Enzyme Defect Research Articles

Prevalence of Glucose-6-Phosphate Dehydrogenase deficiency among Hausa and Fulani ethnics in Kano, North-West Nigeria

Background: Glucose-6-phosphate dehydrogenase catalyzes the rate-determining step in the pentose phosphate pathway. Its activity is a key determinant of the reduced nicotinamide adenine dinucleotide phosphate to oxidized nicotinamide adenine dinucleotide phosphate (NADPH-to-NADP+) ratio in the cytoplasm and thus contributes to the replenishment of the antioxidant glutathione system. Glucose-6-phosphate dehydrogenase deficiency is a common X-linked human enzyme defect of red blood cells. Individuals with this gene defect appear normal until exposed to oxidative stress which induces haemolysis but it is infrequently taken into consideration in health practices. Aim: The present work was designed to determine the frequency of occurrence of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency among Hausa and Fulani ethnics. Material and methods: Cross-sectional comparative study in which 5 mL of venous blood samples were collected from 1000 individuals (596 males and 404 females) all of whom gave informed consent were screened for G-6-PD-deficiency by qualitative methaemoglobin reduction and quantitative enzyme activity measurement techniques. Results: Glucose-6-phoshate dehydrogenase deficiency was found in 194 (19.4%) of participants studied. Of these 10.3% were Fulani and 22.2% were Hausa with mean G-6-PD enzyme activity of 2.5 IU/gHb in a range from 1.4 to 2.4 IU/gHb. G-6-PD deficiency was found in 121 of 596 (20.3%) males and 73 of 404 (18.1%) females screened. Conclusions: These results suggest high occurrence of phenotypic G-6-PD deficiency in Hausa ethnics of Kano with lower enzyme status among males living with G-6-PD deficiency.

Prevalence Of Glucose-6-Phosphate Dehydrogenase Deficiency in People Living in Katsina Metropolis

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most prevalent human enzyme defects observed in malaria endemic areas. The study included 212 participants, with 142 (67%) male and 70 (33%) female, aged between14 to 59 with a mean age of 24.20±9.35 years. The study was granted ethical approval by the Katsina State Ministry of Health. Each subject gave his/her written informed permission after counseling. G6PD deficiency, malaria parasite, reduced glutathione (GSH), glutathione peroxidase (GPx), packed cell volume (PCV) and hemoglobin (Hb) were assayed using standard procedures. Among the 212 participants tested in the study, 40 (19%) were found to be G6PD deficient and 172 (81%) participants were normal. Among 212 participants tested, 75 (35.4%) were infected with malaria parasite out of which 13 (17.3%) were G6PD deficient and 62 (82.7%) were normal. Among the 40 G6PD deficient subjects, 33 (82.5%) were male while 7 (17.5%) were females. GSH concentration was lowered significantly in G6PD-deficient subjects compared to the normal subjects. GPx, Hb and PCV showed no significant difference (p>0.05) in the deficient subjects compared to the normal subjects. The results of this study suggest that G6PD deficiency is quite common with a prevalence of 19% in the population of Katsina metropolis. The data also shows that males are more likely to be affected by G6PD deficiency than females.

Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.

Compilation of Genotype and Phenotype Data in GCDH-LOVD for Variant Classification and Further Application.

Glutaric aciduria type 1 (GA-1) is a rare but treatable autosomal-recessive neurometabolic disorder of lysin metabolism caused by biallelic pathogenic variants in glutaryl-CoA dehydrogenase gene (GCDH) that lead to deficiency of GCDH protein. Without treatment, this enzyme defect causes a neurological phenotype characterized by movement disorder and cognitive impairment. Based on a comprehensive literature search, we established a large dataset of GCDH variants using the Leiden Open Variation Database (LOVD) to summarize the known genotypes and the clinical and biochemical phenotypes associated with GA-1. With these data, we developed a GCDH-specific variation classification framework based on American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines. We used this framework to reclassify published variants and to describe their geographic distribution, both of which have practical implications for the molecular genetic diagnosis of GA-1. The freely available GCDH-specific LOVD dataset provides a basis for diagnostic laboratories and researchers to further optimize their knowledge and molecular diagnosis of this rare disease.

