Two Novel Mutations in the SLC25A4 Gene in a Patient with Mitochondrial Myopathy.

In a 28-year-old male with a mild mitochondrial myopathy manifesting as exercise intolerance and early signs of cardiomyopathy without muscle weakness or ophthalmoplegia, we identified two novel mutations in the SLC25A4 gene: c.707G>C in exon 3 (p.(R236P)) and c.116_137del in exon 2 (p.(Q39Lfs*14)). Serum lactate levels at rest were elevated (12.7 mM). Both the patient's father and brother were heterozygous carriers of the c.707G>C mutation and were asymptomatic. The second mutation causes a 22 bp deletion leading to a frame shift likely giving rise to a premature stop codon and nonsense-mediated decay (NMD). The segregation of the mutations could not be tested directly as the mother had died before. However, indirect evidence from NMD experiments showed that the two mutations were situated on two different alleles in the patient. This case is unique compared to other previously reported patients with either progressive external ophthalmoplegia (PEO) or clear hypertrophic cardiomyopathy with exercise intolerance and/or muscle weakness carrying recessive mutations leading to a complete absence of the SLC25A4 protein. Most likely in our patient, although severely reduced, SLC25A4 is still partially present and functional.

Defective NDUFA9 as a novel cause of neonatally fatal complex I disease.

BACKGROUND: Mitochondrial disorders are associated with abnormalities of the oxidative phosphorylation (OXPHOS) system and cause significant morbidity and mortality in the population. The extensive clinical and genetic heterogeneity of these disorders due to a broad variety of mutations in several hundreds of candidate genes, encoded by either the mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), impedes a straightforward genetic diagnosis. A new disease gene is presented here, identified in a single Kurdish patient born from consanguineous parents with neonatally fatal Leigh syndrome and complex I deficiency. METHODS AND RESULTS: Using homozygosity mapping and subsequent positional candidate gene analysis, a total region of 255.8 Mb containing 136 possible mitochondrial genes was identified. A pathogenic mutation was found in the complex I subunit encoding the NDUFA9 gene, changing a highly conserved arginine at position 321 to proline. This is the first disease-causing mutation ever reported for NDUFA9. Complex I activity was restored in fibroblasts of the patient by lentiviral transduction with wild type but not mutant NDUFA9, confirming that the mutation causes the complex I deficiency and related disease. CONCLUSIONS: The data show that homozygosity mapping and candidate gene analysis remain an efficient way to detect mutations even in small consanguineous pedigrees with OXPHOS deficiency, especially when the enzyme deficiency in fibroblasts allows appropriate candidate gene selection and functional complementation.

Large scale mtDNA sequencing reveals sequence and functional conservation as major determinants of homoplasmic mtDNA variant distribution.

The mitochondrial DNA (mtDNA) is highly variable, containing large numbers of pathogenic mutations and neutral polymorphisms. The spectrum of homoplasmic mtDNA variation was characterized in 730 subjects and compared with known pathogenic sites. The frequency and distribution of variants in protein coding genes were inversely correlated with conservation at the amino acid level. Analysis of tRNA secondary structures indicated a preference of variants for the loops and some acceptor stem positions. This comprehensive overview of mtDNA variants distinguishes between regions and positions which are likely not critical, mainly conserved regions with pathogenic mutations and essential regions containing no mutations at all.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome.

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

The unfolding clinical spectrum of POLG mutations.

BACKGROUND: Mutations in the DNA polymerase-gamma (POLG) gene are a major cause of clinically heterogeneous mitochondrial diseases, associated with mtDNA depletion and multiple deletions. OBJECTIVE: To determine the spectrum of POLG mutations in our Dutch patient cohort, to evaluate the pathogenicity of novel mutations, and to establish genotype-phenotype correlations. RESULTS: The authors identified 64 predominantly recessive mutations in 37 patients from a total of 232 patients, consisting of 23 different mutations. The substitution p.A467T was most frequently observed (n = 23), but was as frequent in childhood cases as in adult cases. Five new pathogenic recessive mutations, p.Lys925ArgfsX42, p.R275X, p.G426S, p.A804T and p.R869Q were identified. The known dominant chronic progressive external ophthalmoplegia (CPEO) mutation p.R943H was for the first time associated with premature ovarian failure as well. In 19 patients the authors identified only a single recessive mutation, or a sequence variant with unclear clinical significance. The data substantiate earlier observations that in POLG patients a fatal status epilepticus and liver failure can be triggered by sodium valproate. It is therefore important to exclude POLG mutations before administering this treatment. CONCLUSION: The clinical features of the patient are the most important features to select putative POLG mutation carriers and not the presence of mtDNA deletions or OXPHOS (oxidative phosphorylation) activity. The authors conclude that POLG mutations are an important cause of heterogeneous mitochondrial pathology and that more accurate genotype-phenotype correlations allow a more rapid genetic diagnosis and improved prognosis for mutation carriers.

