IVEN: A quantitative tool to describe 3D cell position and neighbourhood reveals architectural changes in FGF4-treated preimplantation embryos.

Architectural changes at the cellular and organism level are integral and necessary to successful development and growth. During mammalian preimplantation development, cells reduce in size and the architecture of the embryo changes significantly. Such changes must be coordinated correctly to ensure continued development of the embryo and, ultimately, a successful pregnancy. However, the nature of such transformations is poorly defined during mammalian preimplantation development. In order to quantitatively describe changes in cell environment and organism architecture, we designed Internal Versus External Neighbourhood (IVEN). IVEN is a user-interactive, open-source pipeline that classifies cells into different populations based on their position and quantifies the number of neighbours of every cell within a dataset in a 3D environment. Through IVEN-driven analyses, we show how transformations in cell environment, defined here as changes in cell neighbourhood, are related to changes in embryo geometry and major developmental events during preimplantation mammalian development. Moreover, we demonstrate that modulation of the FGF pathway alters spatial relations of inner cells and neighbourhood distributions, leading to overall changes in embryo architecture. In conjunction with IVEN-driven analyses, we uncover differences in the dynamic of cell size changes over the preimplantation period and determine that cells within the mammalian embryo initiate growth phase only at the time of implantation.

Clinical Trials on Advanced Therapy Investigational Medicinal Products in Spain (2004-2022): Experience and Challenges for the Future.

Clinical investigation is the basis for establishing how useful advanced therapy investigational medicinal products (ATiMP) are for the treatment of serious diseases.In Spain, clinical trials (CT) on ATiMP need to follow the general European legislation on CT with medicinal products plus some specific legislation and guidance depending on the type of ATiMP.This chapter describes the characteristics of CT on ATiMP authorized in Spain in the period 2004-2022 and the legislation applicable along this period. There are clear differences in the clinical trials conducted by non commercial and commercial sponsors: the first have been more involved in CT on somatic cell therapy medicinal products (sCTMP) and tissue-engineered products (TEP), while the second drive more the CT on gene therapy medicinal products (GTMP) in the last years. Difficulties of budget and resources especially by non-commercial sponsors to meet the regulatory requirements are highlighted. The importance of complying with transparency rules with respect to CT on ATiMP is also discussed.

Galactokinase deficiency: lessons from the GalNet registry.

PURPOSE: Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype. METHODS: Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020. RESULTS: Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial. CONCLUSION: The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.

Small aminothiol compounds improve the function of Arg to Cys variant proteins: effect on the human cystathionine ß-synthase p.R336C.

The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.

Sweet and sour: an update on classic galactosemia.

Classic galactosemia is a rare inherited disorder of galactose metabolism caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme of the Leloir pathway. It presents in the newborn period as a life-threatening disease, whose clinical picture can be resolved by a galactose-restricted diet. The dietary treatment proves, however, insufficient in preventing severe long-term complications, such as cognitive, social and reproductive impairments. Classic galactosemia represents a heavy burden on patients' and their families' lives. After its first description in 1908 and despite intense research in the past century, the exact pathogenic mechanisms underlying galactosemia are still not fully understood. Recently, new important insights on molecular and cellular aspects of galactosemia have been gained, and should open new avenues for the development of novel therapeutic strategies. Moreover, an international galactosemia network has been established, which shall act as a platform for expertise and research in galactosemia. Herein are reviewed some of the latest developments in clinical practice and research findings on classic galactosemia, an enigmatic disorder with many unanswered questions warranting dedicated research.

The spectrum of pyruvate oxidation defects in the diagnosis of mitochondrial disorders.

Pyruvate oxidation defects (PODs) are among the most frequent causes of deficiencies in the mitochondrial energy metabolism and represent a substantial subset of classical mitochondrial diseases. PODs are not only caused by deficiency of subunits of the pyruvate dehydrogenase complex (PDHC) but also by various disorders recently described in the whole pyruvate oxidation route including cofactors, regulation of PDHC and the mitochondrial pyruvate carrier. Our own patients from 2000 to July 2014 and patients identified by a systematic survey of the literature from 1970 to July 2014 with a pyruvate oxidation disorder and a genetically proven defect were included in the study (n=628). Of these defects 74.2% (n=466) belong to PDHC subunits, 24.5% (n=154) to cofactors, 0.5% (n=3) to PDHC regulation and 0.8% (n=5) to mitochondrial pyruvate import. PODs are underestimated in the field of mitochondrial diseases because not all diagnostic centres include biochemical investigations of PDHC in their routine analysis. Cofactor and transport defects can be missed, if pyruvate oxidation is not measured in intact mitochondria routinely. Furthermore deficiency of the X-chromosomal PDHA1 can be biochemically missed depending on the X-inactivation pattern. This is reflected by an increasing number of patients diagnosed recently by genetic high throughput screening approaches. PDHC deficiency including regulation and import affect mainly the glucose dependent central and peripheral nervous system and skeletal muscle. PODs with combined enzyme defects affect also other organs like heart, lung and liver. The spectrum of clinical presentation of PODs is still expanding. PODs are a therapeutically interesting group of mitochondrial diseases since some can be bypassed by ketogenic diet or treated by cofactor supplementation. PDHC kinase inhibition, chaperone therapy and PGC1α stimulation is still a matter of further investigations.