Use of nebulized ketamine for subarachnoid block in paediatric patient with g6pd deficiency: A case report

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest human enzyme defect with male predominance. Patients with Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency may face acute haemolysis precipitated by infection, certain medications and some of the anaesthetic drugs. Ketamine helps in providing anxiolysis, analgesia and sedation. Here, we report successful use of nebulized ketamine in a child to give subarachnoid block with G6PD deficiency undergoing below umbilical procedure.

Exercise induced muscle weakness in a young adult: McArdle’s disease unusual presentation

McArdle’s disease (Glycogen storage disease type V) is a rare inherited autosomal recessive disease involving defect in enzyme, glycogen phosphorylase (PYGM) which results in accumulation of glycogen mainly affecting skeletal muscles. It commonly presents in childhood and rarely in adults with symptoms like exercise intolerance, muscle weakness, cramps and fatigue. Herein, we report an unusual case of a 22 years old male in Pakistan with probable McArdle’s Disease presenting with repeated episodes of generalized cramping muscle pain, exercise intolerance and haematuria. The diagnostic approach to identifying this disease as well as the differentials of other rare types of skeletal muscle disorders that should be kept in mind while dealing with a similar clinical picture, irrespective of the age of presentation, have been discussed. Keywords: Glycogen Storage Disease Type V, Myoglobinuria.

A premature stop codon in the CYP2C19 gene may explain the unexpected sensitivity of vultures to diclofenac toxicity

The unintended environmental exposure of vultures to diclofenac has resulted in the deaths of millions of old-world vultures on the Asian subcontinent. While toxicity has been since associated with a long half-life of elimination and zero order metabolism, the actual constraint in biotransformation is yet to be clarified. For this study we evaluated if the evident zero order metabolism could be due to defects in the CYP2C9/2C19 enzyme system. For this, using whole genome sequencing and de-novo transcriptome alignment, the vulture CYP2C19 open reading frame was identified through Splign analysis. The result sequence analysis revealed the presence of a premature stop codon on intron 7 of the identified open reading frame. Even if the stop codon was not present, amino acid residue analysis tended to suggest that the enzyme would be lower in activity than the equivalent human enzyme, with differences present at sites 105, 286 and 289. The defect was also conserved across the eight non-related vultures tested. From these results, we conclude that the sensitivity of the old-world vultures to diclofenac is due to the non-expression of a viable CYP2C19 enzyme system. This is not too dissimilar to the effects seen in certain people with a similar defective enzyme.

Landscape of Adrenal Tumours in Patients with Congenital Adrenal Hyperplasia.

Our aim is to update the topic of adrenal tumours (ATs) in congenital adrenal hyperplasia (CAH) based on a multidisciplinary, clinical perspective via an endocrine approach. This narrative review is based on a PubMed search of full-length, English articles between January 2014 and July 2023. We included 52 original papers: 9 studies, 8 case series, and 35 single case reports. Firstly, we introduce a case-based analysis of 59 CAH-ATs cases with four types of enzymatic defects (CYP21A2, CYP17A1, CYP17B1, and HSD3B2). Secondarily, we analysed prevalence studies; their sample size varied from 53 to 26,000 individuals. AT prevalence among CAH was of 13.3-20%. CAH prevalence among individuals with previous imaging diagnosis of AT was of 0.3-3.6%. Overall, this 10-year, sample-based analysis represents one of the most complex studies in the area of CAH-ATs so far. These masses should be taken into consideration. They may reach impressive sizes of up to 30-40 cm, with compressive effects. Adrenalectomy was chosen based on an individual multidisciplinary decision. Many tumours are detected in subjects with a poor disease control, or they represent the first step toward CAH identification. We noted a left lateralization with a less clear pathogenic explanation. The most frequent tumour remains myelolipoma. The risk of adrenocortical carcinoma should not be overlooked. Noting the increasing prevalence of adrenal incidentalomas, CAH testing might be indicated to identify non-classical forms of CAH.

