Fredoen Valianpour

X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): An update†

X‐linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM302060, BTHS) is a disorder with mitochondrial functional impairments and 3‐methylglutaconic aciduria that maps to Xq28. The associated G4.5 or TAZ gene has been identified but the encoded proteins have not yet been characterized. Following the prediction that the gene encodes one or more acyltransferases, lipid studies have shown a deficiency of cardiolipin, especially its tetralinoleoyl form (L4‐CL). Deficiency of L4‐CL was subsequently demonstrated in a variety of tissues, and determination in thrombocytes or cultured skin fibroblasts is now the most specific biochemical test available. BTHS is the first identified inborn error of metabolism that directly affects cardiolipin, a component of the inner mitochondrial membrane, necessary for proper functioning of the electron transport chain. We report here the finding of deficient docosahexaenoic acid and arachidonic acid in a proportion of patients with BTHS. The initial impression of a uniformly lethal infantile disease has to be modified. Age distribution in 54 living patients ranges between 0 and 49 years and peaks around puberty. Mortality is the highest in the first 4 years. The apex of the survival curve around puberty and the emergence of adults may reflect a dynamic shift towards increased survival. This trend is exemplified in a large pedigree previously published.

Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome

In eukaryotic cells, the acyl species of the phospholipid cardiolipin (CL) are more highly unsaturated than those of the other membrane phospholipids. Defective acylation of CL with unsaturated fatty acids and decreased total CL are associated with Barth syndrome, an X‐linked cardio‐ and skeletal myopathy attributed to a defect in the gene G4.5 (also known as tafazzin). We constructed a yeast mutant (taz1) containing a null mutation in the homologue of the human G4.5 gene. The yeast taz1Δ mutant was temperature sensitive for growth in ethanol as sole carbon source, but grew normally on glucose or glycerol plus ethanol. Total CL content was reduced in the taz1Δ mutant, and monolyso‐CL accumulated. The predominant CL acyl species found in wild‐type cells, C18:1 and C16:1, were markedly reduced in the mutant, whereas CL molecules containing saturated fatty acids were present. Interestingly, CL synthesis increased in the mutant, whereas expression of the CL structural genes CRD1 and PGS1 did not, suggesting that de novo biosynthetic enzyme activities are regulated by CL acylation. These results indicate that the taz1Δ mutant is an excellent genetic tool for the study of CL remodelling and may serve as a model system for the study of Barth syndrome.

Only One Splice Variant of the Human TAZ Gene Encodes a Functional Protein with a Role in Cardiolipin Metabolism

Barth syndrome (BTHS) is an X-linked recessive disorder caused by mutations in the TAZ gene and is characterized by cardiomyopathy, short stature, neutropenia, and 3-methylglutaconic aciduria. Recently it was found that BTHS patients exhibit a profound cardiolipin deficiency although the biosynthetic capacity to synthesize this lipid from its precursor phosphatidylglycerol is entirely normal. Like BTHS patients, a Saccharomyces cerevisiae strain, in which the yeast orthologue of the human TAZ gene has been disrupted, exhibits an abnormal cardiolipin profile as determined by tandem mass spectrometry. Additionally, this yeast strain grows poorly on non-fermentable carbon sources. We have used both properties of this yeast disruptant as a read-out system to test the physiological functionality of each of 12 different splice variants that have been reported for the human TAZ gene. Our results demonstrate that only the splice variant lacking exon 5 was able to complement the retarded growth of the yeast disruptant on selective plates and restore the cardiolipin profile to the wild type pattern. We conclude that this splice variant most likely represents the only physiologically important mRNA, at least with regard to cardiolipin metabolism.

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.

Linoleic acid supplemention of Barth syndrome fibroblasts restores cardiolipin levels implications for treatment

The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients.