H. Smeets

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.

Use of variable simple sequence motifs as genetic markers: application to study of myotonic dystrophy

SummaryAmong the many classes of repetitive elements present in the human genome, the ubiquitous “simple sequence motifs” (SSMs) composed of [A]n, [TG]n, [AG]n or codon-tandem repeats form a major source of genetic variation. Here we report a detailed molecular-genetic study of a “variable simple sequence motif” (VSSM) in the apolipoprotein C2 (apoC2) gene, which maps to the 19q13.2 region in the vicinity of the myotonic dystrophy (DM) locus. By combining in vitro DNA-amplification using the polymerase chain reaction and high-resolution gel electrophoresis, we could demonstrate a high degree of allelic variation with at least ten alleles, which differ in the number of repeated [TG] or [AG] dinucleotide units. Similar results were found for the somatostatin I gene locus. To evaluate the usefulness of SSM-length polymorphisms as genetic markers, the apoC2-VSSM was employed for linkage analysis in DM families. Our results establish that the orientation of the apolipoprotein gene cluster on 19q is cenapoE-apoC2-ter and indicate that the many thousands of structurally similar VSSMs in the human genome represent a rich source of highly informative genetic and diagnostic markers.

Presymptomatic diagnosis of myotonic dystrophy.

The discovery of an expanded (CTG)n repeat sequence in myotonic dystrophy (DM) has greatly improved our ability to detect DM gene carriers who have few or none of the classical signs of this disorder. We report here our experience with two such groups of gene carriers. We used a PCR based protocol that should be especially sensitive to small increases in CTG triplet number which might escape detection by conventional Southern blot analysis. Our analyses show that on 100 non-DM chromosomes the number of CTG triplets ranged from five to 37. We then studied 17 obligate gene carriers aged 55 years and over who showed no muscle weakness. All of the gene carriers in this group showed a relatively small increase in the number of CTG triplets (52 to 90 CTG triplets) with limited somatic mosaicism. We subsequently studied 11 subjects (aged 19 to 36 years) who had previously been identified as gene carriers by genetic linkage studies, but who lacked diagnostic signs. In this prospectively studied group, nine subjects showed an expanded allele, confirming the earlier prediction from linked genetic markers. The other two subjects had only two normal alleles and no expanded allele. Revision of the clinical data casts doubt on the original diagnosis of DM in their families. Preferential amplification of the normal non-expanded allele was noted in three asymptomatic gene carriers in this study (as well as in two of their clinically affected relatives). We caution that, at least in our hands, the DM mutation can be confidently excluded by this PCR based method only if both normal alleles have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical Activity Is the Key Determinant of Skeletal Muscle Mitochondrial Function in Type 2 Diabetes

CONTEXT Conflicting data exist on mitochondrial function and physical activity in type 2 diabetes mellitus (T2DM) development. OBJECTIVE The aim was to assess mitochondrial function at different stages during T2DM development in combination with physical exercise in longstanding T2DM patients. DESIGN AND METHODS We performed cross-sectional analysis of skeletal muscle from 12 prediabetic 11 longstanding T2DM male subjects and 12 male controls matched by age and body mass index. INTERVENTION One-year intrasubject controlled supervised exercise training intervention was done in longstanding T2DM patients. MAIN OUTCOME MEASUREMENTS Extensive ex vivo analyses of mitochondrial quality, quantity, and function were collected and combined with global gene expression analysis and in vivo ATP production capacity after 1 yr of training. RESULTS Mitochondrial density, complex I activity, and the expression of Krebs cycle and oxidative phosphorylation system-related genes were lower in longstanding T2DM subjects but not in prediabetic subjects compared with controls. This indicated a reduced capacity to generate ATP in longstanding T2DM patients only. Gene expression analysis in prediabetic subjects suggested a switch from carbohydrate toward lipid as an energy source. One year of exercise training raised in vivo skeletal muscle ATP production capacity by 21 ± 2% with an increased trend in mitochondrial density and complex I activity. In addition, expression levels of β-oxidation, Krebs cycle, and oxidative phosphorylation system-related genes were higher after exercise training. CONCLUSIONS Mitochondrial dysfunction is apparent only in inactive longstanding T2DM patients, which suggests that mitochondrial function and insulin resistance do not depend on each other. Prolonged exercise training can, at least partly, reverse the mitochondrial impairments associated with the longstanding diabetic state.

