Myopathy due to carnitine palmitoyltransferase II deficiency: updating genetic aspects of the first publication in Brazil.

Carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive inherited disorder related to lipid metabolism affecting skeletal muscle. The first cases of CPT II deficiency causing myopathy were reported in 1973. In 1983, Werneck et al published the first two Brazilian patients with myopathy due to CPT II deficiency, where the biochemical analysis confirmed deficient CPT activity in the muscle of both cases. Over the past 40 years since the pioneering publication, clinical phenotypes and genetic loci in the CPT2 gene have been described, and pathogenic mechanisms have been better elucidated. Genetic analysis of one of the original cases disclosed compound heterozygous pathogenic variants (p.Ser113Leu/p.Pro50His) in the CPT2 gene. Our report highlights the historical aspects of the first Brazilian publication of the myopathic form of CPT II deficiency and updates the genetic background of this pioneering publication.

Deficiência de carnitina palmitoiltransferase II (CPT II) é uma desordem de herança autossômica recessiva relacionada com o metabolismo do lipídio afetando músculo esquelético. Os primeiros dois casos de deficiência de CPT II causando miopatia foram relatados em 1973. Em 1983, Werneck et al. publicaram os primeiros pacientes brasileiros com miopatia por deficiência de CPT II, nos quais a análise bioquímica confirmou a atividade deficiente da CPT nos músculos em ambos os casos. Após 40 anos desde a publicação pioneira, fenótipos clínicos e loci genético no gene CPT2 foram descritos, bem com os mecanismos patológicos foram melhor elucidados. A análise genética de um dos casos da publicação original apresentou variantes patogênicas em heterozigose composta (p.Ser113Leu/p.Pro50His) no gene CPT2. O nosso relato destaca os aspectos históricos da primeira publicação brasileira da forma miopática da deficiência de CPT II e atualiza as bases genéticas dessa publicação pioneira.

Severe neonatal MEGDHEL syndrome with a homozygous truncating mutation in SERAC1.

In the diagnostic work-up of a newborn infant with a metabolic crisis, lethal multiorgan failure on day six of life, and increased excretion of 3-methylglutaconic acid, we found using whole genome sequencing a homozygous SERAC1 mutation indicating MEGDHEL syndrome (3-methylglutaconic aciduria with deafness-dystonia, hepatopathy, encephalopathy, and Leigh-like syndrome). The SERAC1 protein is located at the contact site between mitochondria and the endoplasmic reticulum (ER) and is crucial for cholesterol trafficking. Our aim was to investigate the effect of the homozygous truncating mutation on mitochondrial structure and function. In the patient fibroblasts, no SERAC1 protein was detected, the mitochondrial network was severely fragmented, and the cristae morphology was altered. Filipin staining showed uneven localization of unesterified cholesterol. The calcium buffer function between cytoplasm and mitochondria was deficient. In liver mitochondria, complexes I, III, and IV were clearly decreased. In transfected COS-1 cells the mutant protein with the a 45-amino acid C-terminal truncation was distributed throughout the cell, whereas wild-type SERAC1 partially colocalized with the mitochondrial marker MT-CO1. The structural and functional mitochondrial abnormalities, caused by the loss of SERAC1, suggest that the crucial disease mechanism is disrupted interplay between the ER and mitochondria leading to decreased influx of calcium to mitochondria and secondary respiratory chain deficiency.

Homeostatic Regulation of Glucocorticoid Receptor Activity by Hypoxia-Inducible Factor 1: From Physiology to Clinic.

Glucocorticoids (GCs) represent a well-known class of lipophilic steroid hormones biosynthesised, with a circadian rhythm, by the adrenal glands in humans and by the inter-renal tissue in teleost fish (e.g., zebrafish). GCs play a key role in the regulation of numerous physiological processes, including inflammation, glucose, lipid, protein metabolism and stress response. This is achieved through binding to their cognate receptor, GR, which functions as a ligand-activated transcription factor. Due to their potent anti-inflammatory and immune-suppressive action, synthetic GCs are broadly used for treating pathological disorders that are very often linked to hypoxia (e.g., rheumatoid arthritis, inflammatory, allergic, infectious, and autoimmune diseases, among others) as well as to prevent graft rejections and against immune system malignancies. However, due to the presence of adverse effects and GC resistance their therapeutic benefits are limited in patients chronically treated with steroids. For this reason, understanding how to fine-tune GR activity is crucial in the search for novel therapeutic strategies aimed at reducing GC-related side effects and effectively restoring homeostasis. Recent research has uncovered novel mechanisms that inhibit GR function, thereby causing glucocorticoid resistance, and has produced some surprising new findings. In this review we analyse these mechanisms and focus on the crosstalk between GR and HIF signalling. Indeed, its comprehension may provide new routes to develop novel therapeutic targets for effectively treating immune and inflammatory response and to simultaneously facilitate the development of innovative GCs with a better benefits-risk ratio.