"Losing the tombola": a case study describing the use of community consultation in designing the study protocol for a randomised controlled trial of a mental health intervention in two conflict-affected regions.

BACKGROUND: Community consultation is increasingly recommended, and in some cases, required by ethical review boards for research that involves higher levels of ethical risk such as international research and research with vulnerable populations. In designing a randomised control trial of a mental health intervention using a wait list control, we consulted the community where the research would be undertaken prior to finalising the study protocol. The study sites were two conflict-affected locations: Grozny in the Chechen Republic and Kitchanga in eastern Democratic Republic of Congo. METHODS: Group discussions with a range of community members were held in both study sites. Facilitators used a prepared set of questions to guide the discussions and to solicit feedback on the value of the research as well as on the study design. Specific questions were asked about enablers and barriers to participation in the research. RESULTS: Six groups were held in Grozny and thirteen in Kitchanga. The majority of individuals and groups consulted supported the research, and understood the purpose. In Grozny, the main concern raised was the length of the waiting period. Barriers to both waiting and returning for follow up were identified. In Kitchanga, there was a strong reaction against the wait list control and against randomisation. The consultations provided information on unanticipated harms to the community, allowing changes to the study design to mitigate these harms and increase acceptability of the study. It also served to inform the community of the study, and through engaging with them early, helped promote legitimacy and joint responsibility. CONCLUSION: Community consultation prior to finalising the study design for a mental health intervention trial in two humanitarian settings proved feasible. Our experience reinforces the importance of community consultation before the study design is finalised and the importance of broad consultation that includes both community leaders and the potential study participants.

Insights into the regulatory domain of cystathionine Beta-synthase: characterization of six variant proteins.

Cystathionine beta-synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S-adenosylmethionine (SAM), which binds to its C-terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C-terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C-terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM-binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C-terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C-terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.

Reduced response of Cystathionine Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients.

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.

A frequent splicing mutation and novel missense mutations color the updated mutational spectrum of classic galactosemia in Portugal.

Classic galactosemia is an autosomal recessive disorder caused by deficient galactose-1-phosphate uridylyltransferase (GALT) activity. Patients develop symptoms in the neonatal period, which can be ameliorated by dietary restriction of galactose. Many patients develop long-term complications, with a broad range of clinical symptoms whose pathophysiology is poorly understood. The high allelic heterogeneity of GALT gene that characterizes this disorder is thought to play a determinant role in biochemical and clinical phenotypes. We aimed to characterize the mutational spectrum of GALT deficiency in Portugal and to assess potential genotype-phenotype correlations. Direct sequencing of the GALT gene and in silico analyses were employed to evaluate the impact of uncharacterized mutations upon GALT functionality. Molecular characterization of 42 galactosemic Portuguese patients revealed a mutational spectrum comprising 14 nucleotide substitutions: ten missense, two nonsense and two putative splicing mutations. Sixteen different genotypic combinations were detected, half of the patients being p.Q188R homozygotes. Notably, the second most frequent variation is a splicing mutation. In silico predictions complemented by a close-up on the mutations in the protein structure suggest that uncharacterized missense mutations have cumulative point effects on protein stability, oligomeric state, or substrate binding. One splicing mutation is predicted to cause an alternative splicing event. This study reinforces the difficulty in establishing a genotype-phenotype correlation in classic galactosemia, a monogenic disease whose complex pathogenesis and clinical features emphasize the need to expand the knowledge on this "cloudy" disorder.

Sulphur Amino Acids: How Important is it to Set Reference Ranges for Each Population?

