Categorized Genetic Analysis in Childhood-Onset Cardiomyopathy.

BACKGROUND: Childhood-onset cardiomyopathy is a heterogeneous group of conditions the cause of which is largely unknown. The influence of consanguinity on the genetics of cardiomyopathy has not been addressed at a large scale. METHODS: To unravel the genetic cause of childhood-onset cardiomyopathy in a consanguineous population, a categorized approach was adopted. Cases with childhood-onset cardiomyopathy were consecutively recruited. Based on the likelihood of founder mutation and on the clinical diagnosis, genetic test was categorized to either (1) targeted genetic test with targeted mutation test, single-gene test, or multigene panel for Noonan syndrome, or (2) untargeted genetic test with whole-exome sequencing or whole-genome sequencing. Several bioinformatics tools were used to filter the variants. RESULTS: Two-hundred five unrelated probands with various forms of cardiomyopathy were evaluated. The median age of presentation was 10 months. In 30.2% (n=62), targeted genetic test had a yield of 82.7% compared with 33.6% for whole-exome sequencing/whole-genome sequencing (n=143) giving an overall yield of 53.7%. Strikingly, 96.4% of the variants were homozygous, 9% of which were found in 4 dominant genes. Homozygous variants were also detected in 7 novel candidates (ACACB, AASDH, CASZ1, FLII, RHBDF1, RPL3L, ULK1). CONCLUSIONS: Our work demonstrates the impact of consanguinity on the genetics of childhood-onset cardiomyopathy, the value of adopting a categorized population-sensitive genetic approach, and the opportunity of uncovering novel genes. Our data suggest that if a founder mutation is not suspected, adopting whole-exome sequencing/whole-genome sequencing as a first-line test should be considered.

Identification of new biomarkers of pyridoxine-dependent epilepsy by GC/MS-based urine metabolomics.

α-Aminoadipic semialdehyde and its cyclic form (Δ1-piperideine-6-carboxylate) accumulate in patients with α-aminoadipic semialdehyde dehydrogenase (AASADH; antiquitin; ALDH7A1) deficiency. Δ1-Piperideine-6-carboxylate is known to react with pyridoxal 5'-phosphate (PLP) to form a Knoevenagel condensation product, resulting in pyridoxine-dependent epilepsy. Despite dramatic clinical improvement following pyridoxine supplementation, many patients still suffer some degree of intellectual disability due to delayed diagnosis. In order to expedite the diagnosis of patients with suspected AASADH deficiency and minimize the delay in treatment, we used gas chromatography-mass spectrometry-based metabolomics to search for potentially diagnostic biomarkers in urine from four patients with ALDH7A1 mutations, and identified Δ2-piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate as candidate biomarkers. In a patient at postnatal day six, but before pyridoxine treatment, Δ2-piperideine-6-carboxylate and pipecolate were present at very high concentrations, indicating that these compounds may be good biomarkers for untreated AASADH deficiency patients. On the other hand, following pyridoxine/PLP treatment, 6-oxopipecolate was shown to be greatly elevated. We suggest that noninvasive urine metabolomics screening for Δ2-piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate will be useful for prompt and reliable diagnosis of AASADH deficiency in patients within any age group. The most appropriate combination among Δ2-piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate as biomarkers for AASADH deficiency patients appears to depend on the age of the patient and whether pyridoxine/PLP supplementation has been implemented. We anticipate that the present bioanalytical information will also be useful to researchers studying glutamate, proline, lysine and ornithine metabolism in mammals and other organisms.

Dose-response of different dietary leucine levels on growth performance and amino acid metabolism in piglets differing for aminoadipate-semialdehyde synthase genotypes.

