Clinical and molecular characterization of adult patients with late-onset MTHFR deficiency.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency usually presents as a severe neonatal disease. This study aimed to characterize natural history, biological and molecular data, and response to treatment of patients with late-onset MTHFR deficiency. The patients were identified through the European Network and Registry for Homocystinuria and Methylation Defects and the Adult group of the French Society for Inherited Metabolic Diseases; data were retrospectively colleted. To identify juvenile to adult-onset forms of the disease, we included patients with a diagnosis established after the age of 10 years. We included 14 patients (median age at diagnosis: 32 years; range: 11-54). At onset (median age: 20 years; range 9-38), they presented with walking difficulties (n = 8), cognitive decline (n = 3) and/or seizures (n = 3), sometimes associated with mild mental retardation (n = 6). During the disease course, symptoms were almost exclusively neurological with cognitive dysfunction (93%), gait disorders (86%), epilepsy (71%), psychiatric symptoms (57%), polyneuropathy (43%), and visual deficit (43%). Mean diagnostic delay was 14 years. Vascular events were observed in 28% and obesity in 36% of the patients. One patient remained asymptomatic at the age of 55 years. Upon treatment, median total homocysteine decreased (from 183 µmol/L, range 69-266, to 90 µmol/L, range 20-142) and symptoms improved (n = 9) or stabilized (n = 4). Missense pathogenic variants in the C-terminal regulatory domain of the protein were over-represented compared to early-onset cases. Residual MTHFR enzymatic activity in skin fibroblasts (n = 4) was rather high (17%-58%). This series of patients with late-onset MTHFR deficiency underlines the still unmet need of a prompt diagnosis of this treatable disease.

Exome Sequencing and the Management of Neurometabolic Disorders.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Clinical or ATPase domain mutations in ABCD4 disrupt the interaction between the vitamin B12-trafficking proteins ABCD4 and LMBD1.

Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.

High-resolution capillary zone electrophoresis for transferrin glycoform analysis associated with congenital disorders of glycosylation.

High-resolution capillary zone electrophoresis is used to assess the transferrin profile in serum of patients with eight different congenital disorders of glycosylation that represent type I, type II, and mixed type I/II disorders. Capillary zone electrophoresis data are compared to patterns obtained by gel isoelectric focusing. The high-resolution capillary zone electrophoresis method is shown to represent an effective tool to assess the diversity of transferrin patterns. Hypoglycosylated disialo-, monosialo-, and asialo-transferrin in type I cases can be distinguished from the corresponding underdesialylated transferrin glycoforms present in type II disorders. The latter can be separated from and detected ahead of their corresponding hypoglycosylated forms of type I patients. Both types of glycoforms are detected in sera of mixed type I/II patients. The assay has the potential to be used as screening method for congenital disorders of glycosylation. It can be run with a few microliters of serum when microvials are used.

Novel Mouse Models of Methylmalonic Aciduria Recapitulate Phenotypic Traits with a Genetic Dosage Effect.

Methylmalonic aciduria (MMAuria), caused by deficiency of methylmalonyl-CoA mutase (MUT), usually presents in the newborn period with failure to thrive and metabolic crisis leading to coma or even death. Survivors remain at risk of metabolic decompensations and severe long term complications, notably renal failure and neurological impairment. We generated clinically relevant mouse models of MMAuria using a constitutive Mut knock-in (KI) allele based on the p.Met700Lys patient mutation, used homozygously (KI/KI) or combined with a knockout allele (KO/KI), to study biochemical and clinical MMAuria disease aspects. Transgenic Mut(ki/ki) and Mut(ko/ki) mice survive post-weaning, show failure to thrive, and show increased methylmalonic acid, propionylcarnitine, odd chain fatty acids, and sphingoid bases, a new potential biomarker of MMAuria. Consistent with genetic dosage, Mut(ko/ki) mice have lower Mut activity, are smaller, and show higher metabolite levels than Mut(ki/ki) mice. Further, Mut(ko/ki) mice exhibit manifestations of kidney and brain damage, including increased plasma urea, impaired diuresis, elevated biomarkers, and changes in brain weight. On a high protein diet, mutant mice display disease exacerbation, including elevated blood ammonia, and catastrophic weight loss, which, in Mut(ki/ki) mice, is rescued by hydroxocobalamin treatment. This study expands knowledge of MMAuria, introduces the discovery of new biomarkers, and constitutes the first in vivo proof of principle of cobalamin treatment in mut-type MMAuria.