A knock-in rat model unravels acute and chronic renal toxicity in glutaric aciduria type I.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an autosomal recessive inborn error of metabolism caused by deficiency of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH). The principal clinical manifestation in GA-I patients is striatal injury most often triggered by catabolic stress. Early diagnosis by newborn screening programs improved survival and reduced striatal damage in GA-I patients. However, the clinical phenotype is still evolving in the aging patient population. Evaluation of long-term outcome in GA-I patients recently identified glomerular filtration rate (GFR) decline with increasing age. We recently created the first knock-in rat model for GA-I harboring the mutation p.R411W (c.1231 C>T), corresponding to the most frequent GCDH human mutation p.R402W. In this study, we evaluated the effect of an acute metabolic stress in form of high lysine diet (HLD) on young Gcdhki/ki rats. We further studied the chronic effect of GCDH deficiency on kidney function in a longitudinal study on a cohort of Gcdhki/ki rats by repetitive 68Ga-EDTA positron emission tomography (PET) renography, biochemical and histological analyses. In young Gcdhki/ki rats exposed to HLD, we observed a GFR decline and biochemical signs of a tubulopathy. Histological analyses revealed lipophilic vacuoles, thinning of apical brush border membranes and increased numbers of mitochondria in proximal tubular (PT) cells. HLD also altered OXPHOS activities and proteome in kidneys of Gcdhki/ki rats. In the longitudinal cohort, we showed a progressive GFR decline in Gcdhki/ki rats starting at young adult age and a decline of renal clearance. Histopathological analyses in aged Gcdhki/ki rats revealed tubular dilatation, protein accumulation in PT cells and mononuclear infiltrations. These observations confirm that GA-I leads to acute and chronic renal damage. This raises questions on indication for follow-up on kidney function in GA-I patients and possible therapeutic interventions to avoid renal damage.

Primary Generalized Glucocorticoid Resistance and Hypersensitivity Syndromes: A 2021 Update.

Glucocorticoids are the final products of the neuroendocrine hypothalamic-pituitary-adrenal axis, and play an important role in the stress response to re-establish homeostasis when it is threatened, or perceived as threatened. These steroid hormones have pleiotropic actions through binding to their cognate receptor, the human glucocorticoid receptor, which functions as a ligand-bound transcription factor inducing or repressing the expression of a large number of target genes. To achieve homeostasis, glucocorticoid signaling should have an optimal effect on all tissues. Indeed, any inappropriate glucocorticoid effect in terms of quantity or quality has been associated with pathologic conditions, which are characterized by short-term or long-lasting detrimental effects. Two such conditions, the primary generalized glucocorticoid resistance and hypersensitivity syndromes, are discussed in this review article. Undoubtedly, the tremendous progress of structural, molecular, and cellular biology, in association with the continued progress of biotechnology, has led to a better and more in-depth understanding of these rare endocrinologic conditions, as well as more effective therapeutic management.

Utility of Gene Panels for the Diagnosis of Inborn Errors of Metabolism in a Metabolic Reference Center.

