Lethal Capecitabine Toxicity in Patients With Complete Dihydropyrimidine Dehydrogenase Deficiency Due to Ultra-Rare DPYD Variants.

A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD.

Functional analysis of thirty-four suspected pathogenic missense variants in ALDH5A1 gene associated with succinic semialdehyde dehydrogenase deficiency.

Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.

An overview of combined D-2- and L-2-hydroxyglutaric aciduria: functional analysis of CIC variants.

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.

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.

Novel cases of D-2-hydroxyglutaric aciduria with IDH1 or IDH2 mosaic mutations identified by amplicon deep sequencing.

BACKGROUND: Mosaic IDH1 mutations are described as the cause of metaphyseal chondromatosis with increased urinary excretion of D-2-hydroxyglutarate (MC-HGA), and mutations in IDH2 as the cause of D-2-hydroxyglutaric aciduria (D-2HGA) type II. Mosaicism for IDH2 mutations has not previously been reported as a cause of D-2HGA. Here we describe three cases: one MC-HGA case with IDH1 mosaic mutations, and two D-2HGA type II cases. In one D-2HGA case we identified mosaicism for an IDH2 mutation as the genetic cause of this disorder; the other D-2HGA case was caused by a heterozygous IDH2 mutation, while the unaffected mother was a mosaic carrier. METHODS: We performed amplicon deep sequencing using the 454 GS Junior platform, next to Sanger sequencing, to identify and confirm mosaicism of IDH1 or IDH2 mutations in MC-HGA or D-2HGA, respectively. RESULTS AND CONCLUSIONS: We identified different mutant allele percentages in DNA samples derived from different tissues (blood vs fibroblasts). Furthermore, we found that mutant allele percentages of IDH1 decreased after more passages had occurred in fibroblast cell cultures. We describe a method for the detection and validation of mosaic mutations in IDH1 and IDH2, making quantification with laborious cloning techniques obsolete.

Deficiency in SLC25A1, encoding the mitochondrial citrate carrier, causes combined D-2- and L-2-hydroxyglutaric aciduria.

The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are found elevated not only in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations cause one variant of 2-hydroxyglutaric aciduria. Complementary to these findings, we now report recessive mutations in SLC25A1, the mitochondrial citrate carrier, in 12 out of 12 individuals with combined D-2- and L-2-hydroxyglutaric aciduria. Impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 in fibroblasts harboring certain mutations, suggest that SLC25A1 deficiency is pathogenic. Our results identify defects in SLC25A1 as a cause of combined D-2- and L-2-hydroxyglutaric aciduria.

Characterization and crystallization of a novel haemoglobinase from pathogenic Escherichia coli.

A haemoglobin-degrading enzyme from pathogenic Escherichia coli has been cloned, expressed and purified to homogeneity. The pure protein proteolyses haemoglobin and binds haem. In vivo, its role is to remove haem from haemoglobin and pass it to the bacteria, allowing them to overcome the limiting concentration of iron available in the body. The protein has been crystallized using polyethylene glycol to give crystals in a hexagonal space group with unit-cell parameters a = b = 114.6, c = 434.3 A. X-ray data have been collected to 2.5 A resolution. This is the first member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family of autotransporter proteins to be crystallized.