INTRODUCTION: Metabolism of sulfur amino acids requires an optimal interplay between nutritional demand, enzymes, transporters, and adequate dietary intake of B vitamins. Insufficient intake and excess are detrimental, and concentrations depend on health status. However, plasma aminothiol concentrations, previously reported in healthy subjects using highly sensitive methods, vary considerably, and age- and gender differences were observed. Therefore, defining age- and gender-specific ranges for each population is crucial to evaluate the meaning of plasma thiol redox state in health and disease. METHODS: A healthy Portuguese pediatric population (n=90), aged 9- (n=38) and 17-year-old (n=52), was evaluated. Plasma aminothiols, total homocysteine (tHcy), cysteine (tCys), glutathione (tGSH) and γ-glutamylcysteine (tγ-Glu-Cys), were analysed as SBD-F derivatives by HPLC with fluorescence detection. RESULTS/CASE REPORT: Mean plasma concentrations (SD) for the 9- and the 17-year-old groups, were as following: tHcy = 4.58 (0.98); 8.13 (3.27) µM, p <0.001; tCys = 207.34 (32.07); 198.59 (21.24) µM, p = 0.274; tGSH = 4.54 (1.08); 5.20 (1.84) µM, p = 0.123 and tγ-Glu-Cys = 1.47 (0.30); 1.06 (0.28) µM, p < 0.001, respectively. No statistically significant differences were found between males and females in the 9-year-old group. However, in the 17-year-old group, significant differences between genders were observed for tHcys (p < 0.001) and tγ-Glu-Cys (p = 0.039), with males presenting the highest concentrations. When correlating the four thiols' plasma concentrations, only the precursors of glutathione, tγ-Glu-Cys and tCys, were positively correlated (r = 0.450, p < 0.001). CONCLUSION: Our results showed significant differences in tHcy and tγ-Glu-Cys levels across both age groups, which increased and decreased with age, respectively. It is interesting to highlight that in the 17-year-old group, tHcy and tγ-Glu-Cys levels were higher in males than in females. These observations showed that age and gender influence plasma levels of thiols, which may impact cellular oxidative status. In conclusion, setting age and gender distinct ranges for each specific population is of utmost importance for understanding disease mechanisms and the effectiveness of therapeutic interventions.

Natural history of three late-diagnosed classic Galactosemia patients.

The authors report the natural history of three patients with late-diagnosed Classic Galactosemia (CG) (at 16, 19 and 28 years). This was due to a combination of factors: absence of neonatal screening, absence of some typical acute neonatal symptoms, and negative galactosemia screening. This report underlines the value of neonatal screening and the importance of further diagnostic testing in case of late-onset manifestations.

Brain function in classic galactosemia, a galactosemia network (GalNet) members review.

Classic galactosemia (CG, OMIM #230400, ORPHA: 79,239) is a hereditary disorder of galactose metabolism that, despite treatment with galactose restriction, affects brain function in 85% of the patients. Problems with cognitive function, neuropsychological/social emotional difficulties, neurological symptoms, and abnormalities in neuroimaging and electrophysiological assessments are frequently reported in this group of patients, with an enormous individual variability. In this review, we describe the role of impaired galactose metabolism on brain dysfunction based on state of the art knowledge. Several proposed disease mechanisms are discussed, as well as the time of damage and potential treatment options. Furthermore, we combine data from longitudinal, cross-sectional and retrospective studies with the observations of specialist teams treating this disease to depict the brain disease course over time. Based on current data and insights, the majority of patients do not exhibit cognitive decline. A subset of patients, often with early onset cerebral and cerebellar volume loss, can nevertheless experience neurological worsening. While a large number of patients with CG suffer from anxiety and depression, the increased complaints about memory loss, anxiety and depression at an older age are likely multifactorial in origin.

Evaluation of Mitochondrial Function on Pyruvate Dehydrogenase Complex Deficient Patient-derived Cell Lines.

INTRODUCTION: Pyruvate Dehydrogenase Complex (PDC) is a pivotal gatekeeper between cytosolic glycolysis and mitochondrial oxidative phosphorylation, playing important role in aerobic energy metabolism. Most PDC deficiency, cases being caused by mutations in PDHA1 encoding the α subunit of the rate-limiting E1 enzyme, which is characterized by abnormal phenotypes caused by energy deprivation at peripheral/central nervous systems and muscular tissues. This study aims to evaluate the potential therapeutic effect of arginine and thiamine in ameliorating mitochondrial function in patient-derived cultured cells. MATERIALS AND METHODS: PDC-deficient cell lines, carrying three different PDHA1 variants, were cultured in the absence and presence of arginine and/or thiamine at therapeutical levels, 4 mM and 100 µM, respectively. Mitochondrial bioenergetics profile was evaluated using the Seahorse extracellular flux analyzer. RESULTS: In physiological conditions, control cells presented standard values for all parameters evaluating the mitochondrial function, no differences being observed after supplementation of culture medium with therapeutic levels of arginine and/or thiamine. However, PDC-PDHA1 deficient cell lines consumed less oxygen than the control cells, but arginine and thiamine supplementation increased the basal respiration for values similar or higher than the control cell line. Moreover, arginine and thiamine treatment highlighted an inefficient oxidative phosphorylation carried out by PDC-deficient cell lines. Finally, this treatment showed an increased oxygen consumption by enzymes other than those in the respiratory chain, thus proving the dependence of these mutant cell lines on cytosolic sources for ATP production, namely glycolysis. CONCLUSIONS: This study showed that arginine and thiamine, at therapeutical levels, increase the basal oxygen consumption rate of PDC-deficient cell lines, as well as their ATP-linked respiration. This parameter measures the capacity of the cell to meet its energetic demands and, therefore, its increase reveals a higher electron flow through the respiratory chain, which is coupled to elevated oxidative phosphorylation, thus indicating an overall increased robustness in mitochondrial- related bioenergetics.