Dose-response studies of dietary leucine (Leu) in weaners are needed for a proper diet formulation. Dietary Leu effect was assessed in a 3-weeks dose-response trial with a 2 (genotype) x 5 (diets) factorial arrangement on one-hundred weaned pigs (9 to 20 kg body weight (BW)). Pigs differed for a polymorphism at the aminoadipate-semialdehyde synthase (AASS) gene, involved in lysine (Lys) metabolism. Pigs received experimental diets (d7 to d28) differing for the standardized ileal digestible (SID) Leu:Lys: 70%, 85%, 100%, 115%, 130%. Daily feed intake (ADFI), daily gain (ADG) and feed:gain (F:G) in all pigs and ADG and F:G in two classes of BW were analyzed using regression analysis with curvilinear-plateau (CLP) and linear quadratic function (LQ) models. Amino acid (AA) concentrations in plasma, liver, muscle and urine were determined. AASS genotype did not affect the parameters. Dietary Leu affected performance parameters, with a maximum response for ADG and F:G between 100.5% and 110.7% SID Leu:Lys, higher than the usually recommended one, and between 110.5% and 115.4% and between 94.9% and 110.2% SID Leu:Lys for ADG for light and heavy pigs respectively. AA variations in tissues highlighted Leu role in protein synthesis and its influence on the other branched chain AAs.

Virulence, Host-Selective Toxin Production, and Development of Three Cochliobolus Phytopathogens Lacking the Sfp-Type 4'-Phosphopantetheinyl Transferase Ppt1.

The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.

Activity of α-Aminoadipate Reductase Depends on the N-Terminally Extending Domain.

L-α-Aminoadipic acid reductases catalyze the ATP- and NADPH-dependent reduction of L-α-aminoadipic acid to the corresponding 6-semialdehyde during fungal L-lysine biosynthesis. These reductases resemble peptide synthetases with regard to their multidomain composition but feature a unique domain of elusive function--now referred to as an adenylation activating (ADA) domain--that extends the reductase N-terminally. Truncated enzymes based on NPS3, the L-α-aminoadipic acid reductase of the basidiomycete Ceriporiopsis subvermispora, lacking the ADA domain either partially or entirely were tested for activity in vitro, together with an ADA-adenylation didomain and the ADA domainless adenylation domain. We provide evidence that the ADA domain is required for substrate adenylation: that is, the initial step of the catalytic turnover. Our biochemical data are supported by in silico modeling that identified the ADA domain as a partial peptide synthetase condensation domain.

Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer's disease using pI shift and label-free quantification without enrichment.

Posttranslational modifications modulate protein function in cells. Global analysis of multiple posttranslational modifications can provide insight into physiology and disease, but presents formidable challenges. In the present study, we used a technique that does not require target enrichment to analyze alterations in the phosphorylation and ubiquitination of proteins from patients with Alzheimer's disease (AD). Guided by our previous findings, we applied three strategies to further our understanding of the dysregulation of posttranslationally modified proteins. We first identified phosphorylation sites by determining peptide pI shifts using OFFGEL. Second, using tandem mass spectrometry, we determined the ubiquitination status of the proteins using an assay for a trypsin digestion remnant of ubiquitination (Gly-Gly). Third, for large-scale discovery, we quantified the global differences in protein expression. Of the proteins expressed in AD tissue at levels of 2.0 or greater compared with controls, 60 were phosphorylated and 56 were ubiquitinated. Of the proteins expressed at levels of 0.5 or lower compared with controls, 81 were phosphorylated and 56 were ubiquitinated. Approximately 98 % of the phosphopeptides exhibited a pI shift. We identified 112 new phosphorylation sites (51.38 %), and 92 new ubiquitination sites (96.84 %). Taken together, our findings suggest that analysis of the alterations in posttranslationally modified proteins may contribute to understanding the pathogenesis of AD and other diseases.

Functional and phylogenetic divergence of fungal adenylate-forming reductases.

A key step in fungal L-lysine biosynthesis is catalyzed by adenylate-forming L-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized L-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.

Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a ß-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.

Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates ß-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates ß-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled ß-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for ß-alanine, and a KM of ~ 5 µm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that ß-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a ß-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

Sensitive detection of whole-genome differentiation among closely-related species of the genus Fusarium using DNA-DNA hybridization and a microplate technique.