Next-generation sequencing (NGS) technologies have been proposed as a first-line test for the diagnosis of inborn errors of metabolism (IEM), a group of genetically heterogeneous disorders with overlapping or nonspecific phenotypes. Over a 3-year period, we prospectively analyzed 311 pediatric patients with a suspected IEM using four targeted gene panels. The rate of positive diagnosis was 61.86% for intermediary metabolism defects, 32.84% for complex molecular defects, 19% for hypoglycemic/hyperglycemic events, and 17% for mitochondrial diseases, and a conclusive molecular diagnosis was established in 2-4 weeks. Forty-one patients for whom negative results were obtained with the mitochondrial diseases panel underwent subsequent analyses using the NeuroSeq panel, which groups all genes from the individual panels together with genes associated with neurological disorders (1870 genes in total). This achieved a diagnostic rate of 32%. We next evaluated the utility of a tool, Phenomizer, for differential diagnosis, and established a correlation between phenotype and molecular findings in 39.3% of patients. Finally, we evaluated the mutational architecture of the genes analyzed by determining z-scores, loss-of-function observed/expected upper bound fraction (LOEUF), and haploinsufficiency (HI) scores. In summary, targeted gene panels for specific groups of IEMs enabled rapid and effective diagnosis, which is critical for the therapeutic management of IEM patients.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Glucocorticoid resistance conferring mutation in the C-terminus of GR alters the receptor conformational dynamics.

The glucocorticoid receptor is a key regulator of essential physiological processes, which under the control of the Hsp90 chaperone machinery, binds to steroid hormones and steroid-like molecules and in a rather complicated and elusive response, regulates a set of glucocorticoid responsive genes. We here examine a human glucocorticoid receptor variant, harboring a point mutation in the last C-terminal residues, L773P, that was associated to Primary Generalized Glucocorticoid Resistance, a condition originating from decreased affinity to hormone, impairing one or multiple aspects of GR action. Using in vitro and in silico methods, we assign the conformational consequences of this mutation to particular GR elements and report on the altered receptor properties regarding its binding to dexamethasone, a NCOA-2 coactivator-derived peptide, DNA, and importantly, its interaction with the chaperone machinery of Hsp90.

Consolidating biallelic SDHD variants as a cause of mitochondrial complex II deficiency.

Isolated mitochondrial complex II deficiency is a rare cause of mitochondrial respiratory chain disease. To date biallelic variants in three genes encoding mitochondrial complex II molecular components have been unequivocally associated with mitochondrial disease (SDHA/SDHB/SDHAF1). Additionally, variants in one further complex II component (SDHD) have been identified as a candidate cause of isolated mitochondrial complex II deficiency in just two unrelated affected individuals with clinical features consistent with mitochondrial disease, including progressive encephalomyopathy and lethal infantile cardiomyopathy. We present clinical and genomic investigations in four individuals from an extended Palestinian family with clinical features consistent with an autosomal recessive mitochondrial complex II deficiency, in which our genomic studies identified a homozygous NM_003002.3:c.[205 G > A];[205 G > A];p.[(Glu69Lys)];[(Glu69Lys)] SDHD variant as the likely cause. Reviewing previously published cases, these findings consolidate disruption of SDHD function as a cause of mitochondrial complex II deficiency and further define the phenotypic spectrum associated with SDHD gene variants.

Progressive cerebellar atrophy in a patient with complex II and III deficiency and a novel deleterious variant in SDHA: A Counseling Conundrum.

BACKGROUND: Complex II is an essential component of the electron transport chain, linking it with the tricarboxylic acid cycle. Its four subunits are encoded in the nuclear genome, and deleterious variants in these genes, including SDHA (OMIM 600857), are associated with a wide range of symptoms including neurological disease, cardiomyopathy, and neoplasia (paraganglioma-pheochromocytomas (PGL/PCC), and gastrointestinal stromal tumors). Deleterious variants of SDHA are most frequently associated with Leigh and Leigh-like syndromes. METHODS AND RESULTS: Here, we describe a case of a 9-year-old boy with tremor, nystagmus, hypotonia, developmental delay, significant ataxia, and progressive cerebellar atrophy. He was found to have biallelic variants in SDHA, a known pathogenic variant (c.91C>T (p.R31*)), and a variant of unknown significance (c.454G>A (p.E152K)). Deficient activity of complexes II and III was detected in fibroblasts from the patient consistent with a diagnosis of a respiratory chain disorder. CONCLUSION: We, therefore, consider whether c.454G>A (p.E152K) is, indeed, a pathogenic variant, and what implications it has for family members who carry the same variant.

Successful orthotopic heart transplantation in CPTII deficiency.