Gene therapy for neurodegenerative disorders in children: dreams and realities

Gene therapy encompasses the administration of biological medicinal products containing recombinant nucleic acids, mainly DNA, with the aim of treating or curing diseases. This represents a unique therapeutic strategy to reach the brain, in order to prevent or halt a neurodegenerative process. During the past decade, active multidisciplinary research has started to solve many issues for gene therapy in neurodegenerative disorders in terms of vectors, modes of administration, and expression of the therapeutic DNA. The engineering of hematopoietic stem cells (HSC) with lentivirus vectors for ex vivo gene therapy has demonstrated efficiency in reaching the brain through their transformation into microglial/macrophages cells with a long-term gene expression of the therapeutic vector as an alternative to autologous HSC transplants. Two drugs based on this strategy have been approved to date. The first is for metachromatic leukodystrophy (MLD), a severe lysosomal storage disease, and provides high levels of the deficient enzyme; the second one is for cerebral forms of X-linked adrenoleukodystrophy (X-ALD), and works by halting the neuroinflammation process. However, due to the long-lasting effect of the procedure, the therapy is applicable only to pre- or pauci/oligo-symptomatic patients. In vivo gene therapy via direct injection into the brain or the cerebrospinal fluid, but also by intravenous injection, represents a more efficient approach; however, many challenges remain to be solved despite the approval of two drugs: one for the early infantile form of spinal muscular atrophy (SMA), in which the gene product injected intravenously is able to prevent spinal motoneuron neurodegeneration. The second one, for aromatic L-amino acid decarboxylase (AADC) deficiency, provides the defective enzyme to the basal ganglia via intraparenchymal injection. The production of vectors able to reach the brain target cells with a sufficiently high expression remains a major bottleneck. In parallel, efforts must continue in order to better define (i) the natural history and clinical outcomes of many neurodegenerative disorders with childhood onset, and (ii) the mechanisms involved in the neurodegenerative process. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

Clinical Approaches for Mitochondrial Diseases.

Mitochondria are subcontractors dedicated to energy production within cells. In human mitochondria, almost all mitochondrial proteins originate from the nucleus, except for 13 subunit proteins that make up the crucial system required to perform 'oxidative phosphorylation (OX PHOS)', which are expressed by the mitochondria's self-contained DNA. Mitochondrial DNA (mtDNA) also encodes 2 rRNA and 22 tRNA species. Mitochondrial DNA replicates almost autonomously, independent of the nucleus, and its heredity follows a non-Mendelian pattern, exclusively passing from mother to children. Numerous studies have identified mtDNA mutation-related genetic diseases. The consequences of various types of mtDNA mutations, including insertions, deletions, and single base-pair mutations, are studied to reveal their relationship to mitochondrial diseases. Most mitochondrial diseases exhibit fatal symptoms, leading to ongoing therapeutic research with diverse approaches such as stimulating the defective OXPHOS system, mitochondrial replacement, and allotropic expression of defective enzymes. This review provides detailed information on two topics: (1) mitochondrial diseases caused by mtDNA mutations, and (2) the mechanisms of current treatments for mitochondrial diseases and clinical trials.

FRI430 A Case Of Elevated Sex Hormone-binding Globulin Secondary To Variegate Porphyria In A Patient With Hypogonadotropic Hypogonadism

