A novel VWF variant associated with type 2 von Willebrand disease in German Wirehaired Pointers and German Shorthaired Pointers.

Von Willebrand disease (VWD), caused by deficiency of the von Willebrand factor (VWF), is the most common bleeding disorder in humans and dogs. The complete cDNA encoding VWF of a German Wirehaired Pointer with type 2 VWD was sequenced, and we found four variants that alter the amino acid sequence. These variants were: c.1657T>G corresponding to p.Trp553Gly; c.1777G>A (p.Glu593Lys); c.4937A>G (p.Asn1646Ser) and c.5544G>A (p.Met1848Ile). A haplotype of the c.1657G, c.1777A and c.4937G alleles co-segregated with the VWF antigen level in a four-generation pedigree with the disease. Healthy dogs of the breed were found that were homozygous for the c.1777A or the c.5544A allele, indicating that these variants do not cause VWD. Dogs that were homozygous for the c.4937G allele and had no signs of a bleeding disorder were observed in the Chinese Crested dog breed. Thus, only the c.1657G variant was found in the homozygous state exclusively in VWD affecteds, and this variant is the strongest candidate to be the cause of VWD type 2 in the German Wirehaired Pointer breed. A screen of German Shorthaired Pointers indicated that the variant also segregates with VWD in this breed.

Mutations in the vasopressin type 2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (DIR) is an X-linked disorder characterized by insensitivity of the distal nephron for the pituitary hormone, vasopressin. The genetic map location of the DIR gene on chromosome Xq28 coincides with the physical map location of the functional vasopressin renal V2-type receptor. Recently, the human and rat cDNAs for the vasopressin V2 receptor (AVPR2) have been identified. We show here that the structural AVPR2 gene is localized between DXS52 and G6PD, which is within the genetic map location of DIR. We also tested eight X-linked DIR probands and their families for mutations in one of the most conserved extracellular regions of AVPR2: in three of them, we have identified point mutations resulting in non-conservative amino acid substitutions which cosegregated with DIR in all families.

Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A.

Genetic and metabolic studies have been done on a large kindred in which several males are affected by a syndrome of borderline mental retardation and abnormal behavior. The types of behavior that occurred include impulsive aggression, arson, attempted rape, and exhibitionism. Analysis of 24-hour urine samples indicated markedly disturbed monoamine metabolism. This syndrome was associated with a complete and selective deficiency of enzymatic activity of monoamine oxidase A (MAOA). In each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a termination codon. Thus, isolated complete MAOA deficiency in this family is associated with a recognizable behavioral phenotype that includes disturbed regulation of impulsive aggression.

Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.

Concentration of urine in mammals is regulated by the antidiuretic hormone vasopressin. Binding of vasopressin to its V2 receptor leads to the insertion of water channels in apical membranes of principal cells in collecting ducts. In nephrogenic diabetes insipidus (NDI), the kidney fails to concentrate urine in response to vasopressin. A male patient with an autosomal recessive form of NDI was found to be a compound heterozygote for two mutations in the gene encoding aquaporin-2, a water channel. Functional expression studies in Xenopus oocytes revealed that each mutation resulted in nonfunctional water channel proteins. Thus, aquaporin-2 is essential for vasopressin-dependent concentration of urine.

Benign familial hematuria due to mutation of the type IV collagen alpha4 gene.

Benign familial hematuria (BFH) is characterized by autosomal dominant inheritance, thinning of the glomerular basement membrane (GBM) and normal renal function. It is frequent in patients with persistent microscopic hematuria, but cannot be clinically differentiated from the initial stages of Alport syndrome, a severe GBM disorder which progresses to renal failure. We present here linkage of benign familial hematuria with the COL4A3 and COL4A4 genes at 2q35-37 (Zmax = 3.58 at theta = 0.0). Subsequently, a glycine to glutamic acid substitution was identified in the collagenous region of the COL4A4 gene. We conclude that type IV collagen defects cause both benign hematuria and Alport syndrome. Furthermore, our data suggest that BFH patients can be carriers of autosomal recessive Alport syndrome.

