ins): del AAG1060-62 (del Lys-354), ins AGC after C1203 (ins Ser after Cys-401), and one splice-site mutation at the border of intron A to exon 3: g/G283-->a/G. Although the enzymatic properties are substantially changed in all PK mutations, only two affected amino acid positions are in or close to the active site. Mutations C1594-->T, G994-->A, del AAG1060-62 and the splice-site mutation g/G283-->a/G have been detected in two different patients each. Mutation G1529-->A was found in five different alleles. Haplotype analysis with the A/C polymorphism at position 1705 gave evidence for a single origin of this most frequent mutation in PKD as suggested by Baronciani and Beutler (Proc Natl Acad Sci USA 90:4324, 1993). Carrier detection and prenatal diagnosis are now feasible for the affected families.

Monitoring genomic alterations with a panel of oligonucleotide probes specific for various simple repeat motifs.

Germline and somatic instability of the human genome was studied, using synthetic oligonucleotides specific for simple repeat motifs. The following probes were used: (GTG)5, (GACA)4, (GATA)4, (CT)8, (TTAGGG)3, (GT)8, (GAA)6 and (GGAT)4. Each of them is unique with respect to the target regions recognized in the genome. Thus compilation of the various fingerprint data provides a complex map of the genome (and its deviations). While the fingerprints of differentiated somatic tissues never showed any alterations, in tumor tissues (namely gliomas) many changes could be detected. Most of the latter reflect secondary karyological aberrations. In nearly one third of the gliomas, drastically amplified and apparently monomorphic DNA fragments were identified. This marker should make it possible to deal with causal pathogenetic mechanisms as well as novel diagnostic strategies.