Molecular analysis of hemophilia A mutations in the Finnish population

We have examined the Finnish hemophilia A population for factor VIII gene mutations. This study included 83 unrelated patients and revealed 10 mutations associated with hemophilia. Using cloned cDNA, genomic, and oligonucleotide probes, we have identified three classes of mutations: five mutations causing the loss of TaqI restriction sites, a point mutation resulting in a new TaqI site, and four partial gene deletions. Although exons 5 and 6 were involved in three of the four partial gene deletions, the extent of the DNA lost differs in each case. The fourth deletion was located entirely within intron 1 and segregated with the disease in a large hemophilia pedigree. There was no history of hemophilia in eight of the 10 families. The origin of the mutation was determined in six of these pedigrees, two of which showed evidence for maternal mosaicism.     

Deficient mitochondrial Ca(2+) buffering in the Cln8(mnd) mouse model of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) are a group of genetic childhood-onset progressive brain diseases characterized by a decline in mental and motor capacities, epilepsy, visual loss and premature death. Using patch clamp, fluorescence imaging and caged Ca²⁺ photolysis, we evaluated the mechanisms of neuronal Ca²⁺ clearance in Cln8^mnd mice, a model of the human NCL caused by mutations in the CLN8 gene. In Cln8^mnd hippocampal slices, Ca²⁺ clearance efficiency in interneurons and, to some extent, principal neurons declined with age. In cultured Cln8^mnd hippocampal neurons, clearance of large Ca²⁺ loads was inefficient due to impaired mitochondrial Ca²⁺ uptake. In contrast, neither Ca²⁺ uptake by sarco/endoplasmic reticulum Ca²⁺ ATPase, nor Ca²⁺ extrusion through plasma membrane was affected by the Cln8 mutation. Excitotoxic glutamate challenge caused Ca²⁺ deregulation more readily in Cln8^mnd than in wt neurons. We propose that neurodegeneration in human CLN8 disorders is primarily caused by reduced mitochondrial Ca²⁺ buffering capacity.     

PME of Unverricht-Lundborg type in the Mediterranean region: linkage and linkage disequilibrium confirm the assignment to the EPM1 locus

Seven phenotypically homogeneous Mediterranean myoclonus families were studied using DNA markers from the genetically defined EPM1 region on chromosome 21. No recombinations between the disease phenotype and the markers studied were detected. Within the EPM1 region, the highest lod score value of 5.07 (at = 0.00) was reached at locus PFKL. Significant allelic association (P = 0.02) between the disease mutation and PFKL was detected suggesting a founder effect in Mediterranean myoclonus. However, haplotype data using four marker loci residing within 300kb of each other and of EPM1 suggest the occurrence of more than one mutation. The data are compatible with Mediterranean myoclonus being caused by mutations in the EPM1 gene and strengthen the concept that a large subset of progressive myoclonus epilepsies conforms with Unverricht-Lundborg disease and that this subset is an etiologically homogeneous entity.     

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.     

Delineation of Cohen syndrome following a large-scale genotype-phenotype screen

Cohen syndrome is an autosomal recessive condition associated with developmental delay, facial dysmorphism, pigmentary retinopathy, and neutropenia. The pleiotropic phenotype, combined with insufficient clinical data, often leads to an erroneous diagnosis and has led to confusion in the literature. Here, we report the results of a comprehensive genotype-phenotype study on the largest cohort of patients with Cohen syndrome assembled to date. We found 22 different COH1 mutations, of which 19 are novel, in probands identified by our diagnostic criteria. In addition, we identified another three novel mutations in patients with incomplete clinical data. By contrast, no COH1 mutations were found in patients with a provisional diagnosis of Cohen syndrome who did not fulfill the diagnostic criteria ("Cohen-like" syndrome). This study provides a molecular confirmation of the clinical phenotype associated with Cohen syndrome and provides a basis for laboratory screening that will be valuable in its diagnosis.     

Mass spectrometric analysis reveals changes in phospholipid, neutral sphingolipid and sulfatide molecular species in progressive epilepsy with mental retardation, EPMR, brain: a case study

Progressive epilepsy with mental retardation, EPMR, belongs to a group of inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The CLN8 gene that underlies EPMR encodes a novel transmembrane protein that localizes to the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment. Recently, CLN8 was linked to a large eukaryotic protein family of TLC (TRAM, Lag1, CLN8) domain homologues with postulated functions in lipid synthesis, transport or sensing. By using liquid chromatography/mass spectrometry we analysed molecular species of major phosholipid and simple sphingolipid classes from cerebral samples of two EPMR patients representing a progressive and advanced state of the disease. The progressive state brain showed reduced levels of ceramide, galactosyl- and lactosylceramide and sulfatide as well as a decrease in long fatty acyl chain containing molecular species within these classes. Among glycerophospholipid classes, an increase in species containing polyunsaturated acyl chains was detected especially in phosphatidylserines and phosphatidylethanolamines. By contrast, saturated and monounsaturated species were overrepresented among phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol classes in the advanced state sample. The observed changes in brain sphingo- and phospholipid molecular profiles may result in altered membrane stability, lipid peroxidation, vesicular trafficking or neurotransmission and thus may contribute to the progression of the molecular pathogenesis of EPMR.     

Assignment of the mulibrey nanism gene to 17q by linkage and linkage-disequilibrium analysis.

Mulibrey nanism (MUL) is an autosomal recessive disorder with unknown basic metabolic defect. It is characterized by growth failure of prenatal onset, characteristic dysmorphic features, constrictive pericardium, hepatomegaly as a consequence of constrictive pericardium, yellowish dots in the ocular fundi, and J-shaped sella turcica. Hypoplasia of various endocrine glands, causing hormone deficiencies, is common. Here we report the assignment of the MUL gene, by linkage analysis in Finnish families, to a 7-cM region flanked by D17S1799 and D17S948 on chromosome 17q. Multipoint linkage analysis gave a maximum LOD score of 5.01 at loci D17S1606-D17S1853 and at D17S1604. The estimate of the critical MUL region was further narrowed to within approximately 250 kb of marker D17S1853 by linkage disequilibrium analysis. Positional candidate genes that belong to the growth hormone and homeobox B gene clusters were excluded. These data confirm the autosomal recessive inheritance of MUL and allow highly focused attempts to clone the gene.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.