Carnitine palmitoyl transferase II (CPT II) catalyzes the release of activated long-chain fatty acids from acylcarnitines into mitochondria for subsequent fatty acid oxidation. Depending on residual enzyme activity, deficiency of this enzyme leads to a spectrum of symptoms from early onset hypoglycemia, hyperammonemia, cardiomyopathy and death to onset of recurrent rhabdomyolysis in adolescents and young adults. We present a case of successful orthotopic heart transplantation in a patient with severe infantile onset cardiomyopathy due to CPT II deficiency identified through newborn screening. Excellent cardiac function is preserved 12 years post-transplantation; however, the patient has developed intermittent episodes of hyperammonemia and rhabdomyolysis later in childhood and early adolescence readily resolved with intravenous glucose. Successful heart transplant in this patient demonstrates the feasibility of this management option in patients with even severe forms of long chain fatty acid oxidation disorders.

Inborn disorders of the malate aspartate shuttle.

Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+ /NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox-dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3-phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.

Molecular and metabolic bases of tetrahydrobiopterin (BH4) deficiencies.

Tetrahydrobiopterin (BH4) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.

ß3-Adrenoreceptors as ROS Balancer in Hematopoietic Stem Cell Transplantation.

In the last decades, the therapeutic potential of hematopoietic stem cell transplantation (HSCT) has acquired a primary role in the management of a broad spectrum of diseases including cancer, hematologic conditions, immune system dysregulations, and inborn errors of metabolism. The different types of HSCT, autologous and allogeneic, include risks of severe complications including acute and chronic graft-versus-host disease (GvHD) complications, hepatic veno-occlusive disease, lung injury, and infections. Despite being a dangerous procedure, it improved patient survival. Hence, its use was extended to treat autoimmune diseases, metabolic disorders, malignant infantile disorders, and hereditary skeletal dysplasia. HSCT is performed to restore or treat various congenital conditions in which immunologic functions are compromised, for instance, by chemo- and radiotherapy, and involves the administration of hematopoietic stem cells (HSCs) in patients with depleted or dysfunctional bone marrow (BM). Since HSCs biology is tightly regulated by oxidative stress (OS), the control of reactive oxygen species (ROS) levels is important to maintain their self-renewal capacity. In quiescent HSCs, low ROS levels are essential for stemness maintenance; however, physiological ROS levels promote HSC proliferation and differentiation. High ROS levels are mainly involved in short-term repopulation, whereas low ROS levels are associated with long-term repopulating ability. In this review, we aim summarize the current state of knowledge about the role of ß3-adrenoreceptors (ß3-ARs) in regulating HSCs redox homeostasis. ß3-ARs play a major role in regulating stromal cell differentiation, and the antagonist SR59230A promotes differentiation of different progenitor cells in hematopoietic tumors, suggesting that ß3-ARs agonism and antagonism could be exploited for clinical benefit.

Treatment efficacy of high-dose creatine supplementation in a child with creatine transporter (SLC6A8) deficiency.

BACKGROUND: Creatine transporter deficiency is an inborn error of metabolism caused by a deficiency in the creatine transporter protein encoded by the SLC6A8 gene. Previous treatment with creatine supplementation, either alone or in combination with creatine precursors (arginine or glycine), has been attempted; the efficacy of therapy, however, remains controversial. METHODS AND RESULTS: To analyze the treatment efficacy of high-dose creatine supplementation on creatine transporter deficiency, we reported a child diagnosed with creatine transporter deficiency, who was treated with a conventional dose of creatine (400 mg/kg/d) for 1 month, then twice the dose (800 mg/kg/d) for 2 months, and finally 3 times the dose (1200 mg/kg/d) for 3 months. The patient tolerated the treatment well and showed improvements in muscle mass and strength when the creatine dose was gradually increased to 1200 mg/kg/d. However, when assessed by proton magnetic resonance spectroscopy (H-MRS), the brain creatine concentration did not increase, and there was no improvement in speech and neurodevelopmental symptoms. CONCLUSION: We conclude that high-dose creatine supplementation (1200 mg/kg/d) alone improved muscular symptoms, but did not improve cognitive symptoms and brain creatine concentration assessed using H-MRS. Therefore, new treatment strategies are required for the management of creatine transporter deficiency.

Considerations of expanded carrier screening: Lessons learned from combined malonic and methylmalonic aciduria.

