Clinical Utility of LCT Genotyping in Children with Suspected Functional Gastrointestinal Disorder

Genetic testing is a good predictor of lactase persistence (LP) in specific populations but its clinical utility in children is less clear. We assessed the role of lactose malabsorption in functional gastrointestinal disorders (FGID) in children and the correlation between the lactase non-persistence (LNP) genotype and phenotype, based on exhaled hydrogen and gastrointestinal symptoms, during a hydrogen breath test (HBT). We also evaluate dairy consumption in this sample. We conducted a 10-year cross-sectional study in a cohort of 493 children with suspected FGID defined by Roma IV criteria. Distribution of the C/T-13910 genotype was as follows: CC, 46.0%; TT, 14.4% (LP allele frequency, 34.1%). The phenotype frequencies of lactose malabsorption and intolerance were 36.3% and 41.5%, respectively. We observed a strong correlation between genotype and both lactose malabsorption (Cramér’s V, 0.28) and intolerance (Cramér’s V, 0.54). The frequency of the LNP genotype (p = 0.002) and of malabsorption and intolerance increased with age (p = 0.001 and 0.002, respectively). In 61% of children, evaluated dairy consumption was less than recommended. No association was observed between dairy intake and diagnosis. In conclusion, we found a significant correlation between genotype and phenotype, greater in older children, suggesting that the clinical value of genetic testing increases with ageS

Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit

Biochemistry; Molecular medicineBioquímica; Medicina molecularBioquímica; Medicina molecularImbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance.We thank Dr, Luke Formosa (Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for his valuable advice and assistance on NDUFA10 molecular studies and Dr. Francesc Canals and his team (Proteomics Laboratory, Vall d’Hebron Institute of Oncology [VHIO], Universitat Autònoma de Barcelona, Barcelona, Spain) for their assistance with LC-MS/MS analyses. This work was supported by the Spanish Ministry of Industry, Economy and Competitiveness [grants BFU2014-52618-R, SAF2017-87506, and PID2020-112929RB-I00 to Y.C.], by the Spanish Instituto de Salud Carlos III [grants PI21/00554 and PMP15/00025 to R.M.], co-financed by the European Regional Development Fund (ERDF), and by an NHMRC Project grant to M.R. (GNT1164459)

Identification of a Novel Variant in EARS2 Associated with a Severe Clinical Phenotype Expands the Clinical Spectrum of LTBL

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient’s fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient’s clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this diseaseThis study was supported with a competitive PhD grant from a pre-Doctoral scholarship for research groups of the Health Research Institute of Santiago (IDIS)S

Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit

Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance

Identification and Characterization of New Variants in FOXRED1 Gene Expands the Clinical Spectrum Associated with Mitochondrial Complex I Deficiency

Complex I (nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase) is the largest complex of the mitochondrial oxidative phosphorylation system (OXPHOS) system. Forty-four subunits encoded in nuclear and mitochondrial genomes compose this multiprotein complex, its assembly being a highly complex process involving at least 15 additional nuclear encoded assembly factors. Complex I deficiency is a mitochondrial disorder usually associated with early-onset severe multisystem disorders characterized by highly variable clinical manifestations. Flavin adenine dinucleotide (FAD)-dependent oxidoreductase domain-containing protein 1 (FOXRED1) is a complex I assembly factor. To date, only five patients with mitochondrial complex I deficiency due to mutations in FOXRED1 have been characterized. Here, we describe a child with ataxia, epilepsy and psychomotor developmental delay carrying two heterozygous FOXRED1 variants, c.920G>A (p.Gly307Glu) and c.733+1G>A. We demonstrate the molecular mechanism supporting the pathogenicity of the FOXRED1 variants, showing a clear deficiency of complex I activity. The reduction in the steady-state level of complex I holoenzyme in patient fibroblasts, confirmed the pathogenicity of the variants and showed the molecular mechanism behind their pathogenicity. A comparison of the clinical presentation of the index case with the previously described cases allowed deepening our knowledge about the clinical variability associated with FOXRED1 defectsThis study was supported with a competitive Ph.D. grant by Pre-Doctoral scholarship, for research groups of the Health Research Institute of Santiago (IDIS)S

The search for an autoimmune origin of psychotic disorders: prevalence of autoantibodies against hippocampus antigens, glutamic acid decarboxylase and nuclear antigens