Altered myocardial gene expression reveals possible maladaptive processes in heterozygous and homozygous cardiac myosin-binding protein C knockout mice.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by left ventricular hypertrophy (LVH) predominantly affecting the interventricular septum. Cardiac myosin-binding protein C (cMyBP-C) mutations are common causes of FHC. Gene expression profiling was performed in left ventricles of 9-week-old wild-type mice, heterozygous cMyBP-C KO mice displaying asymmetric septal hypertrophy, and homozygous mice developing eccentric LVH. Knocking out one or two cMyBP-C genes leads primarily to gene expression changes indicating an increased energy demand, activation of the JNK and p38 parts of the MAPK pathway and deactivation of the ERK part, and induction of apoptosis. Altered gene expression for processes related to cardiac structure, contractile proteins, and protein turnover was also identified. Many of the changes were more pronounced in the homozygous KO mice. These alterations point to physiological and pathological adaptations in the prehypertrophic heterozygous KO mice and the hypertrophic homozygous mice.

Early and transient gene expression changes in pressure overload-induced cardiac hypertrophy in mice.

Cardiac hypertrophy is an important risk factor for cardiac morbidity and mortality. To unravel the underlying pathogenic genetic pathways, we hybridized left ventricular RNA from Transverse Aortic Constriction mice at 48 h, 1 week, and 2, 3, and 8 weeks after surgery to microarrays containing a 15K fetal cDNA collection. Key processes involved an early restriction in the expression of metabolic genes, accompanied by increased expression of genes related to growth and reactivation of fetal genes. Most of these genes returned to basal expression levels during the later, compensated hypertrophic phase. Our findings suggest that compensated hypertrophy in these mice is established by rapid adaptation of the heart at the cost of gene expression associated with metabolic activity, with only temporary expression of possible maladaptive processes. Therefore, the transient early changes may reflect a beneficial response to pressure overload, as deterioration of cardiac hemodynamic function or heart failure does not occur.

Increased risk for cardiorespiratory failure associated with the A3302G mutation in the mitochondrial DNA encoded tRNALeu(UUR) gene.

Screening the mitochondrial DNA of a 64-year-old woman with mitochondrial myopathy revealed 76% of the tRNA(Leu(UUR)) A3302G mutation in muscle. Muscle of her affected son carried 96% mutated mitochondrial DNA. Both patients were biopsied twice, showing isolated complex I deficiency in the son's first biopsy, additional increased (within normal range) complex II + III activities in his second biopsy, combined complex I, II + III deficiency in mothers first biopsy and additional complex IV deficiency in her second biopsy. After a stay in the mountains, the son died of cardiac arrhythmia. The A3302G mutation has been reported before and is associated with mitochondrial myopathy and cardiorespiratory failure. Pathogenesis is explained by abnormal mtRNA processing, which was also reported for the adjacent C3303T mutation associated with cardiomyopathy and/or skeletal myopathy. Our findings suggest that a high mutation load of the A3302G mutation can lead to fatal cardiorespiratory failure, likely triggered by low environmental oxygen pressure and exercise.

Methods in molecular cardiology: microarray technology.

It has become more and more evident that changes in expression levels of genes can play an important role in cardiovascular diseases. Specific gene expression profiles may explain, for example, the pathophysiology of myocardial hypertrophy and pump failure and may provide clues for therapeutic interventions. Knowledge of gene expression patterns can also be applied for diagnostic and prognostic purposes, in which differences in gene activity can be used for classification. DNA microarray technology has become the method of choice to simultaneously study the expression of many different genes in a single assay. Each microarray contains many thousands of different DNA sequences attached to a glass slide. The amount of messenger RNA, which is a measure of gene activity, is compared for each gene on the microarray by labelling the mRNA with different fluorescently labelled nucleotides (Cy3 or Cy5) for the test and reference samples. After hybridisation to the microarray the relative amounts of a particular gene transcript in the two samples can be determined by measuring the signal intensities for the fluorescent groups (Cy3 and Cy5) and calculating signal ratios. This paper describes the development of in-house microarray technology, using commercially available cDNA collections. Several technical approaches will be compared and an overview of the pitfalls and possibilities will be presented. The technology will be explained in the context of our project to determine gene expression differences between normal, hypertrophic and failing heart.

Integrating images into a central medical information system.

Of the information items that must be easily available to the different actors involved in the care process, radiological images are not the least important. While until recently it was not feasible to include these into the medical information system, this situation has changed. Still, emphasis in PACS (Picture Archiving and Communication Systems) is primarily on the technological aspects. In this paper, in contrast, we stress the importance of integration of images into the overall workflow and into the overall medical record. We do so using illustrations from the PACS project of the University Hospitals Leuven. We briefly indicate that tight integration at the user interface level is needed, and that this requires more than standardized communication between subsystems.