High- and low-uptake forms of beta-hexosaminidase in human platelets. Selective retention of the high-uptake form during stimulation with thrombin.

Human platelets are rich in beta-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from alpha-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37 degrees C) induces the secretion of 100% of the contents of alpha- and dense granules, but only 40-60% of total beta-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellularly. There is no selective recapture or plasma membrane binding by platelets of secreted beta-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH4Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all beta-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.

Influence of heat treatment on rabbit liver ferritin.

Ferritin was purified from rabbit livers either by heat treatment and immunoaffinity chromatography, or by immunoaffinity chromatography alone. The immunoreactivity of ferritin with antibodies raised against heat-treated ferritin was significantly higher for heat-treated preparations than for non-heated preparations. The amount of ferritin protein could be estimated with equal reliability by the assay according to Lowry et al. and by nitrogen determination. Heat treatment favoured the L-subunit-rich ferritin fraction, as measured by densitometric scanning of SDS gradient-pore polyacrylamide gels. Amino acid analysis showed small changes in the amounts of valine, isoleucine and histidine in the heat-treated ferritin, possibly due to selective partial degradation of H-subunit-rich forms of ferritin. These results illustrate that heat treatment, which is a commonly used step in most purification procedures, induces partial denaturation of the ferritin molecules.

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

Non-pruritic granuloma in Norwegian forest cats.

The eosinophilic granuloma complex is a group of skin disorders common in cats. This paper describes the clinical, haematological and histopathological features of 17 related Norwegian forest cats, six of which had a linear granuloma on the caudal thigh, three of which also had a granuloma on the lower lip, and one of which had a granuloma in combination with an indolent ulcer. The high prevalence of the disease in this population is suggestive of a genetic background.

Autosomal recessive nephrogenic diabetes insipidus caused by an aquaporin-2 mutation.

Vasopressin V2 receptors, expressed from an x-chromosomal gene, are involved in antidiuresis, but also in release of coagulation factor VIII and von Willebrand factor (vWF). The present study describes autosomal recessive nephrogenic diabetes insipidus (NDI) in a large cluster of patients in Israel's Lower-Galilee. Evidence for an intact V2 receptor was concluded by their normal increase in factor VIII and vWF after desmopressin infusion. Thus, in these patients a defect in the pathway beyond the V2 receptor was suspected. The recent cloning of the human Aquaporin-2 gene enabled us to test this gene as a candidate for such a postreceptor defect. Direct sequencing of the Aquaporin-2 gene revealed a G298T substitution causing a Gly100Stop nonsense mutation in the third transmembrane region. Because this putative disease-causing mutation was identified in index patients of different families, we suggest that all patients are descendants of a common ancestor. Thus, this new entity is characterized by an autosomal recessive NDI. The differential response of clotting factors and urine osmolality to desmopressin may provide a simple tool for clinical diagnosis of a V2-postreceptor defect. The early stop-codon of Aquaporin-2 results in complete resistance to vasopressin antidiuretic effect.

Mutation detection in the aspartoacylase gene in 17 patients with Canavan disease: four new mutations in the non-Jewish population.

Canavan disease is a severe progressive autosomal recessive disorder, which is characterised by spongy degeneration of the brain. The disease is caused by mutations in the aspartoacylase gene. Two different mutations were reported on 98% of the alleles of Ashkenazi Jewish patients, in which population the disease is highly prevalent. In non-Jewish patients of European origin, one mutation (914C > A) is found in 50% of the alleles, the other alleles representing all kinds of different mutations. We here describe the results of the mutation analysis in 17 European, non-Jewish patients. Ten different mutations were found, of which four had not been described before (H21P, A57T, R168H, P181T). A deletion of exon4, which until now had only been described once, was revealed in all five alleles of Turkish origin tested, indicating that this is a founder effect in the Turkish population.

Clinical symptoms of adult metachromatic leukodystrophy and arylsulfatase A pseudodeficiency.