Abstract Disclosure: J. Law: None. C. Yip: Research Investigator; Self; Eli Lilly & Company. Background: Acute porphyria is a group of disorders characterized by enzymatic defects in the heme biosynthetic pathway. The liver is the source of both acute intermittent porphyria (AIP) and variegate porphyria. AIP is known to cause an elevation in sex hormone-binding globulin (SHBG) levels, likely secondary to impaired hepatic function and hepatocyte abnormalities1. We describe a case of elevated SHBG secondary to variegate porphyria, in a male with hypogonadotropic hypogonadism. Case Report: A 44-year-old male presented with an elevated SHBG and symptoms of hypogonadism with low libido, weakness, and fatigue. Fasting morning bloodwork revealed an elevated SHBG at 89 nmol/l (13.5- 71.0), a normal total testosterone level at 12.95 nmol/l (8-32), and a low bioavailable testosterone at 0.82 nmol/l (2.5-10), with confirmatory repeat blood work. His history was significant for longstanding variegate porphyria, manifesting as frequent attacks of abdominal pain. He was on opioids for greater then 10 years for his chronic pain, as well as metoclopramide. On assessment of causes for his elevated SHBG, his TSH was normal at 1.49 mIU/L (0.35-4.30). He did not have cirrhosis, hepatitis, hereditary hemochromatosis, and was not on anticonvulsants. No other cause was found, and his elevated SHBG was deemed secondary to his variegate porphyria. His normal total testosterone level was due to his elevated SHBG, but as mentioned, his bioavailable testosterone was low. His LH and FSH were both inappropriately normal, with his LH being 4.67 IU/L (0.6-12.0) and his FSH 4.7 IU/L (1.0-12.0), in keeping with hypogonadotropic hypogonadism. He went through normal pubertal development and had no anosmia. Workup revealed an elevated prolactin level of 73.8 ug/l (3.5-19.4). He had no galactorrhea or gynecomastia and an MRI sella revealed a normal pituitary gland. His elevated prolactin level was determined to be secondary to his opioid medication and/or metoclopramide use. His hypogonadotropic hypogonadism was deemed secondary to his opioid medications and/or hyperprolactinemia. Conclusion: This case demonstrated variegate porphyria as a likely cause of elevated SHBG, similar to AIP. In patients with variegate porphyria and symptoms of hypogonadism, the SHBG should be measured and if it is elevated, the bioavailable or free testosterone levels should be used in assess instead of relying on the total testosterone levels. As has been previously shown, this case also illustrated the use of opioid medications as a cause of hypogonadotropic hypogonadism.

SAT252 Homozygous CYP24A1 Gene Mutation Induced Hypercalcemia During Pregnancy

Abstract Disclosure: A.R. Ankireddypalli: None. H. Alameddine: None. Introduction: The approach and management of hypercalcemia in pregnancy can be challenging. Pregnancy challenges the calcium homeostasis with physiological increase in 1,25 dihydroxy Vitamin D levels by kidneys and placental 1-alpha hydroxylase, however it rarely causes hypercalcemia. In PTH independent hypercalcemia, disorders in vitamin D metabolism should be considered. Here we report a case of PTH independent hypercalcemia due to autosomal recessive homozygous CYP24A1 gene mutation. Case: A 25-year-old primigravid female presented to the clinic at 26 weeks of gestation for management of hypercalcemia. At 25 weeks gestation, laboratory workup revealed calcium: 13.9 mg/dL, albumin: 2.9 g/dL, PTH: < 6 pg/mL, vitamin D: 45.17ng/mL, creatinine: 1.02 mg/dL, alkaline phosphatase (ALP): 117mcg/L. Since getting pregnant she reported nausea and vomiting. She reported excessive thirst, memory loss, fatigue, exertional dyspnea and 10 lbs weight loss. She denied any personal history of kidney stones or family history of calcium or bone disorders. She stopped her prenatal vitamins at the time of hypercalcemia diagnosis. Her work-up was suggestive of PTH independent hypercalcemia. Differential diagnosis included granulomatous disease, malignancy, benign PTHrP secreting tumors, vitamin D metabolism dysfunction and milk-alkali syndrome. Further work-up revealed ionized calcium: 6.2 mg/dL, PTHrP: 5.3 pmol/L, bone specific ALP: 11.8 mcg/L, 25 hydroxy vitamin D: 132 mcg/L, 1, 25 dihydroxy vitamin D: > 200 pg/mL, 24,25 dihydroxy vitamin D: 1.16 ng/mL, 25-hydroxy vitamin D to 24,25 dihydroxy vitamin D ratio: 75.0, which was indicative of CYP24A1 mutation, an enzymatic defect in vitamin D metabolism. Molecular testing confirmed autosomal recessive homozygous CYP24A1 gene mutation. With elevated 24,25 dihydroxy vitamin D levels, it was unlikely that her 1,25 dihydroxy vitamin D was elevated due to malignancy or granulomatous disease. Treatment with prednisone 60 mg daily for 1 month did not improve her calcium levels. She was recommended to limit her calcium intake, avoid vitamin D supplements or sun exposure. At 38 weeks, she delivered a healthy baby without neonatal complication. Postpartum day 2, her labs revealed calcium: 10.7mg/dL with albumin: 2.7mg/dL. Discussion: Diagnosis of CY24A1 gene mutation associated hypercalcemia is based on increased 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D ratio and confirmed by genetic analysis. Recommendations are adequate hydration, limiting calcium intake, avoiding vitamin D supplements and preventing sun exposure. It is important to monitor for neonatal hypocalcemia and worsening of maternal hypercalcemia during breastfeeding. We report this case to raise awareness of this rare cause of hypercalcemia in pregnancy and the importance of checking calcium and vitamin D levels prior to vitamin D supplementation. Presentation: Saturday, June 17, 2023