We developed a new system for detection of whole-genome differentiation using DNA-DNA hybridization, and tested its sensitivity with three closely-related Fusarium species. We compared DNA-DNA relatedness to nucleotide sequence homologies of five genetic regions between each of five strains of three Fusarium species. DNA-DNA relatedness by our system was 16.2-86.6%. Sequence homologies of 18S rDNA, rDNA cluster region from ITS1 to 28S rDNA, ß-tub, EF-1α and lys2 were 100.0, 99.0-100.0, 96.7-100.0, 95.1-99.4, and 94.7-100.0%, respectively. Our system could clearly detect differentiation between closely-related fungal species which have very similar morphological-characteristics, and exhibit little diagnoses in nucleotide sequences. Our results suggest that this system is a good tool for identification and phylogenetic analysis of closely-related fungal species.

Urinary AASA excretion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency.

Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

Evaluation of genetic markers for identifying isolates of the species of the genus Fusarium.

BACKGROUND: Members of the genus Fusarium are well known as one of the most important plant pathogens causing food spoilage and loss worldwide. Moreover, they are associated with human and animal diseases through contaminated foods because they produce mycotoxins. To control fungal hazards of plants, animals and humans, there is a need for a rapid, easy and accurate identification system of Fusarium isolates with molecular methods. RESULTS: To specify genes appropriate for identifying isolates of various Fusarium species, we sequenced the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5.8S rDNA, 28S rDNA, ß-tubulin gene (ß-tub), and aminoadipate reductase gene (lys2), and subsequently calculated the nucleotide sequence homology with pair-wise comparison of all tested strains and inferred the ratio of the nucleotide substitution rates of each gene. Inter-species nucleotide sequence homology of ß-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in a database have a high possibility of identifying unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter-species pair-wise combinations that had 100% homologies. Other markers often showed 100% homology in inter-species pair-wise combinations. The nucleotide substitution rate of lys2 was the highest among the six genes. CONCLUSION: The lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study.

Differential expression of adipose tissue proteins between obesity-susceptible and -resistant rats fed a high-fat diet.

One of the major questions in the field of obesity is why some humans become obese (obesity prone, OP) and others resist the development of obesity (obesity resistant, OR) when exposed to a high-calorie diet, which has not been completely studied. Therefore, in the present study, in order to gain insight into the molecular mechanisms underlying this propensity, we have performed a comparative analysis of protein expression profiles in white adipose tissue (WAT) and brown adipose tissue (BAT) of rats fed a high-fat diet by 2-DE and MALDI-TOF-MS. Protein mapping of homogenates revealed significant alterations to a number of proteins; 60 and 70 proteins were differentially regulated in BAT and WAT, respectively. For careful interpretation of proteomic results, we categorized the identified proteins into two groups by analysis of both average spot density of pooled six rat adipose tissues and individual spot density of each adipose tissue of six rats as a function of body weight. One of the most striking findings of this study was that significant changes of Ehd1 and laminin receptor in BAT as well as antiquitin, DJ-1 protein, and paraoxonase 2 in WAT were found for the first time in obese rats. In addition, we confirmed the increased expression of some thermogenic enzymes and decreased lipogenic enzymes in adipose tissues of OR rats by immunoblot analysis. To our knowledge, this is the first proteomic study of profiling of protein modulation in OP and OR rats, thereby providing the first global evidence for different propensities to obesity between OP and OR rats.

Metabolism of lysine in alpha-aminoadipic semialdehyde dehydrogenase-deficient fibroblasts: evidence for an alternative pathway of pipecolic acid formation.

The mammalian degradation of lysine is believed to proceed via two distinct routes, the saccharopine and the pipecolic acid routes, that ultimately converge at the level of alpha-aminoadipic semialdehyde (alpha-AASA). alpha-AASA dehydrogenase-deficient fibroblasts were grown in cell culture medium supplemented with either L-[alpha-(15)N]lysine or L-[epsilon-(15)N]lysine to explore the exact route of lysine degradation. L-[alpha-(15)N]lysine was catabolised into [(15)N]saccharopine, [(15)N]alpha-AASA, [(15)N]Delta(1)-piperideine-6-carboxylate, and surprisingly in [(15)N]pipecolic acid, whereas L-[epsilon-(15)N]lysine resulted only in the formation of [(15)N]saccharopine. These results imply that lysine is exclusively degraded in fibroblasts via the saccharopine branch, and pipecolic acid originates from an alternative precursor. We hypothesize that pipecolic acid derives from Delta(1)-piperideine-6-carboxylate by the action of Delta(1)-pyrroline-5-carboxylic acid reductase, an enzyme involved in proline metabolism.

