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.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

BACKGROUND AND AIMS: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). MATERIALS AND METHODS: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. RESULTS: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. CONCLUSION: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

Polar metabolomics in human muscle biopsies using a liquid-liquid extraction and full-scan LC-MS.

We describe here a user-friendly analysis protocol for semi-targeted polar metabolomics in human muscle biopsies using Zwitterionic Hydrophilic Interaction Liquid Chromatography and high-resolution full-scan mass spectrometry. Previously, this protocol has been used for Caenorhabditis elegans. Here we show that it can be successfully applied to human muscle biopsies with minor adjustments. Summarized instructions for other matrices are also provided. As peak integration in metabolomics can be challenging, we provide expected retention times and extensive peak descriptions to aid this process. For complete details on the use and execution of this protocol, please refer to Molenaars et al. (2021).

Identification of three novel pathogenic mutations in cystathionine beta-synthase gene of Pakistani intellectually disabled patients.

BACKGROUND: Classical homocystinuria (HCU) is an autosomal recessive inborn error of metabolism, which is caused by the cystathionine-ß-synthase (CBS: encoded by CBS) deficiency. Symptoms of untreated classical HCU patients include intellectual disability (ID), ectopia lentis and long limbs, along with elevated plasma methionine, and homocysteine. METHODS: A total of 429 ID patients (age range: 1.6-23 years) were sampled from Northern areas of Punjab, Pakistan. Biochemical and genetic analyses were performed to find classical HCU disease in ID patients. RESULTS: Biochemically, nine patients from seven unrelated families were identified with high levels of plasma methionine and homocysteine. Targeted exonic analysis of CBS confirmed seven causative homozygous mutations; of which three were novel missense mutations (c.451G>T; p.Gly151Trp, c.975G>C; p.Lys325Asn and c.1039 + 1G>T splicing), and four were recurrent variants (c.451 + 1G>A; IVS4 + 1 splicing, c.770C>T; p.Thr257Met, c.808_810del GAG; p.Glu270del and c.752T>C; p.Leu251Pro). Treatment of patients was initiated without further delay with pyridoxine, folic acid, cobalamin, and betaine as well as dietary protein restriction. The immediate impact was noticed in behavioral improvement, decreased irritability, improved black hair color, and socialization. Overall, health outcomes in this disorder depend on the age and symptomatology at the time of treatment initiation. CONCLUSIONS: With personalized treatment and care, such patients can reach their full potential of living as healthy a life as possible. This screening study is one of the pioneering initiatives in Pakistan which would help to minimize the burden of such treatable inborn errors of metabolism in the intellectually disabled patients.

Is impaired energy production a novel insight into the pathogenesis of pyridoxine-dependent epilepsy due to biallelic variants in ALDH7A1?

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. METHODS: We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. RESULTS: We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. DISCUSSION: We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.

Preemptive screening of DPYD as part of clinical practice: high prevalence of a novel exon 4 deletion in the Finnish population.

Capecitabine is a fluoropyrimidine that is widely used as a cancer drug for the treatment of patients with a variety of cancers. Unfortunately, early onset, severe or life-threatening toxicity is observed in 19-32% of patients treated with capecitabine and 5FU. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the degradation of 5FU and a DPD deficiency has been shown to be a major determinant of severe fluoropyrimidine-associated toxicity. DPD is encoded by the DPYD gene and some of the identified variants have been described to cause DPD deficiency. Preemptive screening for DPYD gene alterations enables the identification of DPD-deficient patients before administering fluoropyrimidines. In this article, we describe the application of upfront DPD screening in Finnish patients, as a part of daily clinical practice, which was based on a comprehensive DPYD gene analysis, measurements of enzyme activity and plasma uracil concentrations. Almost 8% of the patients (13 of 167 patients) presented with pathogenic DPYD variants causing DPD deficiency. The DPD deficiency in these patients was further confirmed via analysis of the DPD activity and plasma uracil levels. Interestingly, we identified a novel intragenic deletion in DPYD which includes exon 4 in four patients (31% of patients carrying a pathogenic variant). The high prevalence of the exon 4 deletion among Finnish patients highlights the importance of full-scale DPYD gene analysis. Based on the literature and our own experience, genotype preemptive screening should always be used to detect DPD-deficient patients before fluoropyrimidine therapy.