The etiology of psychotic disorders is still unknown, but in a subgroup of patients symptoms might be caused by an autoimmune reaction. In this study, we tested patterns of autoimmune reactivity against potentially novel hippocampal antigens. Serum of a cohort of 621 individuals with psychotic disorders and 257 controls were first tested for reactivity on neuropil of rat brain sections. Brain reactive sera (67 diseased, 27 healthy) were further tested for antibody binding to glutamic acid decarboxylase (GAD) isotype 65 and 67 by cell-based assay (CBA). A sub-cohort of 199 individuals with psychotic disorders and 152 controls was tested for the prevalence of anti-nuclear antibodies (ANA) on HEp2-substrate as well as for reactivity to double-stranded DNA, ribosomal P (RPP), and cardiolipin (CL). Incubation of rat brain with serum resulted in unidentified hippocampal binding patterns in both diseased and control groups. Upon screening with GAD CBA, one of these patterns was identified as GAD65 in one individual with schizophrenia and also in one healthy individual. Two diseased and two healthy individuals had low antibody levels targeting GAD67 by CBA. Antibody reactivity on HEp-2-substrate was increased in patients with schizoaffective disorder, but only in 3 patients did antibody testing hint at a possible diagnosis of systemic lupus erythematosus. Although reactivity of serum to intracellular antigens might be increased in patients with psychotic disorder, no specific targets could be identified. GAD antibodies are very rare and do not seem increased in serum of patients with psychotic disorders.HEALTH-F2-2010-241909info:eu-repo/semantics/publishedVersio

Limited dCTP Availability Accounts for Mitochondrial DNA Depletion in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored

Enfermedades mitocondriales: identificación y caracterización de mutaciones en los genes POLG y mtRNA Lys

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina,Departamento de Bioquímica. Fecha de lectura: 19 de Febrero de 200

Clinical Utility of LCT Genotyping in Children with Suspected Functional Gastrointestinal Disorder

Genetic testing is a good predictor of lactase persistence (LP) in specific populations but its clinical utility in children is less clear. We assessed the role of lactose malabsorption in functional gastrointestinal disorders (FGID) in children and the correlation between the lactase non-persistence (LNP) genotype and phenotype, based on exhaled hydrogen and gastrointestinal symptoms, during a hydrogen breath test (HBT). We also evaluate dairy consumption in this sample. We conducted a 10-year cross-sectional study in a cohort of 493 children with suspected FGID defined by Roma IV criteria. Distribution of the C/T-13910 genotype was as follows: CC, 46.0%; TT, 14.4% (LP allele frequency, 34.1%). The phenotype frequencies of lactose malabsorption and intolerance were 36.3% and 41.5%, respectively. We observed a strong correlation between genotype and both lactose malabsorption (Cramér’s V, 0.28) and intolerance (Cramér’s V, 0.54). The frequency of the LNP genotype (p = 0.002) and of malabsorption and intolerance increased with age (p = 0.001 and 0.002, respectively). In 61% of children, evaluated dairy consumption was less than recommended. No association was observed between dairy intake and diagnosis. In conclusion, we found a significant correlation between genotype and phenotype, greater in older children, suggesting that the clinical value of genetic testing increases with age

Spotlight on the relevance of mtDNA in cancer

The potential role of the mitochondrial genome has recently attracted interest because of its high mutation frequency in tumors. Different aspects of mtDNA make it relevant for cancer‘s biology, such as it encodes a limited but essential number of genes for OXPHOS biogenesis, it is particularly susceptible to mutations, and its copy number can vary. Moreover, most ROS in mitochondria are produced by the electron transport chain. These characteristics place the mtDNA in the center of multiple signaling pathways, known as mitochondrial retrograde signaling, which modifies numerous key processes in cancer. Cybrid studies support that mtDNA mutations are relevant and exert their effect through a modification of OXPHOS function and ROS production. However, there is still much controversy regarding the clinical relevance of mtDNA mutations. New studies should focus more on OXPHOS dysfunction associated with a specific mutational signature rather than the presence of mutations in the mtDNA.Work in the authors’ laboratories is supported by “Instituto de Salud Carlos III” [PI13/01806 and PIE14/0064 to M.P., PI10/0703 and PI13/00556 to R.G. and PI04/1001 to M.A.F.M.]; “Comunidad Autónoma de Madrid” [S2010/BMD-2402 to R.G.]; “Fundación Mutua Madrileña” [10.04.02.0064 to M.A.F.M.].Peer Reviewe