A variant of uncertain significance in SDHAF1, the succinate dehydrogenase chaperone protein, in an adult patient with spastic paraparesis and leukoencephalopathy.

Succinate dehydrogenase (SDH), or respiratory complex II, consists of four nuclear-encoded subunits. The chaperone protein succinate dehydrogenase assembly factor 1 (SDHAF1) plays an essential role in the assembly of SDH, and in the incorporation of iron-sulfur clusters into the SDHB subunit. SDHB couples the oxidation of succinate to fumarate with the reduction of ubiquinone (coenzyme Q) to ubiquinol. Previously reported mutations in SDHAF1 have been associated with infantile leukoencephalopathy. We report an adult case with a homozygous variant of uncertain significance (VUS) mutation in SDHAF1, presenting with dementia, spastic paraparesis, and cardiomyopathy, initially diagnosed as multiple sclerosis.

Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis.

Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.

Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region beta-chain T-cell repertoire and mediates an HLA-DR-restricted immune response.

Inherited susceptibility to rheumatoid arthritis is associated with genes encoding the human major histocompatibility complex class II molecule HLA-DR4. To study the immune function of HLA-DR4 and attempt to generate a murine model of rheumatoid arthritis we have produced triple transgenic mice expressing HLA-DRA*0101, -DRB1*0401, and human CD4. The expression of the HLA transgenes is driven by the promoter of the murine major histocompatibility complex class II I-E alpha gene and was found on murine cells that normally display major histocompatibility complex class II molecules. The expression of the human CD4 transgene is driven by the murine CD3 delta-promoter, and therefore its gene product was found on cells that express murine CD3. In contrast to other HLA-DR and HLA-DQ transgenic mouse lines, the transgenes are functional in our mice. In H-2 I-E-negative transgenic mice, T cells expressing variable region beta chain (V beta) 3, 5, 6, 7, 9, 11, 12, or 13 were either absent or significantly reduced, in contrast to H-2 I-E-negative nontransgenic littermates. In addition, the peptide antigen influenza A virus hemagglutinin 307-319, which binds to the HLA-DRA*0101/-DRB1*0401 heterodimer with high affinity and induces an HLA-DR-restricted and CD4+ T-cell response in humans, also induced a T-cell response in the triple transgenic mice but not in nontransgenic littermates. Thus, these transgenic mice should permit extensive testing of the antigen-presentation capabilities of the HLA-DRA*0101/-DRB1*0401 molecule.

Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: implications in induction of autoimmunity.

The precise mechanisms of failure of immunological tolerance to self proteins are not known. Major histocompatibility complex (MHC) susceptibility alleles, the target peptides, and T cells with anti-self reactivity must be present to cause autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a murine model of a human autoimmune disease, multiple sclerosis. In EAE, residues 1-11 of myelin basic protein (MBP) are the dominant disease-inducing determinants in PL/J and (PL/J x SJL/J)F1 mice. Here we report that a six-residue peptide (five of them native) of MBP can induce EAE. Using peptide analogues of the MBP-(1-11) peptide, we demonstrate that only four native MBP residues are required to stimulate MBP-specific T cells. Therefore, this study demonstrates lower minimum structural requirements for effective antigen presentation by MHC class II molecules. Many viral and bacterial proteins share short runs of amino acid similarity with host self proteins, a phenomenon known as molecular mimicry. Since a six-residue peptide can sensitize MBP-specific T cells to cause EAE, these results define a minimum sequence identity for molecular mimicry in autoimmunity.

Genetic factors in sleep disorders.

Several sleep disorders have a genetic basis. These conditions include the narcoleptic syndrome, sleep walking, periodic movements in sleep, circadian delay syndromes and familial insomnia. These disorders illustrate different control mechanisms involved in sleep and wakefulness, including those determining the prevalence and timing of NREM and REM activity, somatomotor inhibition and excitation, autonomic discharge, and the circadian framework of sleep. The genetic defect in narcolepsy has been localised to the short arm of chromosome 6, but the chromosomal localisations of the genetic basis for the other disorders are not known. Also, with the possible exception of acetylcholine, no definite neurotransmitter involved in any aspect of sleep regulation has been positively identified and the biochemical defect in narcolepsy is not known.

Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients.

We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation.

MHC class II sequences of an HLA-DR2 narcoleptic.

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 beta chains, along with DQw1 alpha and beta chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3' untranslated region of one of the DR2 beta genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 alpha or beta from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ alpha. Partial protein sequences of both DQ alpha and beta from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR beta or DQ alpha/beta sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.

Selegiline in narcolepsy.

We examined the effect of the specific monoamine oxidase-B (MAO-B) inhibitor selegiline (deprenyl, Eldepryl), 20-30 mg p.o. daily, in 21 subjects with the narcoleptic syndrome for 4 weeks. Selegiline was compared to no treatment (7 subjects) or conventional central stimulant drugs, including dexamphetamine or mazindol (14 subjects). Severity and frequency of narcolepsy, accessory symptoms, and effects of selegiline on mood were measured. Selegiline, as well as causing MAO-B inhibition, is interconverted to amphetamine. Urinary amphetamine and methamphetamine excretion were determined in 18 subjects after 4 weeks on selegiline and the results were compared with amphetamine excretion in subjects on dexamphetamine. The effect of selegiline, 20-30 mg p.o., on alertness and mood was similar to that of dexamphetamine in the same dosage, with comparable sympathomimetic side effects. Selegiline, 20 mg p.o., caused a subjective increase in alertness for 4-8 h. Mean urinary amphetamine excretion on dexamphetamine, 15-70 mg daily (mean 29 mg) at pH 5.6-6.6, was 5,184 micrograms/24 h, and on selegiline, 20-30 mg daily (mean 22.5), was 4,127 micrograms/24 h. We conclude that selegiline, 20-30 mg daily, requires further evaluation in narcolepsy.