Abstract
Mitochondrial flavin adenine dinucleotide (FAD) transporter deficiencies are new entities recently reported to cause a neuro-myopathic phenotype. We report three patients from two unrelated families who presented primarily with hypoketotic hypoglycemia. They all had acylcarnitine profiles suggestive of multiple acyl-CoA dehydrogenase deficiency (MADD) with negative next-generation sequencing of electron-transfer flavoprotein genes (ETFA, ETFB, and ETFDH). Whole exome sequencing revealed a homozygous c.272āGā>āT (p.Gly91Val) variant in exon 2 of the SLC25A32 gene. The three patients shared the same variant, and they all demonstrated similar clinical and biochemical improvement with riboflavin supplementation. To date, these are the first patients to be reported with hypoketotic hypoglycemia without the neuromuscular phenotype previously reported in patients with SLC25A32 deficiency.
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Introduction
Riboflavin (Vitamin B2) is precursor of two essential cofactors namely flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which are utilized in numerous enzymatic reactions and in a wide range of metabolic pathways including the mitochondrial electron transport chain, mitochondrial and peroxisomal fatty acid Ī²-oxidation, and branched-chain amino acids catabolism [1].
Primary defects of flavocoenzyme metabolism include genetically inherited disorders involving enzymes in the synthetic pathway of essential cofactors FMN and FAD and disorders of riboflavin or flavocoenzyme transport. Secondary flavocoenzyme defects refer to the functional disruption of cofactor-dependent reactions. Both primary and secondary flavocoenzyme deficiencies may present with abnormal biochemical features including characteristic elevations of plasma acylcarnitines and urinary organic acidsĀ abnormalities. Riboflavin therapy will be beneficial in both; hence early recognition of these disorders is clinically important [2, 3].
Mitochondrial FAD transporter deficiencies are entities that were first described in 2016 primarily in association with a variable myopathic presentation. Two patients have been reported so far. The first was a 14-year-old girl with riboflavin-responsive recurrent exercise intolerance and biochemical features of multiple acyl-CoA dehydrogenase deficiency (MAAD) [4]. The second patient, 51 years old at the time of reporting, presented at 3 years of age with muscle weakness following an episode of influenza, and subsequently had progressive exercise intolerance in childhood, together with neurological symptoms including early-onset ataxia, myoclonus, dysarthria, and dysphagia [5].
Both patients had a dramatic improvement in the clinical and biochemical abnormalities following oral riboflavin supplementation, including improved exercise tolerance.
We report three patients from two unrelated Omani families who presented primarily with hypoketotic hypoglycemia. They all had an acylcarnitine profile suggestive of MADD with negative next-generation sequencing of ETFA, ETFB, and ETFDH genes. A homozygous c.272āGā>āT (p.Gly91Val) variant was detected in exon 2 of the SLC25A32 gene. The variant is shared by the three patients reported.
Materials
Patient 1
This is a 25-year-old man who developed an episode of vomiting and hypoglycemia at the age of 6 months. Since then, he had recurrent admissions with episodes of vomiting and lethargy triggered by infections or febrile illness. He had no symptoms in between those episodes, and they became less frequent as he grew up. He was less tolerant to fasting, but never had any exercise intolerance, myalgia, or evidence of rhabdomyolysis (change in urine color). Family history was remarkable for consanguinity and a younger sister who died at the age of seven months while asleep during an intercurrent illness. She had a documented episode of hypoglycemia prior to this incidence.
Blood tests during an episode of hypoglycemia showed normal blood gas, normal ammonia with mildly elevated lactic acid at 5āmmol/L (0.5ā1.6āmmol/l). His creatine kinase (CK) was normal as well. Initial acylcarnitine profile showed significantly increased short-chain, medium-chain acylcarnitines, and to a lesser extent long-chain acylcarnitines and glutarylcarnitines. Urine organic acids showed moderately increased urinary excretion of 2-hydroxyglutaric, ketoglutaric, ethylmalonic acids, and isovalerylglycine. Those results were thought to be suggestive of a diagnosis of MADD and hence he was started on riboflavin and carnitine supplementation. His free and total carnitines were significantly reduced at 3 umol/L (reference range; 30ā50āumol/l) and 4āummol/L (reference range; 43ā65āumol/L), respectively. Currently, he takes riboflavin at a dose of 50āmg twice daily, carnitine at 1āg three times daily and Co-enzyme Q10 at a dose of 60āmg two times daily. There were no admissions with hypoglycemia episodes since then, he is now able to fast for more than 14āh, and his biochemical abnormalities including lactate have completely normalized. Echocardiography was normal. He presently runs an active lifestyle with regular strenuous exercises with no physical or neurological limitations.
Last physical examination at the age of 25 years showed a well-appearing young man, with a weight of 76.4 height of 162ācm. There was no hepatomegaly and the rest of the examination including neurological examination was unremarkable.
Next-generation sequencing with deletion/duplication analysis was carried out for electron-transfer flavoprotein genes (ETFA and ETFB) and the electron-transfer flavoprotein dehydrogenase-related gene (ETFDH) and the result showed no clinically significant DNA variants. Whole exome sequencing and mitochondrial DNA analysis, was done at Breda Genetics Laboratory in Italy and a homozygous c.272āGā>āT (p.Gly91Val) variant was detected in exon 2 of the SLC25A32 gene (NM_030780).