Modern anatomical and physiological bases for maintaining the transparency of the corneal stroma

The article presents a literature review of the modern concept of anatomical and physiological structure and functioning of the cornea. The strict morphological structure and corneal tissue homeostasis ensure its transparency. Studying the mechanisms that regulate the constancy of the corneal tissue internal environment allows us to get closer to understanding the prospects forregenerative therapy for the corneal stroma pathology. The article discusses in detail the role and functional potential of corneal stromal cells, which are capable of reverse cytologic differentiation, which primarily ensures the maintenance of tissue homeostasis and corneal transparency. The functional activity of corneal cells can change for a number of reasons, which may be exogenous, iatrogenic (trauma, infection, etc.) or endogenous. Endogenous causes include: cell autoregulation pathologies (for example, enzyme defects); defects in transport systems leading to tissue hypoxia; disorders of the neuro-humoral regulation of trophism. The physical reason forthe violation of the corneal transparency is an increase in the light scattering. The article presents five main causes of increased light scattering in the opaque cornea, and also provides an overview of the main substances – components and products of cellular synthesis of corneal stromal cells: cytokines and growth factors (complex of the signal molecule and the SDF1/CXCR4 receptor, insulin-like growth factor 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, erythropoietin, neurotrophic factors, etc.). Thus, corneal opacity can be caused by a single pathogenic mechanism or be the result of a complex effect of several factors. The main processes of tissue homeostasis regulation are aimed at maintaining the unique morphological structure of the cornea.

SAT264 Effects Of Coenzymes In Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Model Mice