Multiple phenotypes in phosphoglucomutase 1 deficiency.

BACKGROUND: Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS: Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS: Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS: Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).

Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system.

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing.

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo-transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate-deficient transferrin or by capillary isoelectric focusing in a pH 5-8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.

Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells.

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations.

Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency.

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.

Hepatic glycogen storage disorders: what have we learned in recent years?

PURPOSE OF REVIEW: Glycogen storage disorders (GSDs) are inborn errors of metabolism with abnormal storage or utilization of glycogen. The present review focuses on recent advances in hepatic GSD types I, III and VI/IX, with emphasis on clinical aspects and treatment. RECENT FINDINGS: Evidence accumulates that poor metabolic control is a risk factor for the development of long-term complications, such as liver adenomas, low bone density/osteoporosis, and kidney disease in GSD I. However, mechanisms leading to these complications remain poorly understood and are being investigated. Molecular causes underlying neutropenia and neutrophil dysfunction in GSD I have been elucidated. Case series provide new insights into the natural course and outcome of GSD types VI and IX. For GSD III, a high protein/fat diet has been reported to improve (cardio)myopathy, but the beneficial effect of this dietary concept on muscle and liver disease manifestations needs to be further established in prospective studies. SUMMARY: Although further knowledge has been gained regarding pathophysiology, disease course, treatment, and complications of hepatic GSDs, more controlled prospective studies are needed to assess effects of different dietary and medical treatment options on long-term outcome and quality of life.

Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism.

The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patient's fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.

Characterization of functional domains of the cblD (MMADHC) gene product.

In humans vitamin B12 (cobalamin, Cbl) must be converted into two coenzyme forms, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), in order to maintain intracellular homeostasis of homocysteine and methylmalonic acid, respectively. Previously we have shown that in cblD patients three types of MMADHC mutations exist: 1) null mutations N-terminal to Met116 cause isolated methylmalonic aciduria (cblD-MMA) due to AdoCbl deficiency; 2) null mutations across the C-terminus (p.Y140-R250) cause combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC) due to AdoCbl and MeCbl deficiency; 3) missense mutations in a conserved C-terminal region (p.D246-L259) cause isolated homocystinuria (cblD-HC) due to MeCbl deficiency. To better understand the domain boundaries related to MeCbl formation, we made selected point mutations and C-terminal truncations in MMADHC and tested rescue of MeCbl and AdoCbl synthesis in immortalized cblD-MMA/HC patient fibroblasts. Testing 20 mutations (15 missense and five C-terminal truncations) across p.P154-S287 revealed the presence of a region (p.R197-D226) responsible for MeCbl synthesis, which gave a similar cellular phenotype as cblD-HC. Further, mutation of the polypeptide stretch between the new and patient defined regions (p.D226-D246) and directly C-terminal to the patient region (p.L259-R266), gave cellular phenotypes intermediate to those of cblD-HC and cblD-MMA/HC. Finally, C-terminal truncation of more than 20 amino acids resulted in a cblD-MMA/HC like cellular phenotype, while truncation of between ten and 20 amino acids resulted in a cblD-HC like cellular phenotype. These data suggest that specific regions of MMADHC are involved in differential regulation of AdoCbl and MeCbl synthesis and help better define the boundaries of these regions.

A single mutation in MCCC1 or MCCC2 as a potential cause of positive screening for 3-methylcrotonyl-CoA carboxylase deficiency.

Isolated 3-Methylcrotonyl-CoA carboxylase deficiency (MCC deficiency) is an organic aciduria presenting with a highly variable phenotype and has been part of newborn screening programs in various countries, in particular in the US. Here we present enzymatic and genetic characterisation of 22 individuals with increased 3-hydroxyisovalerylcarnitine and/or 3-methylcrotonylglycine suggesting MCC deficiency, but only partially reduced 3-methylcrotonyl-CoA carboxylase activity. Among these, 21 carried a single mutant allele in either MCCC1 (n=20) or MCCC2 (n=1). Our results suggest that heterozygosity for such a single deleterious mutation may lead to misdiagnosis of MCC deficiency.

Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation.

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patient's fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairments.

Is serum biotinidase enzyme activity a potential marker of perturbed glucose and lipid metabolism?