Myotonic dystrophy is closely linked to the gene for muscle-type creatine kinase (CKMM)

SummaryWe have studied genetic linkage between the gene for creatine kinase muscle type (CKMM) and the gene for myotonic dystrophy (DM). In a panel of 65 myotonic dystrophy families from Canada and the Netherlands, a maximum lod score (Zmax) of 22.8 at a recombination frequency (Θ) of 0.03 was obtained. Tight linkage was also demonstrated for CKMM and the gene for apolipoprotein C2 (ApoC2). This establishes CKMM as a useful marker for myotonic dystrophy.

A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19

Employing 16 polymorphic DNA markers as well as the chromosome 19 centromere heteromorphism, we have performed a genetic linkage study in 26 families with myotonic dystrophy. Fourteen of these markers had been assigned previously to one of five different intervals of the 19cen-19q13.2 segment by using somatic cell hybrids. For the long arm of chromosome 19, a genetic map that encompasses 9 polymorphic markers and the DM gene has been constructed. Our studies indicate that the DM and CKMM genes map distal to the ApoC2-ApoE gene cluster and to the anonymous polymorphic markers D19S15 and D19S16, but proximal to the D19S22 marker. The orientation of DM and CKMM remains to be determined.

A mitochondrial tRNAVal 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 tRNAVal 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 tRNAVal mutation is associated with the MELAS syndrome.

A PCR test for progressive external ophthalmoplegia and Kearns-Sayre syndrome on DNA from blood samples

Progressive external ophthalmoplegia (PEO) and Kearns-Sayre syndrome (KSS) are caused by deletions in mitochondrial DNA. Identification of these deletions is important for diagnosis, prognosis and genetic counselling. As yet, the most frequently used test is Southern blot analysis of DNA isolated from a muscle biopsy. Here, we describe a sensitive PCR-based test for the identification of these deletions in DNA isolated from blood. The main advantage is that in the majority of cases a muscle biopsy is no longer necessary for the molecular diagnosis of PEO and KSS.

Juvenile-onset Alpers syndrome: interpreting MRI findings

A previously healthy 20-year-old woman was admitted to the department of neurology of our hospital after 2 generalized tonic-clonic seizures in the preceding 2 weeks. Since the first seizure, she had been seeing bright spots. Neurologic examination at admission was normal. EEG showed slowed background activity and continuous epileptic activity in the left occipital lobe. MRI revealed asymmetric but bilateral areas of increased signal intensity in the occipital cortex, most prominent in the left hemisphere (figure, A). During the following weeks, symptoms expanded, as she developed epilepsia partialis continua of her right arm and leg. Repeat brain MRI showed increase and extension of the occipital lesions, now including hyperintensity of the pulvinar region in the left thalamus (figure, B). We considered a diagnosis of juvenile-onset Alpers syndrome, mitochondrial encephalopathy with lactate acidosis and stroke-like episodes, cerebral vasculitis, or Rasmussen encephalitis. Urgent DNA sequencing of all protein encoding exons and at least 40 nucleotides of the flanking introns of the POLG1 gene, the gene encoding the mitochondrial DNA (mtDNA) polymerase γ, confirmed a homozygous c.1399G>A mutation (p.A467T) as the only mutation or variant present in this gene. The family history was negative for disorders possibly related to POLG1 mutations. Despite treatment with several combinations of antiepileptic drugs, avoiding valproic acid, she developed a generalized status epilepticus and was admitted to the ICU. Eight weeks after the initial symptoms, she died of refractory status epilepticus …

OP45 – 3024: Efficient lentiviral vector-mediated hematopoietic stem cell gene therapy in MNGIE mice

Objective Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase (TP) resulting in systemic thymidine (Thd) and deoxyuridine (dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogeneic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Application of lentiviral vector therapy. Methods Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp-/-Upp-/- double knockout mice, a model for MNGIE disease, was investigated. Results At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco=96±4 nmoles/h/mg), compared to a 400-fold increase observed in SFFV recipient mice (450±5) and consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months post treatment) showed on average 1.2-fold TP wild type activity levels in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice, sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). Conclusion Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.