The ACADS gene variation spectrum in 114 patients with short-chain acyl-CoA dehydrogenase (SCAD) deficiency is dominated by missense variations leading to protein misfolding at the cellular level

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited disorder of mitochondrial fatty acid oxidation associated with variations in the ACADS gene and variable clinical symptoms. In addition to rare ACADS inactivating variations, two common variations, c.511C > T (p.Arg171Trp) and c.625G > A (p.Gly209Ser), have been identified in patients, but these are also present in up to 14% of normal populations leading to questions of their clinical relevance. The common variant alleles encode proteins with nearly normal enzymatic activity at physiological conditions in vitro. SCAD enzyme function, however, is impaired at increased temperature and the tendency to misfold increases under conditions of cellular stress. The present study examines misfolding of variant SCAD proteins identified in patients with SCAD deficiency. Analysis of the ACADS gene in 114 patients revealed 29 variations, 26 missense, one start codon, and two stop codon variations. In vitro import studies of variant SCAD proteins in isolated mitochondria from SCAD deficient (SCAD−/−) mice demonstrated an increased tendency of the abnormal proteins to misfold and aggregate compared to the wild-type, a phenomenon that often leads to gain-of-function cellular phenotypes. However, no correlation was found between the clinical phenotype and the degree of SCAD dysfunction. We propose that SCAD deficiency should be considered as a disorder of protein folding that can lead to clinical disease in combination with other genetic and environmental factors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information

Background  

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.     

Circulating immune complexes contain citrullinated fibrinogen in rheumatoid arthritis

Introduction  

There is increasing evidence that autoantibodies and immune complexes (ICs) contribute to synovitis in rheumatoid arthritis (RA), yet the autoantigens incorporated in ICs in RA remain incompletely characterised.     

X chromosome-inactivation patterns confirm the late timing of monoamniotic-MZ twinning.

Autosomal dominant brachydactyly type B (BDB) is characterized by nail aplasia with rudimentary or absent distal and middle phalanges. We describe two unrelated families with BDB. One family is English; the other family is Canadian but of English ancestry. We assigned the BDB locus in the Canadian family to an 18-cM interval on 9q, using linkage analysis (LOD score 3.5 at recombination fraction [theta] 0, for marker D9S938). Markers across this interval also cosegregated with the BDB phenotype in the English family (LOD score 2.1 at straight theta=0, for marker D9S277). Within this defined interval is a smaller (7.5-cM) region that contains 10 contiguous markers whose disease-associated haplotype is shared by the two families. This latter result suggests a common founder among families of English descent that are affected with BDB.     

Bezafibrate activation of PPAR drives disturbances in mitochondrial redox bioenergetics and decreases the viability of cells from patients with VLCAD deficiency

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear.The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial.We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.     

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.