Web-access to a central medical record to improve cooperation between hospital and referring physicians.

We set up Web-access to the central electronic medical record of the University Hospitals Leuven. Purpose was to enable external physicians to more actively participate in the care process as full members of the multidisciplinary treatment team. They get a view on the complete care process in the hospital, and can access results as these become available instead of being provided summary information post factum. This complements traditional electronic exchange between healthcare records. We report on technical setup and results from an extended pilot. We believe that this approach can enrich the discussion on how to provide the "global medical record".

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography.

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1-2 kb, digested in fragments of 90-600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA(Leu(UUR)) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA(Leu(UUR)) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.

Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation.

BACKGROUND: Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family. METHODS: COL4A3 and COL4A4 cDNAs were generated by nested reverse transcription-polymerase chain reaction and were analyzed by DNA sequence analysis. Denaturating high-performance liquid chromatography (DHPLC) was used for mutation and segregation analysis at the genomic DNA level. RESULTS: In the AD-AS family, a splice site mutation resulting in skipping of exon 21 of the COL4A3 gene was detected. The mutation does not alter the reading frame and is predicted to result in a COL4A3 chain with an internal deletion. CONCLUSION: As the NC domain is intact, this chain may be incorporated and distort the collagen triple helix, thereby causing the dominant effect of the mutation. The finding of a specific COL4A3 mutation in AD-AS completes the spectrum of type IV collagen mutations in all genetic forms of AS.

Using WWW and JAVA for image access and interactive viewing in an integrated PACS.

We start from the observations that (1) potential benefits from PACS can only be realized if PACS and HIS are integrated into a 'multimedia medical information system', and (2) that this also requires integration at the level of the user interface. The user interface should allow integrated interaction with information from different subsystems, of which PACS is one. However, in the real world, different information systems are constructed using different technologies. Moreover, radiological image viewing is a highly interactive task that puts a particular burden on the graphical user interface. We describe our experiences in applying technology emerging around the 'World Wide Web' (WWW) for interactive access to an integrated PACS/HIS. We illustrate the use of Web browsers to access new medical services, touch technologies for interactive access to HIS-PACS information, and emphasize the potential of JAVA applets. We argue that JAVA may become an important tool for providing highly interactive user interfaces to larger multimedia information systems. We discuss Web technology in the general context of HIS/PACS integration.

Integrating digital ICU viewing into the global working environment.

We illustrate that to benefit from the advantages of Picture Archiving and Communication Systems (PACS) for the Intensive Care Unit (ICU), the PACS must be strongly integrated within the overall working environment. This includes adaptation of the PACS toward specific working patterns and integrating it with the Hospital Information System (HIS). This is reflected in our prototype system in different ways. The user interface of the viewing station is centered around often used patterns in ICU viewing. Information about bed occupancy is retrieved from the HIS and exploited in the viewing station. A digital connection between the phosphorplate scanner and the HIS ensures that images are correctly related to other patient information and to previous images. Using minor adaptations to the existing HIS, PACS and HIS have been made to cooperate in integrated presentation of images and radiological reports, as a step towards a multi-media medical information system. We discuss the relation between PACS and the global information environment, emphasizing organizational issues rather than technological aspects.

Design for user efficiency in a dedicated ICU viewing station.

The intensive care unit (ICU) is one application where significant benefit is expected from the use of digital technology in the acquisition, management and presentation of images. However, the potential benefits should not be outweighed by disadvantages of current digital technology. One of the bottlenecks is the efficiency of image viewing using a workstation, especially if this viewing station is implemented using affordable commonly available hardware. In this paper we describe the design concepts of a relatively low-cost but efficient viewing station for chest images, and discuss clinical experience with this system at an ICU ward. The user interface has been optimized towards the specific patterns of ICU image viewing. By anticipating user requests and preparing images during idle times of the computer, the mean image access time could be reduced by a factor of 4, while most images could be presented instantaneously. Information from the hospital information system (HIS) is exploited in the user interface, and a simplified PAC-HIS coupling has been implemented for the simultaneous presentation of images and reports.

Failure of anterior cruciate-ligament reconstruction using tendon xenograft.

From January 1981 to September 1983 forty knees had replacement of a torn anterior cruciate ligament with a tendon xenograft. In six of the first thirty knees that were operated on severe synovitis developed within eight months after the operation, and total synovectomy and removal of the graft was required. Because of this complication we changed the recommended rinsing procedure for the graft in the last ten knees, and no synovitis occurred in these. Approximately half of all of the grafts ruptured between twelve and twenty months after the operation.