Patient 2
This is a 4 year and 3-month old female child who was born to non-consanguineous Omani parents with unremarkable family history otherwise. She was born full-term via vaginal delivery with a good Apgar score. She had normal growth and development. At the age of one year, she presented with acute gastroenteritis and was found incidentally to have hepatomegaly and mildly elevated aminotransferases. Diagnostic work-up was negative for infectious mononucleosis and hepatitis. Her abdominal ultrasound showed a mildly enlarged liver with normal echogenicity. She also had photosensitivity since early infancy, but her eye exam was normal.
Subsequently, she had recurrent hospital admissions with hypoketotic hypoglycemia triggered by febrile illness associated with vomiting. The lowest blood sugar reading was 0.7āmmol and she only had trace ketonuria then. She also had associated recurrent lactic acidosis/acidemia with the lactate level being around 2.7ā6.8āmmol/l (0.5ā1.6āmmol/l). There was no evidence of hyperlipidemia or hyperuricemia and she always had normal CK. A next-generation sequencing panel for hypoglycemia that included Fructose 1,6 bisphophatase deficiency was outsourced to Fulgent Diagnostics laboratory with negative results for any causative variants.
Plasma acylcarnitine showed increased butyrylcarnitine (C4) and urine organic acids showed elevated methylsuccinic acid, methylfumaric acid, and ethylmalonic acid excretion. Ethylmalonic aciduria was ruled out with negative sequencing and deletion/duplication testing of ETHE1. Whole-exome sequencing outsourced to Medical Neurogenetics, Atlanta, USA showed a homozygous c.272āGā>āT (p.Gly91Val) variant in SLC25A23.
This patient initially was empirically managed with intravenous dextrose during acute febrile illness and hospital admissions and was kept on supplementations of uncooked corn starch (UCCS) at 1.5āg/kg/day at home with avoidance of fasting. After she was diagnosed with SLC25A32-related disorder, UCCS was stopped, and she was started on carnitine at 90āmg/Kg/day and riboflavin at 100āmg two times daily. She had no recurrence of hypoglycemia since then.
Patient 3
This is the brother of patient 2. Following the diagnosis of SLC25A32-related disorder in his sister, he was pre-symptomatically screened with acylcarnitine profiling in dried blood spots at the age of 19 months. He was also found to have an acylcarnitine profile suggestive of MADD (TableĀ 1). Urine organic acids in a sample collected while normoglycemic was essentially unremarkable.
The parents did not come to collect prescribed riboflavin and they elected to wait for his sisterās scheduled routine clinic appointment. He presented with symptomatic hypoglycemia in the context of a febrile illness before starting him on the supplementation. He was managed during that episode with intravenous dextrose 10% 0.9%NaCl solution at 1.5 Ć maintenance, and was kept on oral riboflavin at 100āmg per day and carnitine at 100āmg/Kg/day. Testing for the known familial variant confirmed homozygosity for c.272āGā>āT (p.Gly91Val) variant in SLC25A32, previously identified in his sister.
Discussion
Mitochondrial FAD transporter deficiencies are a rare cause of MADD previously reported to cause exercise intolerance and muscle weakness with progressive neurological symptoms which improved after riboflavin supplementation [4, 5]. The clinical delineation in the two previously reported patients reported showed no associated hypoglycemia, which is instead the major presenting complication in the patients reported here. The limited number of cases reported so far argue for the limitation of knowing the full phenotypic spectrum associated with disease-causing variants in implicated genes like SLC25A32 discussed here.
The three patients we report were all found to have a novel homozygous c.272āGā>āT (p.Gly91Val) in SLC25A32. This variant has not been reported yet in public databases like in dbSNP, gnomAD, 1000 Genomes, NHLI Exome Sequencing Project (ESP), or ClinVar. This variant is also absent from in-house 1562 population-specific exomes. The nucleotide position is conserved across 35 mammalian species. In silico analysis indicates that the reported variant is likely damaging (MutationTaster: disease-causing; FATHMM-MKL: damaging; Provean: damaging; DANN: 0.998, range 0ā1, being 1 the most damaging). The three described patients are from two different Omani families and two distant geographical regions arguing for the possibility of a founder effect given the rarity of this variant in the Omani population otherwise. The diagnosis of FAD transporter defect is also supported with the acylcarnitine profiles and urine organic acids patterns suggestive of MADD in the absence of an alternative genetic diagnosis despite WES. The notable clinical response to riboflavin supplementation is also in supportive of this diagnosis.