Abstract Disclosure: Y. Naiki: None. M. Miyado: None. R. Horikawa: None. N. Katsumata: None. S. Takada: None. M. Fukami: None. Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s and they need coenzymes respectively. Microsomal P450s include 21-hydroxylase need P450 oxidoreductase which coded by POR and ferredoxin and ferredoxin reductase, which coded by FDX and FDXR, respectively, work for mitochondrial P450s include 11β-hydroxylase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, and 11β-hydroxylase deficiency (11β-OHD), which caused by mutated CYP11B1 are the top two cause of CAH. CAH are treated with oral steroid supplementation, but optimal control remains difficult. Thus, new therapeutic approaches are still needed. We succeeded gene therapy for model mice of CAH and iPS cell from CAH patients (1). In this study, we examined the effects of induction of coenzyme genes with an AAV vector into a model mouse. A 21OHD mouse were made by breeding with H-2aw18 haplotype mice and treated with subcutaneous corticosteroid injection until 3 weeks of age. A 11β-OHD mouse were made by using the CRISPR/Cas9 method. A type 9 AAV (AAV9) containing Cyp21a cDNA (AAV9-Cyp21a1) was constructed with pAAV-CMV-shuttle. AAV9 containing Por cDNA (AAV9-Por), Cyp11b1 cDNA (AAV9-Cyp11b1), Fdx (AAV9-Fdx), Fdxr (AAV9-Fdxr) were also made. An AAV9-Cyp21a1 and AAV9-Por were mixed and injected into limbs muscles of a 21-OHD mouse and AAV9-Cyp11b1 mixed with AAV9-Fdx and AAV9-Fdxr injected into limbs muscles of 11β-OHD mouse. Serum progesterone (P4) and deoxycorticosterone (DOC) concentrations for 21-OHD mouse and DOC and corticosterone (B) concentrations for 11β-OHD mouse were measured before and after injection. P4 and DOC concentrations were measured by LC-MS/MS. An AAV9-Cyp21a1 and AAV9-Por injected mouse showed improved steroid synthesis up to 7 months after AAV9 induction. Comparing with a mouse injected only AAV9-Cyp21a1, significant reduction of P4/DOC was observed after 4 weeks of injection but there was no difference in the effectiveness of Cyp21a1 and Por combined treatment at the end of observation. Instead of a 21-OHD mouse, a 11β-OHD mouse did not show significant improvement by AAV mixture injected. These results indicate that extra-adrenal induction of Cyp21a1 and Por may ameliorate steroid metabolism in 21-OHD model mice. Although this coenzyme combined therapy showed some effects, but no long-term improvement of the efficacy exhibited. Instead of microsomal P450s, extra-adrenal induction with coenzyme genes did not work enough for mitochondrial P450s defects. Coenzymes of P450s are necessary for their activity, but our results suggest that the coenzyme combined gene therapeutic strategies are needed to be improved. Reference: (1) Naiki et al., Hum Gene Ther. 2022 Aug;33(15-16):801-809. Presentation: Saturday, June 17, 2023

CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum.

In this paper, we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition, the multicellular development is affected as well as growing on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued. The results of the study suggest that this new D. discoideum strain offers a wide range of possibilities to easily explore diverse FA FXN variants. This can facilitate the development of straightforward drug screenings to look for new therapeutic strategies.

Metabolomic analysis of Drosophila melanogaster larvae lacking pyruvate kinase.

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster are complicated by the fact that the fly genome encodes 6 Pyk paralogs whose functions remain poorly defined. To address this issue, we used sequence distance and phylogenetic approaches to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other 5 Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of 2 different Pyk mutant strains revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectedly reveals that pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and protease activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans.

The impact of neonatal 17-hydroxyprogesterone cutoff determination in a public newborn screening program for congenital adrenal hyperplasia in Southern Brazil: 3 years' experience.

Congenital adrenal hyperplasia (CAH) occurs due to enzyme defects in adrenal steroidogenesis. The 21-hydroxylase deficiency accounts for 90-95% of cases, triggering accumulation of 17-hydroxyprogesterone (17-OHP). Early diagnosis through neonatal screening allows adequate treatment and reduced mortality. The purpose of the study was to determine 17-OHP cutoffs for the diagnosis of CAH in a public newborn screening program in Southern Brazil. A retrospective, descriptive, cross-sectional study was conducted to analyze 17-OHP levels in dried blood samples collected on filter paper of 317,745 newborns screened at a public newborn screening center from May 2014 to April 2017. Neonatal 17-OHP was measured in DBS samples using a time-resolved fluoroimmunoassay (GSP® kit 3305-0010; PerkinElmer). Different cutoffs were determined and stratified by birth weight. The incidence of CAH was 1:15,887 live births in the state of Rio Grande do Sul, with 20 cases of classical CAH diagnosed during the study period. Most newborns (80.73%) were white, and the prematurity rate was 9.8% in the study population. The combination of different percentiles, 98.5th for birth weight 2001-2500 g and 99.8th for the other birth weight groups, decreased false-positive results and increased specificity compared with current reference values to identify classical CAH cases. The local 17-OHP cutoffs determined were higher than those currently used by this screening program for all birth weight groups. The calculation of reference values from local population data and the combination of percentiles proved to be a valuable tool for proper diagnosis of CAH and reduction in the number of false positives.