The evolutionary history of lysine biosynthesis pathways within eukaryotes.

Lysine biosynthesis occurs in two ways: the diaminopimelate (DAP) pathway and the alpha-aminoadipate (AAA) pathway. The former is present in eubacteria, plants, and algae, whereas the latter was understood to be almost exclusive to fungi. The recent finding of the alpha-aminoadipate reductase (AAR) gene, one of the core genes of the AAA pathway, in the marine protist Corallochytrium limacisporum was, therefore, believed to be a molecular synapomorphy of fungi and C. limacisporum. To test this hypothesis, we undertook a broader search for the AAR gene in eukaryotes, and also analyzed the distribution of the lysA gene, a core gene of the DAP pathway. We show that the evolutionary history of both genes, AAR and lysA, is much more complex than previously believed. Furthermore, the AAR gene is present in several unicellular opisthokonts, thus rebutting the theory that its presence is a molecular synapomorphy between C. limacisporum and fungi. AAR gene seems to be exclusive of Excavata and Unikonts, whereas the lysA gene is present in several unrelated taxa within all major eukaryotic lineages, indicating a role for several lateral gene transfer (LGT) events. Our data imply that the choanoflagellate Monosiga brevicollis and the "choanozoan" Capsaspora owczarzaki acquired their lysA copies from a proteobacterial ancestor. Overall, these observations represent new evidence that the role of LGT in the evolutionary history of eukaryotes may have been more significant than previously thought.

Cloning, sequencing and characterization of the alpha-aminoadipate reductase gene (LYS2) from Saccharomycopsis fibuligera.

The gene putatively encoding alpha-aminoadipate reductase (AAR) was isolated successfully by degenerate PCR and chromosome walking, based on cassette PCR methods, from the dimorphous yeast Saccharomycopsis fibuligera PD70 and was named SfLYS2. Sequence analysis revealed that it contained a putative open reading frame (ORF) of 4161 bp and encoded a polypeptide of 1386 amino acids. The deduced translation product shared an identity of 53% and 51% to the Lys2p homologues of Candida albicans and Saccharomyces cerevisiae, respectively. An atypical TATA box and a GCN4-box element were found in the 5'-upstream region. Genomic Southern hybridization suggested the presence of a single locus of SfLYS2 in the S. fibuligera genome. Expression of the ORF of SfLYS2 in a lys2(-) strain of S. cerevisiae could functionally complement the lysine mutant of the S. cerevisiae strain. S. fibuligera could use lysine as the sole nitrogen source but its growth was inhibited on the alpha-aminoadipate (AA) medium. Approximately 90% of the mutants of S. cerevisiae resistant to AA are lysine auxotrophs; in contrast all the mutants of S. fibuligera resistant to AA recovered in this work were not lysine auxotrophs.

Antiquitin, a relatively unexplored member in the superfamily of aldehyde dehydrogenases with diversified physiological functions.

Antiquitin is a member of the aldehyde dehydrogenase superfamily. Sequence analyses indicate that the protein is highly conserved from plants to animals. The plant antiquitins are generally believed to play a role in osmoregulation and/or detoxification. The physiological functions of animal antiquitins remain largely elusive, their involvement in a number of human diseases has been implicated.

Pyrroloquinoline quinone nutritional status alters lysine metabolism and modulates mitochondrial DNA content in the mouse and rat.