OBJECTIVE: To determine the clinical symptoms in adult metachromatic leukodystrophy and in adult pseudodeficiency for arylsulfatase A. DESIGN: Case series. SETTING: University hospital. PATIENTS: Twenty-five adult patients with very low arylsulfatase A activity. RESULTS: In 13 patients, a diagnosis of adult metachromatic leukodystrophy was made. The main symptoms were dementia, behavioral abnormalities, ataxia, and polyneuropathy. In 12 patients, a diagnosis of arylsulfatase A pseudodeficiency was made. No characteristic clinical syndrome could be detected in these patients. CONCLUSIONS: Adult metachromatic leukodystrophy is a progressive metabolic disease with symptoms of demyelination of the central and peripheral nervous systems. Diagnosis must be confirmed by determination of arylsulfatase A activity and accumulation of sulfatides. Pseudodeficiency for arylsulfatase A can be confirmed or excluded by means of DNA analysis.

Duplication of the proteolipid protein gene is the major cause of Pelizaeus-Merzbacher disease.

BACKGROUND/OBJECTIVE: Pelizaeus-Merzbacher disease (PMD), an X-linked recessive dysmyelination disorder, is caused by mutations in the proteolipid protein (PLP) gene. However, missense mutations were only found in a fraction of PMD patients, even in families that showed linkage with the PLP locus on Xq22. Here we describe the use of an extended protocol that includes screening for both missense mutations and duplications. METHOD: Two groups of patients were analyzed, one group with 10 independent PMD families and one group with 24 sporadic patients suspected of PMD. Missense mutations in the PLP gene were identified by sequencing. PLP gene duplications were detected by quantitative polymerase chain reaction and/or Southern blot analysis. RESULTS: Sequencing of the PLP gene revealed four mutations in group 1 and one mutation in group 2. However, inclusion of duplication analysis in the screening protocol raised the amount of mutations found in group 1 from 40 to 90%, and in group 2 from 4 to 25%. CONCLUSIONS: These results demonstrate that duplications of the PLP gene are the major cause of PMD. Furthermore, it appears that the phenotype resulting from PLP duplications is relatively mild, and that many probands are nontypical PMD patients.

A mitochondrial tRNA(Val) gene mutation (G1642A) in a patient with mitochondrial myopathy, lactic acidosis, and stroke-like episodes.

We studied a patient with the diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) for mitochondrial DNA mutations in muscle. Established MELAS mutations were excluded. Mitochondrial DNA was further analyzed for mutations in the 22 tRNA genes by single-strand conformation polymorphism (SSCP) analysis; a tRNA(Val) mutation (G1642A) was found. The structure of the altered tRNA, the heteroplasmy, and the absence of the mutation in the mother and in 100 control subjects suggests that the tRNA(Val) mutation is associated with the MELAS syndrome.

Myopathology and a mitochondrial DNA deletion in the Pearson marrow and pancreas syndrome.

A patient with the Pearson marrow and pancreas syndrome is presented. She showed an anaemia with neutropenia and thrombopenia, failure to thrive, diarrhoea, disturbed glucose homeostasis and lactic acidosis. An exocrine pancreatic insufficiency was lacking. The disease followed a fatal course. Biochemical investigations of skeletal muscle revealed a disturbed mitochondrial energy metabolism, while many ultrastructural abnormal features were observed in the muscle tissue. Molecular genetic studies showed a de novo deletion in the mitochondrial DNA (mtDNA), different in size from the already published deletions and flanked by two 4 bp direct repeats, interspaced by 4-5 non-repeated nucleotides. mtDNA from 12 other tissues showed the same deletion in different percentages. No obvious relation between these percentages and tissue dysfunction was found. In spite of an open reading frame of 74 codons, only little transcription product of the genomic region resulting from the deletion was found.

The gene for X-linked myotubular myopathy is located in an 8 Mb region at the border of Xq27.3 and Xq28.