An efficient method for simultaneous species, individual, and sex identification via in-solution single nucleotide polymorphism capture from low-quality scat samples.

Understanding predator population dynamics is important for conservation management because of the critical roles predators play within ecosystems. Noninvasive genetic sampling methods are useful for the study of predators like canids that can be difficult to capture or directly observe. Here, we introduce the FAECES* method (Fast and Accurate Enrichment of Canid Excrement for Species* and other analyses) which expands the toolbox for canid researchers and conservationists by using in-solution hybridization sequence capture to produce single nucleotide polymorphism (SNP) genotypes for multiple canid species from scat-derived DNA using a single enrichment. We designed a set of hybridization probes to genotype both coyotes (Canis latrans) and kit foxes (Vulpes macrotis) at hundreds of polymorphic SNP loci and we tested the probes on both tissues and field-collected scat samples. We enriched and genotyped by sequencing 52 coyote and 70 kit fox scats collected in and around a conservation easement in the Nevada Mojave Desert. We demonstrate that the FAECES* method produces genotypes capable of differentiating coyotes and kit foxes, identifying individuals and their sex, and estimating genetic diversity and effective population sizes, even using highly degraded, low-quantity DNA extracted from scat. We found that the study area harbours a large and diverse population of kit foxes and a relatively smaller population of coyotes. By replicating our methods in the future, conservationists can assess the impacts of management decisions on canid populations. The method can also be adapted and applied more broadly to enrich and sequence multiple loci from any species of interest using scat or other noninvasive genetic samples.

Phenylalanine hydroxylase deficiency: molecular epidemiology and predictable BH4-responsiveness in South Portugal PKU patients.

Hyperphenylalaninemia (HPA, OMIM #261600), which includes phenylketonuria (PKU), is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH), being already described more than 600 different mutations. Genotype-phenotype correlation is a useful tool to predict the metabolic phenotype, to establish the better tailored diet and, more recently, to assess the potential responsiveness to BH(4) therapy, a current theme on PKU field. The aim of this study was the molecular analysis of the PAH gene, evaluation of genotype-phenotype relationships and prediction of BH(4)-responsiveness in the HPA population living in South Portugal. We performed the molecular characterization of 83 HPA patients using genomic DNA extracted from peripheral blood samples or Guthrie cards. PAH mutations were scanned by PCR amplification of exons and related intronic boundaries, followed by direct sequence analysis. Intragenic polymorphisms were determined by PCR-RFLP analysis. The results allowed the full characterization of 67 patients. The mutational spectrum encompasses 34 distinct mutations, being the most frequent IVS10nt-11G>A (14.6%), V388M (10.8%), R261Q (8.2%) and R270K (7.6%), which account for 46% of all mutant alleles. Moreover, 12 different haplotypes were identified and most mutations were associated with a single one. Notably, more than half of the 34 mutations belong to the group of more than 70 mutations already identified in BH(4)-responsive patients, according to BIOPKU database. Fifty one different genotypic combinations were found, most of them in single patients and involving a BH(4)-responsive mutation. In conclusion, a significant number (30-35%) of South Portugal PKU patients may potentially benefit from BH(4) therapy which, combined with a less strict diet, or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions.

Structural and functional impact of clinically relevant E1α variants causing pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ßß') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3-100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.

Human testis-specific PDHA2 gene: methylation status of a CpG island in the open reading frame correlates with transcriptional activity.