FAD-linked dehydrogenases are essential in the mitochondrialĀ fatty acid beta-oxidation pathway [6]. Stress or fasting-induced hypoglycemia would thus be an anticipated complication in patients with FAD transport defects given the expected impact of FAD availability to dependent dehydrogenases involved in fatty acid beta-oxidation [7]. The hypoketotic response to hypoglycemia seen in the patients we report agrees with this assumption. Hypoketotic hypoglycemia has not been reported in the previously published patients with SLC25A32-related defects. It is not clear if this complication was overridden by the predominant neuromuscular phenotype, or it was not reported on the assumption of it being a mild secondary or non-specific complication. It would be of interest to know whether patients with SLC25A32-related defects who presented at an older age with neuromuscular symptoms may have had fasting or stress-induced hypoglycemia at younger age enabling this diagnosis earlier. On the other hand, it is uncertain whether the patients we report here at a younger age with hypoketotic hypoglycemia are distant to develop neuromuscular complications related to this diagnosis when they grow older. One questions whether riboflavin supplementation alone would be sufficient to prevent this complication or additional supplementation with folate or formate [8] may be required for prevention of the neuromuscular phenotype associated with defective mitochondrial folate transport [9, 10].
TableĀ 1 summarizes the patients with defects in SLC25A32 presently known, including the patients reported here. There is still not enough data to support the phenotype-genotype correlation in FAD transporter defects because of the limited number of cases reported to date. The variant detected in patient 5 completely abolishes translation and it is therefore highly likely that the protein is either completely absent. Patient 4 was compound heterozygous for nonsense and a missense variant which may explain the milder phenotype compared to patient 5. Patients 1, 2, and 3 are all homozygous for the same missense variant. It is uncertain whether the neuromuscular complications related to defects in SLC25A32 are distant to occur in the absence of at least one truncating variant.
The limited genotypeāphenotype correlations, genetic heterogeneity, and impact of physiological status at the time of samples collection for biochemical testing are recognized challenges while investigating patients with hypoglycemia with absent immediate biochemical or endocrine clues. Whole-exome sequencing may arguably be a cost-effective strategy to identify the underlying genetic defects for these disorders [3].
Finally, we conclude that screening for SLC25A32 defects should be included in the differential diagnosis of patients with hypoketotic hypoglycemia; particularly when the acylcarnitine profile or urine organic acids profiles are suggestive of MADD. More patients would need to be evaluated clinically before the full clinical spectrum of this disorder could be ascertained. Whether riboflavin alone is adequate for the prevention of long-term complications is yet to be answered.
Data availability
Data available on request from the authors
References
Balasubramaniam S, Christodoulou J, Rahman S. Disorders of riboflavin metabolism. J Inherit Metab Dis. 2019;42:608ā19.
Vidailhet M. Inborn metabolic diseases. Arch de PĆ©diatrie. 2016;4:100ā1.
Yıldız Y, Talim B, Haliloglu G, Topaloglu H, AkƧƶren Z, Dursun A, et al. Determinants of riboflavin responsiveness in multiple Acyl-CoA dehydrogenase deficiency. Pediatr Neurol. 2019;99:69ā75.
Schiff M, Veauville-MerlliĆ© A, Su CH, et al. SLC25A32 mutations and riboflavin-responsive exercise intolerance. N. Engl J Med. 2016;374:795ā7.
Hellebrekers DMEI, Sallevelt SCEH, Theunissen TEJ, Hendrickx ATM, Gottschalk RW, Hoeijmakers JGJ, et al. Novel SLC25A32 mutation in a patient with a severe neuromuscular phenotype. Eur J Hum Genet. 2017;25:886ā8. https://doi.org/10.1038/ejhg.2017.62. Available from
Wang Y, Palmfeldt J, Gregersen N, Makhov AM, Conway JF, Wang M, et al. Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex. J Biol Chem. 2019;294:12380ā91.
Mochel F, Slama A, Touati G, Desguerre I, Giurgea I, Rabier D, et al. Respiratory chain defects may present only with hypoglycemia. J Clin Endocrinol Metab. 2005;90:3780ā5.
Kim J, Lei Y, Guo J, Kim SE, Wlodarczyk BJ, Cabrera RM, et al. Formate rescues neural tube defects caused by mutations in Slc25a32. Proc Natl Acad Sci USA. 2018;115:4690ā5.
Findley TO, Tenpenny JC, OāByrne MR, Morrison AC, Hixson JE, Northrup H, et al. Mutations in folate transporter genes and risk for human myelomeningocele. Am J Med Genet Part A. 2017;173:2973ā84.
OāCallaghan B, Bosch AM, Houlden H. An update on the genetics, clinical presentation, and pathomechanisms of human riboflavin transporter deficiency. J Inherit Metab Dis. 2019;42:598ā607.
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Bushra AL Shamsi: drafting the manuscript, Khalid Al-Thihli and Fathiya Al-Murshedi: supervising writing the manuscript and medical care of patients, Asila Al-Habsi: the medical care of patients.
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Given the retrospective nature of this study, and since no identifying data are included in this manuscript ethical approval was not required for the purpose of this study. Clinical consent forms were obtained.
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Al Shamsi, B., Al Murshedi, F., Al Habsi, A. et al. Hypoketotic hypoglycemia without neuromuscular complications in patients with SLC25A32 deficiency. Eur J Hum Genet 30, 976ā979 (2022). https://doi.org/10.1038/s41431-021-00995-7
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DOI: https://doi.org/10.1038/s41431-021-00995-7
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