Rescue of respiratory failure in pulmonary alveolar proteinosis due to pathogenic MARS1 variants.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a heterogeneous condition with more than 100 different underlying disorders that need to be differentiated to target therapeutic options, which are generally limited. METHODS: The clinical course of two brothers with pathogenic variants in the methionyl-tRNA synthetase (MARS)1 gene was compared to previously published patients. Functional studies in patient-derived fibroblasts were performed and therapeutic options evaluated. RESULTS: The younger brother was diagnosed with PAP at the age of 1 year. Exome sequencing revealed the homozygous MARS1 variant p.(Arg598Cys), leading to interstitial lung and liver disease (ILLD). At 2 years of age, following surgery hypoglycemia was detected, the pulmonary condition deteriorated, and the patient developed multiorgan failure. Six therapeutic whole lung lavages (WLL) were necessary to improve respiratory insufficiency. Methionine supplementation was started and a high protein diet ensured, leading to complete respiratory recovery. The older brother, homozygous for the same MARS1 variant, had a long-known distinct eating preference of methionine-rich food and showed a less severe clinical phenotype. Decreased aminoacylation activity confirmed the pathogenicity of p.(Arg598Cys) in vitro. In agreement with our review of currently published ILLD patients, the presence of hepatopathy, developmental delay, muscular hypotonia, and anemia support the multisystemic character of the disease. CONCLUSIONS: Catabolic events can provoke a severe deterioration of the pulmonary situation in ILLD with a need for repetitive WLL. Although the precise role of oral methionine supplementation and high protein intake are unknown, we observed an apparent treatment benefit, which needs to be evaluated systematically in controlled trials.

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.

Bi-allelic TARS Mutations Are Associated with Brittle Hair Phenotype.

Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEß), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.

Cysteinyl-tRNA Synthetase Mutations Cause a Multi-System, Recessive Disease That Includes Microcephaly, Developmental Delay, and Brittle Hair and Nails.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.

IDH1-mutated transgenic zebrafish lines: An in-vivo model for drug screening and functional analysis.

INTRODUCTION: The gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants. MATERIALS AND METHODS: IDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage. RESULTS: In total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity. CONCLUSIONS: We have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.

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.

In vitro modeling of experimental succinic semialdehyde dehydrogenase deficiency (SSADHD) using brain-derived neural stem cells.

We explored the utility of neural stem cells (NSCs) as an in vitro model for evaluating preclinical therapeutics in succinic semialdehyde dehydrogenase-deficient (SSADHD) mice. NSCs were obtained from aldh5a1+/+ and aldh5a1-/- mice (aldh5a1 = aldehyde dehydrogenase 5a1 = SSADH). Multiple parameters were evaluated including: (1) production of GHB (γ-hydroxybutyrate), the biochemical hallmark of SSADHD; (2) rescue from cell death with the dual mTOR (mechanistic target of rapamycin) inhibitor, XL-765, an agent previously shown to rescue aldh5a1-/- mice from premature lethality; (3) mitochondrial number, total reactive oxygen species, and mitochondrial superoxide production, all previously documented as abnormal in aldh5a1-/- mice; (4) total ATP levels and ATP consumption; and (5) selected gene expression profiles associated with epilepsy, a prominent feature in both experimental and human SSADHD. Patterns of dysfunction were observed in all of these parameters and mirrored earlier findings in aldh5a1-/- mice. Patterns of dysregulated gene expression between hypothalamus and NSCs centered on ion channels, GABAergic receptors, and inflammation, suggesting novel pathomechanisms as well as a developmental ontogeny for gene expression potentially associated with the murine epileptic phenotype. The NSC model of SSADHD will be valuable in providing a first-tier screen for centrally-acting therapeutics and prioritizing therapeutic concepts of preclinical animal studies applicable to SSADHD.