DNA methylation is an important epigenetic modification that has profound roles in gene expression and, in particular, is thought to be crucial for regulation of tissue-specific genes in animal cells. The pivotal E(1)alpha subunit of human pyruvate dehydrogenase complex, an essential and rate-limiting enzyme system in energy metabolism, is encoded by two distinct genes: PDHA1 gene, located on chromosome X is expressed in somatic tissues, whereas PDHA2 gene, located on chromosome 4, is exclusively expressed in spermatogenic cells. The objective of this study is to elucidate the role of DNA methylation as an epigenetic mechanism controlling the regulation of PDHA2 gene expression in human tissues, namely its repression in somatic tissues and its activation in testicular germ cells. Genomic DNA was isolated from human somatic tissues (circulating lymphocytes and gastric cells) and from testis, including isolated fractions of haploid and diploid germ cells. After primer design with appropriate software, it was performed the sodium bisulfite PCR sequencing of the PDHA2 promoter and coding regions. Total RNA of the same tissues was isolated, reverse transcribed and PDHA1and PDHA2 transcripts were amplified with specific primers and analysed by agarose gel electrophoresis. The analysis of the genomic sequence of the PDHA2 gene revealed the presence of 61 CpG sites whose distribution matches the criteria for the presence of two CpG islands. Sequence analysis of both CpG islands upon bisulfite treatment displayed several differences, either between islands or among tissues. In particular, the methylation pattern of one of the CpG islands revealed a perfect correlation with transcriptional activity of the PDHA2 gene either in testis or in somatic tissues. Surprisingly, it is the full demethylation of the CpG island located in the coding region that seems to play a crucial role upon PDHA2 gene transcription in testis.

Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods.

BACKGROUND: The most frequently used methods for measuring global DNA methylation are based on two different principles: the use of methylation-sensitive restriction endonucleases followed by analysis of the obtained fragments, or the hydrolysis of genomic DNA followed by specific detection and quantification of the 5-methylcytosine content. We aimed to compare two different methods for evaluation of global DNA methylation: the cytosine extension assay after enzymatic digestion of DNA (Cyt-Ext), and a recently described method using liquid chromatography-electrospray ionization-tandem mass spectrometry after DNA hydrolysis (LC-MS/MS). METHODS: Both approaches were applied to evaluate global DNA methylation in leukocyte DNA from 96 healthy subjects. Calf thymus and pBR322 DNAs were used as hyper- and hypo-methylated references, respectively. RESULTS: Using the Cyt-Ext method, the DNA from healthy individuals showed radiolabel incorporation of 11,312±1600 Dpm/µg DNA, while the LC-MS/MS method showed 4.55±0.1% methylation. Results are shown as mean±SD. The analysis of hypo- and hyper-methylated references showed that both methods are practical for discriminating different levels of methylation. CONCLUSIONS: Cyt-Ext and LC-MS/MS are viable methods in evaluating global DNA methylation status. However, the LC-MS/MS assay allows absolute quantification and displays far superior intra-day precision. Therefore, we consider the later approach to be better for use in global DNA methylation studies.

Pyruvate dehydrogenase complex deficiency: updating the clinical, metabolic and mutational landscapes in a cohort of Portuguese patients.

BACKGROUND: The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate into acetyl-CoA. PDC deficiency can be caused by alterations in any of the genes encoding its several subunits. The resulting phenotype, though very heterogeneous, mainly affects the central nervous system. The aim of this study is to describe and discuss the clinical, biochemical and genotypic information from thirteen PDC deficient patients, thus seeking to establish possible genotype-phenotype correlations. RESULTS: The mutational spectrum showed that seven patients carry mutations in the PDHA1 gene encoding the E1α subunit, five patients carry mutations in the PDHX gene encoding the E3 binding protein, and the remaining patient carries mutations in the DLD gene encoding the E3 subunit. These data corroborate earlier reports describing PDHA1 mutations as the predominant cause of PDC deficiency but also reveal a notable prevalence of PDHX mutations among Portuguese patients, most of them carrying what seems to be a private mutation (p.R284X). The biochemical analyses revealed high lactate and pyruvate plasma levels whereas the lactate/pyruvate ratio was below 16; enzymatic activities, when compared to control values, indicated to be independent from the genotype and ranged from 8.5% to 30%, the latter being considered a cut-off value for primary PDC deficiency. Concerning the clinical features, all patients displayed psychomotor retardation/developmental delay, the severity of which seems to correlate with the type and localization of the mutation carried by the patient. The therapeutic options essentially include the administration of a ketogenic diet and supplementation with thiamine, although arginine aspartate intake revealed to be beneficial in some patients. Moreover, in silico analysis of the missense mutations present in this PDC deficient population allowed to envisage the molecular mechanism underlying these pathogenic variants. CONCLUSION: The identification of the disease-causing mutations, together with the functional and structural characterization of the mutant protein variants, allow to obtain an insight on the severity of the clinical phenotype and the selection of the most appropriate therapy.