Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particularly important in the neonatal period due to ingestion of galactose-containing milk. GALT deficiency results in the genetic disorder classic galactosemia, whose pathophysiology is still not fully elucidated. Whereas classic galactosemia has been hypothesized to result from GALT misfolding, a thorough functional–structural characterization of GALT most prevalent variants was still lacking, hampering the development of alternative therapeutic approaches. The aim of this study was to investigate the structural–functional effects of nine GALT mutations, four of which account for the vast majority of the mutations identified in galactosemic patients. Several methodologies were employed to evaluate the mutations' impact on GALT function, on the protein secondary and tertiary structures, and on the aggregation propensity. The major structural effect concerns disturbed propensity for aggregation, particularly striking for the p.Q188R variant, resulting from the most frequent (∼60%) allele at a worldwide scale. The absence of major effects at the secondary and tertiary structure levels suggests that the disturbed aggregation results from subtle perturbations causing a higher and/or longer exposure of hydrophobic residues in the variants as compared to WT GALT. The results herein described indicate a possible benefit from introducing proteostasis regulators and/or chemical/pharmacological chaperones to prevent the accumulation of protein aggregates, in new avenues of therapeutic research for classic galactosemia.

Classic galactosemia is a rare genetic disease of the galactose metabolism, resulting from deficient activity of galactose-1-phosphate uridylyltransferase (GALT). The current standard of care is lifelong dietary restriction of galactose, which however fails to prevent the development of long-term complications. Structural-functional studies demonstrated that the most prevalent GALT mutations give rise to proteins with increased propensity to aggregate in solution. Arginine is a known stabilizer of aggregationprone proteins, having already shown a beneficial effect in other inherited metabolic disorders.Herein we developed a prokaryotic model of galactose sensitivity that allows evaluating in a cellular context the mutations' impact on GALT function, as well as the potential effect of arginine in functionally rescuing clinically relevant variants.This study revealed that some hGALT variants, previously described to exhibit no detectable activity in vitro, actually present residual activity when determined in vivo.

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.

Human PDHA2 is a testis-specific gene that codes for the E1α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the −122 to −6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2′-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy.

In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820 þ 13A4G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. European Journal of Human Genetics (2015) 23, 500-506; doi:10.1038/ejhg.2014.149; published online 23 July 2014 INTRODUCTIONOver the last years, splicing mutations emerged as an important pathogenic mechanism, underlying 10-30% of genetic diseases (HGMD Professional 2013.1). 1 Splicing accuracy depends not only on the recognition of exon-intron junctions, defined by intronic ciselements: 5 0 splice site, 3 0 splice site, branch site and poly-pyrimidine tract, 2-4 but also on more discrete elements, entitled splicing regulatory elements, which direct the splicing machinery to use the correct splice sites. Exonic and intronic splicing enhancers stimulate splicing and serve as binding sites mainly for serine/arginine-rich proteins. Exonic and intronic splicing silencers repress splicing, and often function by binding proteins from the heterogenous nuclear ribonucleoprotein family. 2,4-6 Although most reported splicing mutations directly abolish an authentic splice site or create a novel one, an increasing number of disease-associated variations that alter splicing enhancers or silencers have been reported. 2,7-9 Each nucleotide modification should be considered a potential candidate for splicing alterations, as not only intronic but also nonsense, missense and silent modifications may impact splicing. 7 Accordingly, constitutive and regulated splicing reactions are considered potential therapeutic targets and novel strategies for their correction are evolving. Among these, antisense oligonucleotides display an exquisi...

our results encourage the use of amino acid supplementation to overcome the metabolic/biochemical changes induced by PDHA1 gene specific mutations associated with mild PDHc phenotypes.

No abstract

This article presents a dataset proving the simultaneous presence of a 5′UTR-truncated PDHA1 mRNA and a full-length PDHA2 mRNA in the somatic cells of a PDC-deficient female patient and all members of her immediate family (parents and brother).We have designed a large set of primer pairs in order to perform detailed RT-PCR assays allowing the clear identification of both PDHA1 and PDHA2 mRNA species in somatic cells. In addition, two different experimental approaches were used to elucidate the copy number of PDHA1 gene in the patient and her mother.The interpretation and discussion of these data, along with further extensive experiments concerning the origin of this altered gene expression and its potential therapeutic consequences, can be found in “Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells” (A. Pinheiro, M.J. Silva, C. Florindo, et al., 2016) [1].