Guanidinoacetate Methyltransferase Activity in Lymphocytes, for a Fast Diagnosis.

INTRODUCTION: Guanidinoacetate methyltransferase (GAMT) deficiency is an inborn error of metabolism (IEM), clinically characterized by intellectual disability, developmental delay, seizures, and movement disorders. Biochemical diagnosis of GAMT deficiency is based on the measurement of creatine and guanidinoacetate in urine, plasma, or CSF and is confirmed genetically by DNA analysis or by enzyme assay in lymphoblasts or fibroblasts. To obtain enough cells, these cells need to be cultured for at least 1 month. A less time-consuming diagnostic functional test is needed, since GAMT deficiency is a candidate for newborn screening (NBS) programs, to be able to confirm or rule out this IEM after an initial positive result in the NBS. METHODS: Stable-isotope-labeled 13C2-guanidinoacetate and 2H3-S-adenosylmethionine (SAM) were used, which are converted by GAMT present in lymphocyte extracts into 2H3-13C2-creatine. The formed 2H3-13C2-creatine was butylated and subsequently measured by liquid chromatography tandem mass-spectrometry (LC-MS/MS). RESULTS: We measured GAMT enzyme activity in lymphocyte extracts of 24 controls, 3 GAMT deficient patients and of 2 parents proven to be carrier. Because GAMT activity decreases when isolation time after venipuncture increases, reference values were obtained for 2 control groups: isolation on the day of venipuncture (27-130 pmol/h/mg) and 1 day afterwards (15-146 pmol/h/mg). Deficient patients had no detectable GAMT activity. The two carriers had GAMT activity within the normal range. CONCLUSION: We designed a fast, less invasive, and valid method to measure GAMT activity in lymphocytes using LC-MS/MS analysis without the need of time-consuming and laborious cell culture.

Gallbladder and the risk of polyps and carcinoma in metachromatic leukodystrophy.

OBJECTIVES: To assess frequency of gallbladder polyposis and carcinoma in metachromatic leukodystrophy (MLD). METHODS: We evaluated 34 patients with MLD (average age 16.7 years, age range 2-39 years) screened for gallbladder abnormalities by ultrasound. In the case of cholecystectomy, findings at pathology were reviewed. RESULTS: Only 8 of 34 patients (23%) had a normal gallbladder at ultrasound. Gallbladder polyps were visible in 8 patients (23%). Cholecystectomy was performed in 11 patients (32%). In these, pathology revealed various abnormalities, including hyperplastic polyps, intestinal metaplasia, prominent Rokitansky-Aschoff sinuses, and sulfatide storage. CONCLUSIONS: Our results demonstrate that gallbladder involvement is the rule rather than the exception in MLD. The high prevalence of hyperplastic polyps, a known precancerous condition, and one death from gallbladder carcinoma at a young age suggest that MLD predisposes to neoplastic gallbladder abnormalities. As novel therapies for this patient group are emerging leading to increased life expectancy, we recommend screening for gallbladder abnormalities by ultrasound in order to prevent early death.

Mutations in TKT Are the Cause of a Syndrome Including Short Stature, Developmental Delay, and Congenital Heart Defects.

Whole-exome sequencing (WES) is increasingly being utilized to diagnose individuals with undiagnosed disorders. Developmental delay and short stature are common clinical indications for WES. We performed WES in three families, using proband-parent trios and two additional affected siblings. We identified a syndrome due to an autosomal-recessively inherited deficiency of transketolase, encoded by TKT, on chromosome 3p21. Our series includes three families with a total of five affected individuals, ranging in age from 4 to 25 years. Two families of Ashkenazi Jewish ancestry were homozygous for an 18 base pair in-frame insertion in TKT. The third family was compound heterozygous for nonsense and missense variants in TKT. All affected individuals had short stature and were developmentally delayed. Congenital heart defects were noted in four of the five affected individuals, and there was a history of chronic diarrhea and cataracts in the older individuals with the homozygous 18 base pair insertion. Enzymatic testing confirmed significantly reduced transketolase activity. Elevated urinary excretion of erythritol, arabitol, ribitol, and pent(ul)ose-5-phosphates was detected, as well as elevated amounts of erythritol, arabitol, and ribitol in the plasma of affected individuals. Transketolase deficiency reduces NADPH synthesis and nucleic acid synthesis and cell division and could explain the problems with growth. NADPH is also critical for maintaining cerebral glutathione, which might contribute to the neurodevelopmental delays. Transketolase deficiency is one of a growing list of inborn errors of metabolism in the non-oxidative part of the pentose phosphate pathway.