X-linked recessive myotubular myopathy (XLMTM) is a rare and severe neonatal neuromuscular disease characterized by muscle weakness, hypotonia, and respiratory problems. Here we report an extensive linkage analysis in two families with XLMTM. Using 18 markers in the Xq27-Xqter region we found a maximum two-point lod score of Z = 4.00 at theta = 0.00 for the marker II-10 (DXS466). Three recombinations were detected between markers and the disease locus. At the distal side of Xq27.3 a recombination was present in between RNI (DXS369) and VK23b (DXS297), another in between VK23b (DXS297) and II-10 (DXS466), and at the proximal side of Xq28 a recombination in between U6.2 (DXS304) and Cpx67 (DXS134). Combining the results of both families we conclude that XLMTM is located in the 8 Mb(11 cM) region between VK23b (DXS297) and Cpx67 (DXS134).

Increased urinary beta-thromboglobulin excretion in diabetes assayed with a modified RIA kit-technique.

A modification of the commercial radioimmunoassay (RIA) kit adapting to the assay of beta-thromboglobulin (beta-TG) in the urine, is described. The urine was concentrated by dialysis against dry Sepharose G 200. 131I-albumin was used as marker. Experiments in which 125I-beta-TG and 131I-albumin were added simultaneously showed a parallel recovery of both markers in the final sample. The sensitivity of the RIA was enhanced by incubation overnight of the urine sample with the antiserum. Increased urinary beta TG values were found in 20 patients with diabetes mellitus as compared to 20 young healthy normals. Increased urinary beta-TG values correlated with decreased creatinine clearance, but the difference between diabetics and normals was still observed when patients with a creatinine clearance of less than 100 ml/min were excluded. The urine beta TG level was correlated with the presence of neovascularization within the diabetic group.

Determination of the density distribution of human platelets--methodological aspects and comparison with other tests for platelet activation.

Stimulation of human platelets to release results in decreased buoyant density. This decreased density provides a tool to detect circulating platelets which have participated in a thrombotic process. Platelet density gradient centrifugation using Stractan was standardized and the effects of anticoagulation, temperature, and osmolarity were investigated. In 7 out of 32 patients with thrombotic disease less dense platelets were found. Platelet activation in the patient group was also indicated by spontaneous aggregation (10/32), decreased circulating platelet aggregate ratios (5/24) and elevated plasma beta-thromboglobulin levels (2/ 11). Several of these tests were also abnormal in diabetes mellitus thrombocytosis, leukaemia and several systemic diseases with thrombotic complications. The platelet density test using Stractan is reproducible and independent of other tests for platelet activation and is therefore potentially a useful extension of platelet function testing in patients with thrombotic disease.

Tests for platelet changes, acute phase reactants and serum lipids in diabetes mellitus and peripheral vascular disease.

Platelets tests, acute phase reactants and serum lipids were measured in patients with diabetes mellitus and patients with peripheral vascular disease. Patients frequently had abnormal platelet tests and significantly increased acute phase reactants and serum lipids, compared to young healthy control subjects. These differences were compared with multidiscriminant analysis. Patients could be separated in part from the control subjects with variables derived from the measurement of acute phase proteins and serum lipids. Platelet test results improved the separation between diabetics and control subjects, but not between patients with peripheral vascular disease and control subjects. Diabetic patients with severe retinopathy frequently had evidence of platelet activation. They also had increased acute phase reactants and serum lipids compared to diabetics with absent or non-proliferative retinopathy. In patients with peripheral vascular disease, only the fibrinogen concentration was related to the degree of vessel damage by arteriography.

Exclusion of the lim homeodomain gene LHX4 as a candidate gene for pituitary dwarfism in German shepherd dogs.

Pituitary dwarfism in the German shepherd dog is an autosomal recessive inherited abnormality. We tested the hypothesis that a variant of the LIM homeodomain gene LHX4 is responsible for the dwarfism phenotype. To this end, we isolated Bacterial Artificial Chromosome clones for the canine LHX4 gene. Southern blotting experiments showed that the LHX4 gene is a single copy gene in the canine genome. A complex CA-repeat was isolated from the BAC clones and was found to be polymorphic in German shepherd dogs. Genotyping 5 litters in which the dwarfism was segregating showed disconcordance between the inheritance of the dwarfism phenotype and the DNA marker. It is concluded that the LHX4 gene does not play a primary role in the pituitary dwarfism in the German shepherd dogs.