BACKGROUND: Expanded carrier screening (ECS) utilizes high-throughput next-generation sequencing to evaluate an individual's carrier status for multiple conditions. Combined malonic and methylmalonic aciduria (CMAMMA) due to ACSF3 deficiency is a rare inherited disease included in such screening panels. Some cases have been reported with metabolic symptoms in childhood yet other cases describe a benign clinical course, suggesting the clinical phenotype is not well defined. METHODS/CASE REPORT: Clinical and laboratory findings during the prenatal period were obtained retrospectively from medical records. RESULTS: A 37-year-old nulliparous woman and her partner were each identified as carriers of ACSF3 variants and presented at 9 weeks gestation for prenatal genetic consultation. The couple received extensive genetic counseling and proceeded with chorionic villus sampling at 11 weeks gestation. Subsequent analysis confirmed that the fetus inherited both parental ACSF variants. The couple was devastated by the results and after reviewing options of pregnancy continuation and termination, they decided to terminate the pregnancy. Following this decision, the patient was diagnosed with acute stress disorder. CONCLUSION: This case highlights how expanded carrier screening adds complexity to reproductive decision-making. Stronger guidelines and additional research are needed to direct and evaluate the timing, composition, and implementation of ECS panels.

Expansion of the ophthalmic phenotype of SPINT2-related syndromic congenital sodium diarrhea.

A 5-year-old girl presented with treatment-refractory dry eye and recurrent episodes of eye pain. She had been previously diagnosed with syndromic congenital sodium diarrhea (SCSD) caused by a pathogenic variant in SPINT2. Her local pediatric ophthalmologist had made the diagnosis of severe dry eye with corneal erosions, based on which, we arranged an eye exam under anesthesia (EUA) and punctal plug placement. Anterior segment optical coherence tomography (OCT) and corneal photographs were taken during the procedure. There are reports describing similar ophthalmic findings in this syndrome. However, to the best of our knowledge, this is the first case report to document OCT imaging and corneal photographs in a patient with SCSD, which we feel expands the ophthalmic phenotype of this rare genetic disorder.

MITOCHONDRIA: Succinate dehydrogenase subunit B-associated phaeochromocytoma and paraganglioma.

Phaeochromocytomas and paragangliomas are rare neuroendocrine tumours. So far, over 20 causative genes have been identified, of which the most frequent and strongest indicator for malignancies are mutations in succinate dehydrogenase subunit B. No curative therapy is available for patients with metastases resulting in poor prognosis. Therapy development has been hindered by lack of suitable model systems. The succinate dehydrogenase complex is located in the inner membrane of the mitochondria and plays a crucial role in the oxidative phosphorylation chain and the tricarboxylic acid-cycle. Succinate dehydrogenase deficiency results in accumulation of the oncometabolite succinate inducing hypoxia inducible factor stabilization, deoxyribonucleic acid and histone methylation inhibition, and impaired production of adenosine triphosphate. It remains unknown which combination of pathways and/or triggers are decisive for metastases development. In this review, the role of mitochondria in malignant succinate dehydrogenase subunit B-associated phaeochromocytomas and paragangliomas and implications for mitochondria as therapeutic target are discussed.

Novel variants in the stem cell niche factor WNT2B define the disease phenotype as a congenital enteropathy with ocular dysgenesis.

WNT2B is a member of the Wnt family, a group of signal transduction proteins involved in embryologic development and stem cell renewal and maintenance. We recently reported homozygous nonsense variants in WNT2B in three individuals with severe, neonatal-onset diarrhea, and intestinal failure. Here we present a fourth case, from a separate family, with neonatal diarrhea associated with novel compound heterozygous WNT2B variants. One of the two variants was a frameshift variant (c.423del [p.Phe141fs]), while the other was a missense change (c.722 G > A [p.G241D]) that we predict through homology modeling to be deleterious, disrupting post-translational acylation. This patient presented as a neonate with severe diet-induced (osmotic) diarrhea and growth failure resulting in dependence on parenteral nutrition. Her gastrointestinal histology revealed abnormal cellular architecture particularly in the stomach and colon, including oxyntic atrophy, abnormal distribution of enteroendocrine cells, and a paucity of colonic crypt glands. In addition to her gastrointestinal findings, she had bilateral corneal clouding and atypical genital development later identified as a testicular 46,XX difference/disorder of sexual development. Upon review of the previously reported cases, two others also had anterior segment ocular anomalies though none had atypical genital development. This growing case series suggests that variants in WNT2B are associated with an oculo-intestinal (and possibly gonadal) syndrome, due to the protein's putative involvement in multiple developmental and stem cell maintenance pathways.