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.

Recessive ITPA mutations cause an early infantile encephalopathy.

OBJECTIVE: To identify the etiology of a novel, heritable encephalopathy in a small group of patients. METHODS: Magnetic resonance imaging (MRI) pattern analysis was used to select patients with the same pattern. Homozygosity mapping and whole exome sequencing (WES) were performed to find the causal gene mutations. RESULTS: Seven patients from 4 families (2 consanguineous) were identified with a similar MRI pattern characterized by T2 signal abnormalities and diffusion restriction in the posterior limb of the internal capsule, often also optic radiation, brainstem tracts, and cerebellar white matter, in combination with delayed myelination and progressive brain atrophy. Patients presented with early infantile onset encephalopathy characterized by progressive microcephaly, seizures, variable cardiac defects, and early death. Metabolic testing was unrevealing. Single nucleotide polymorphism array revealed 1 overlapping homozygous region on chromosome 20 in the consanguineous families. In all patients, WES subsequently revealed recessive predicted loss of function mutations in ITPA, encoding inosine triphosphate pyrophosphatase (ITPase). ITPase activity in patients' erythrocytes and fibroblasts was severely reduced. INTERPRETATION: Until now ITPA variants have only been associated with adverse reactions to specific drugs. This is the first report associating ITPA mutations with a human disorder. ITPase is important in purine metabolism because it removes noncanonical nucleotides from the cellular nucleotide pool. Toxicity of accumulated noncanonical nucleotides, leading to neuronal apoptosis and interference with proteins normally using adenosine triphosphate/guanosine triphosphate, probably explains the disease. This study confirms that combining MRI pattern recognition to define small, homogeneous patient groups with WES is a powerful approach for providing a fast diagnosis in patients with an unclassified genetic encephalopathy.

Estimated carrier frequency of creatine transporter deficiency in females in the general population using functional characterization of novel missense variants in the SLC6A8 gene.

Creatine transporter deficiency (CRTR-D) is an X-linked inherited disorder of creatine transport. All males and about 50% of females have intellectual disability or cognitive dysfunction. Creatine deficiency on brain proton magnetic resonance spectroscopy and elevated urinary creatine to creatinine ratio are important biomarkers. Mutations in the SLC6A8 gene occur de novo in 30% of males. Despite reports of high prevalence of CRTR-D in males with intellectual disability, there are no true prevalence studies in the general population. To determine carrier frequency of CRTR-D in the general population we studied the variants in the SLC6A8 gene reported in the Exome Variant Server database and performed functional characterization of missense variants. We also analyzed synonymous and intronic variants for their predicted pathogenicity using in silico analysis tools. Nine missense variants were functionally analyzed using transient transfection by site-directed mutagenesis with In-Fusion HD Cloning in HeLa cells. Creatine uptake was measured by liquid chromatography tandem mass spectrometry for creatine measurement. The c.1654G>T (p.Val552Leu) variant showed low residual creatine uptake activity of 35% of wild type transfected HeLa cells and was classified as pathogenic. Three variants (c.808G>A; p.Val270Met, c.942C>G; p.Phe314Leu and c.952G>A; p.Ala318Thr) were predicted to be pathogenic based on in silico analysis, but proved to be non-pathogenic by our functional analysis. The estimated carrier frequency of CRTR-D was 0.024% in females in the general population. We recommend functional studies for all novel missense variants by transient transfection followed by creatine uptake measurement by liquid chromatography tandem mass spectrometry as fast and cost effective method for the functional analysis of missense variants in the SLC6A8 gene.

Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.