The CLN6 gene that causes variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a recessively inherited neurodegenerative disease that features blindness, seizures, and cognitive decline, maps to 15q21-23. We have used multiallele markers spanning this approximately 4-Mb candidate interval to reveal a core haplotype, shared in Costa Rican families with vLINCL but not in a Venezuelan kindred, that highlighted a region likely to contain the CLN6 defect. Systematic comparison of genes from the minimal region uncovered a novel candidate, FLJ20561, that exhibited DNA sequence changes specific to the different disease chromosomes: a G-->T transversion in exon 3, introducing a stop codon on the Costa Rican haplotype, and a codon deletion in exon 5, eliminating a conserved tyrosine residue on the Venezuelan chromosome. Furthermore, sequencing of the murine homologue in the nclf mouse, which manifests recessive NCL-like disease, disclosed a third lesion-an extra base pair in exon 4, producing a frameshift truncation on the nclf chromosome. Thus, the novel approximately 36-kD CLN6-gene product augments an intriguing set of unrelated membrane-spanning proteins, whose deficiency causes NCL in mouse and man.
Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.
Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK ␣ and synergistically promote T cell maturation in the thymus. Absence of both DGK␣ and (DGK␣ ؊/؊ ؊/؊ ) results in a severe decrease in the number of CD4 ؉ CD8 ؊ and CD4 ؊ CD8 ؉ single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGK␣ ؊/؊ ؊/؊ mice. The developmental blockage in DGK␣ ؊/؊ ؊/؊ mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.phosphatidic acid ͉ signaling ͉ tumorigenesis D iacylglycerol (DAG) kinases (DGKs) are a family of enzymes that catalyze phosphorylation of DAG, converting it to phosphatidic acid (PA). Ten DGK isoforms have been identified in mammals and are divided into 5 subtypes based on unique structural features (1, 2). Within a single tissue, multiple DGK isoforms can be expressed. A notable feature of the DGK-mediated reaction is that both the substrate, DAG, and the product, PA, can be important second messengers. DAG can associate with Ras guanyl nucleotide-releasing proteins (RasGRPs), protein kinase Cs (PKCs), protein kinase Ds, chimaerins, and Munc-13s through their cysteine-rich (C1) domains (3). PA has been reported to bind to the SH-2 domain containing tyrosine phosphatase-1 (SHP-1), mammalian target of Rapamycin (mTOR), cAMP-specific phosphodiesterase 4 (PDE4), protein phosphatase-1 (PP-1), son of sevenless (Sos), and type I phosphatidylinositol 4-phosphate 5-kinase (PI5K) (4 -8). Through the regulation of the activities and subcellular localizations of these signaling molecules, DAG and PA play critical roles in signaling from many cell surface receptors (3, 4). It has been hypothesized that DGKs act as crucial regulators of receptor signaling and cellular function by modulating DAG and PA concentrations. However, the physiologic importance of most DGKs and the role of DGK-derived PA in receptor signaling have been poorly defined.T cell maturation in the thymus occurs through CD4 Ϫ CD8 Ϫ double-negative (DN) to CD4 ϩ CD8 ϩ double-positive (DP) and finally to the CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ single-positive (SP) stage (9). Engagement of TCR expressed on DP thymocytes with self-peptide presented by MHCs on thymic stromal cells and bone marrow-derived dendritic cells induces intracellular signaling that can lead to either maturation (positive selection) or cell death (negative selection). In general, TCRs with high affinity to self-antigens elicit strong signals directing negative selection, whereas TCRs with low affinity to self-antigens induce...
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