Assessing Glycosphingolipid Profiles in Human Health and Disease Using Non-Imaging MALDI Mass Spectrometry

Glycosphingolipids (GSLs) are a glycolipid subtype which plays vital roles in numerous biological processes, cell–cell interactions, as well as oncogenesis and ontogenesis. They are ubiquitous molecules found mostly in cell membranes. Abnormal expression of GSLs as well as altered molecular structure have been linked with progression of cancer and metastasis and are involved in the pathophysiology of neurodegenerative, autoimmune, and infectious diseases as well as inherited enzyme defects—glycosphingolipidoses. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) plays a leading role in analyzing and characterizing different GSLs, and thus can help to distinguish altered GSL patterns. This review offers insights into the benefits and limitations when using MALDI MS in this field of lipidomic research, with an emphasis on which are the optimal matrices in analyzing GSLs from different tissues (normal and pathological) as well as highlighting GSLs’ particular profiles in various cell cultures, and normal and pathological human tissues obtained by MALDI non-imaging MS (non-IMS). These findings can have implications in further understanding the role of altered GSL expression in various pathological conditions and could be a target for future therapies.

Synthesis and Evaluation of Novel Triaryl Derivatives with Readthrough-Inducing Activity.

The readthrough mechanism, which skips the premature termination codon and restores the biosynthesis of the defective enzyme, is an emerging therapeutic tactic for nonsense mutation-related diseases, such as Hurler syndrome, a type of mucopolysaccharidosis. In the present study, novel triaryl derivatives were synthesized and their readthrough-inducing activities were evaluated by a luciferase reporter assay with a partial α-L-iduronidase (IDUA) DNA sequence containing the Q70X nonsense mutation found in Hurler syndrome and by measuring the enzyme activity of IDUA knockout cells transfected with the mutant IDUA gene. KY-516, a representative compound in which the meta position carboxyl group of the left ring of the clinically used ataluren was converted to the para position sulfamoylamino group, the central ring to triazole, and the right ring to cyanobenzene, exhibited the most potent readthrough-inducing activity in the Q70X/luciferase reporter assay. In Q70X mutant IDUA transgenic cells, KY-516 significantly increased enzyme activity at 0.1 µM. After the oral administration of KY-516 (10 mg/kg), the highest plasma concentration of KY-516 was above 5 µM in rats. These results indicate that KY-516, a novel triaryl derivative, exhibits potent readthrough-inducing activity and has potential as a therapeutic agent for Hurler syndrome.

Omics-Based Approaches for the Characterization of Pompe Disease Metabolic Phenotypes.

Lysosomal storage disorders (LSDs) constitute a large group of rare, multisystemic, inherited disorders of metabolism, characterized by defects in lysosomal enzymes, accessory proteins, membrane transporters or trafficking proteins. Pompe disease (PD) is produced by mutations in the acid alpha-glucosidase (GAA) lysosomal enzyme. This enzymatic deficiency leads to the aberrant accumulation of glycogen in the lysosome. The onset of symptoms, including a variety of neurological and multiple-organ pathologies, can range from birth to adulthood, and disease severity can vary between individuals. Although very significant advances related to the development of new treatments, and also to the improvement of newborn screening programs and tools for a more accurate diagnosis and follow-up of patients, have occurred over recent years, there exists an unmet need for further understanding the molecular mechanisms underlying the progression of the disease. Also, the reason why currently available treatments lose effectiveness over time in some patients is not completely understood. In this scenario, characterization of the metabolic phenotype is a valuable approach to gain insights into the global impact of lysosomal dysfunction, and its potential correlation with clinical progression and response to therapies. These approaches represent a discovery tool for investigating disease-induced modifications in the complete metabolic profile, including large numbers of metabolites that are simultaneously analyzed, enabling the identification of novel potential biomarkers associated with these conditions. This review aims to highlight the most relevant findings of recently published omics-based studies with a particular focus on describing the clinical potential of the specific metabolic phenotypes associated to different subgroups of PD patients.