Glycogen storage diseases (GSDs) belong to the group of inborn errors of carbohydrate metabolism. Hepatic GSDs predominantly involve the liver and most present with hepatomegaly. Biochemically they show known disturbances in glucose and fatty acids metabolism, namely fasting hypoglycaemia and increased triglycerides. Additionally, increased biotinidase (BTD) enzyme activity has been shown to be associated with many GSD types, whereas the mechanism by which BTD enzyme activity is altered remains unknown so far. We aimed to delineate changes in gluconeogenesis and fatty acid synthesis, potentially explaining raised BTD enzyme activity, by using liver (specimens from 2 patients) and serum samples of GSD Ia and GSD IV patients. By expression analysis of genes involved in gluconeogenesis, we ascertained increased levels of phosphoenolpyruvate carboxykinase and fructose-1,6-biphosphatase, indicating an increased flux through the gluconeogenic pathway. Additionally, we found increased gene expression of the biotin-dependent pyruvate and acetyl-CoA carboxylases, providing substrate for gluconeogenesis and increased fatty acid synthesis. We also observed a significant linear correlation between BTD enzyme activity and triglyceride levels in a cohort of GSD Ia patients. The results of this pilot study suggest that enhancement of BTD activity might serve the purpose of providing extra cofactor to the carboxylase enzymes as an adjustment to disturbed glucose and fatty acid metabolism. Future studies involving a higher number of samples should aim at confirming the here proposed mechanism, which extends the application of BTD enzyme activity measurement beyond its diagnostic purpose in suspected GSD, and opens up possibilities for its use as a sensor for increased gluconeogenesis and fatty acid synthesis.

Severe Distal Motor Involvement in a Non-compliant Adult With Biotinidase Deficiency: The Necessity of Life-Long Biotin Therapy.

Biotinidase deficiency is an autosomal recessive disorder in which affected individuals are unable to recycle biotin. Untreated, children usually exhibit hypotonia, seizures, ataxia, developmental delay, and/or hearing loss. Individuals diagnosed by newborn screening have an excellent prognosis with life-long biotin supplementation. We report a young adult diagnosed with profound biotinidase deficiency by newborn screening who was asymptomatic while on therapy. At 18 years of age, 6 months after voluntarily discontinuation of biotin, he developed a progressive distal muscle weakness. Molecular analysis of the BTD gene showed a pathogenic homozygous duplication c.1372_1373dupT p.(Cys458LeufsTer26) (1). Despite 16 months since reintroduction of biotin, muscle strength only partially recovered. Transition to adulthood in chronic metabolic diseases is known to be associated with an increased risk for non-compliance. Neurological findings in this adult are similar to those described in others with adult-onset biotinidase deficiency. Long-term prognosis in non-compliant symptomatic adult with biotinidase deficiency likely depends on the delay and/or severity of intervening symptoms until reintroduction of biotin.

Induced Pluripotent Stem Cells to Understand Mucopolysaccharidosis. I: Demonstration of a Migration Defect in Neural Precursors.

Background: Mucopolysaccharidosis type I-Hurler (MPS1-H) is a severe genetic lysosomal storage disorder due to loss-of-function mutations in the IDUA gene. The subsequent complete deficiency of alpha l-iduronidase enzyme is directly responsible of a progressive accumulation of glycosaminoglycans (GAG) in lysosomes which affects the functions of many tissues. Consequently, MPS1 is characterized by systemic symptoms (multiorgan dysfunction) including respiratory and cardiac dysfunctions, skeletal abnormalities and early fatal neurodegeneration. Methods: To understand mechanisms underlying MPS1 neuropathology, we generated induced pluripotent stem cells (iPSC) from a MPS1-H patient with loss-of-function mutations in both IDUA alleles. To avoid variability due to different genetic background of iPSC, we established an isogenic control iPSC line by rescuing IDUA expression by a lentivectoral approach. Results: Marked differences between MPS1-H and IDUA-corrected isogenic controls were observed upon neural differentiation. A scratch assay revealed a strong migration defect of MPS1-H cells. Also, there was a massive impact of IDUA deficiency on gene expression (340 genes with an FDR <0.05). Conclusions: Our results demonstrate a hitherto unknown connection between lysosomal degradation, gene expression and neural motility, which might account at least in part for the phenotype of MPS1-H patients.