Pyrroloquinoline quinone (PQQ) added to purified diets devoid of PQQ improves indices of perinatal development in rats and mice. Herein, PQQ nutritional status and lysine metabolism are described, prompted by a report that PQQ functions as a vitamin-like enzymatic cofactor important in lysine metabolism (Nature 422 [2003] 832). Alternatively, we propose that PQQ influences lysine metabolism, but by mechanisms that more likely involve changes in mitochondrial content. PQQ deprivation in both rats and mice resulted in a decrease in mitochondrial content. In rats, alpha-aminoadipic acid (alphaAA), which is derived from alpha-aminoadipic semialdehyde (alphaAAS) and made from lysine in mitochondria, and the plasma levels of amino acids known to be oxidized in mitochondria (e.g., Thr, Ser, and Gly) were correlated with changes in the liver mitochondrial content of PQQ-deprived rats, but not PQQ-supplemented rats. In contrast, the levels of NAD dependent alpha-aminoadipate-delta-semialdehyde dehydrogenase (AASDH), a cytosolic enzyme important to alphaAA production from alphaAAS, was not influenced by PQQ dietary status. Moreover, the levels of U26 mRNA were not significantly changed even when diets differed markedly in PQQ and dietary lysine content. U26 mRNA levels were measured, because of U26's proposed, albeit questionable role as a PQQ-dependent enzyme involved in alphaAA formation.

Molecular evidence of fungal signatures in the marine protist Corallochytrium limacisporum and its implications in the evolution of animals and fungi.

Fungi, animals, and single-celled organisms belonging to the choanozoans together constitute the supergroup Opisthokonta. The latter are considered crucial in understanding the evolutionary origin of animals and fungi. The choanozoan Corallochytrium limacisporum is an enigmatic marine protist of considerable interest in opisthokontan evolution. Several isolates of the organism were obtained from a coral reef lagoon in the Lakshadweep group of islands of the Arabian Sea. The capability of these cultures to grow on media containing inorganic nitrogen sources prompted us to examine the possible presence of fungal signatures, namely the enzyme alpha-aminoadipate reductase (alpha-AAR) involved in the alpha-aminoadipate (AAA) pathway for synthesizing lysine and ergosterol, in one of the isolates. These features, as well as the sterol C-14 reductase gene involved in the sterol pathway of animals and fungi, were detected in the organism. Phylogenetic trees based on the alpha-AAR gene suggested that Corallochytrium limacisporum is a sister clade to fungi, while those based on the C-14 reductase gene did not adequately resolve whether the organism was more closely related to fungi or animals. While many studies indicate that Corallochytrium is a sister clade to animals, we suggest that further studies are required to examine whether this protist is in fact more closely related to fungi rather than to animals.

Cloning and characterization of a novel human homolog* of mouse U26, a putative PQQ-dependent AAS dehydrogenase.

Mouse U26 has been defined as a 2-aminoadipic 6-semialdehyde dehydrogenase. It was speculated to be a PQQ-dependent AAS dehydrogenase due to the research of demonstrating PQQ as a new B vitamin. We isolated a novel human cDNA from the human fetal brain cDNA library we constructed. Its deduced protein was most related to mouse U26. Thus, we termed it human U26. This putative protein contains an AMP-binding domain, a Phosphopantetheine-binding domain and six PQQ-binding motifs. Human U26 mRNA is ubiquitously expressed in adult tissues and is highly expressed in colon adenocarcinoma (CX-1) and colon adenocarcinoma (GI-112) cell lines. Further study should be made to clarify the precise function of human U26.

Biochemistry: is pyrroloquinoline quinone a vitamin?

The announcement by Kasahara and Kato of pyrroloquinoline quinone (PQQ) as a 'new' vitamin has received considerable attention. We have since attempted to reproduce the findings on which their conclusion is based, namely that defects in lysine metabolism occur in PQQ-deprived rodents. However, we find that the activity of alpha-aminoadipic acid-delta-semialdehyde (AAS) dehydrogenase in liver and plasma levels of alpha-aminoadipic acid (AAA), both of which act as indicators of lysine degradation in mammals, are not affected by changes in PQQ dietary status. Our results call into question the identification of PQQ as a new vitamin.