Riboflavin metabolism: role in mitochondrial function

Riboflavin Transporter Deficiency Neuronopathy (OMIM #614707, OMIM# 211500, and OMIM #211530) – previously known as Brown-Vialetto-Van Laere and Fazio- Londe syndrome

Human riboflavin transporters, RFVT1, RFVT2, and RFVT3, are encoded by their respective genes, SLC52A1, SLC52A2, and SLC52A3^[18-21]. These transporters with different tissue expressions transport riboflavin across plasma membranes and maintain the supply of flavins to the cells.

Mutations in the SLC52A2 and SLC52A3 have been reported to cause Brown-Vialetto-Van Laere (BVVL) and Fazio-Londe (FL)^[22,23]. Both conditions are now considered as a single disease entity^[24], with progressive sensorimotor and cranial neuropathy in both and absent sensorineural hearing loss in FL as primary manifestations. A new nomenclature was proposed to clarify the specific disease mechanism and both conditions were renamed to riboflavin transporter deficiency (RTD)^[25], with mutations in SLC52A2 and SLC52A3 causing RTD2 and RTD3, respectively^[26]. A recent review identified 109 RTD patients (52 RTD2, 56 RTD3, and 1 RTD2/3)^[26]. The overall mean age of onset of these patients was 5.3 years (range: 0-5 years). The predominant clinical features were hearing loss and muscle weakness. Some characteristics of patients with RTD3 included a later onset of presentation (> 10 years to third decade) and bulbar symptoms. The common presenting features of patients with RTD2, on the other hand, was abnormal gait and/or ataxia^[26]. Plasma acylcarnitine profiles prior to riboflavin supplementation was suggestive of multiple acyl-CoA dehydrogenase defect (MADD)^[22]. Urine organic acids commonly showed ethylmalonic aciduria suggesting impaired fatty acid, methionine, and/or isoleucine oxidation^[26]. Both plasma acylcarnitine and urine organic acids, however, have been observed to be normal in nearly half of RTD patients^[26].

Effective doses of riboflavin varied 10-80 mg/kg/day with over 70% of patients demonstrating improvements in muscle strength, motor abilities, respiratory function, and/or cranial nerve deficits^[26]. The pathomechanism of the specific vulnerability of neurons in RTD might be due to mitochondrial dysfunction and impairment in the clearance of reactive oxygen species^[26].

Haploinsufficiency of the SLC52A1 (OMIM#615026) due to maternal microdeletion and heterozygous intronic variant has been reported to cause a transient riboflavin responsive neonatal multiple acyl-CoA dehydrogenase deficiency that resolved with oral supplementation of riboflavin^[27-29].

Mitochondrial FAD transporter deficiency (OMIM #616839)

SLC25A32 encoding the mitochondrial FAD transporter has been described in two patients with biallelic mutations to date^[30,31]. The first, a 14-year-old girl presented with riboflavin-responsive recurrent exercise intolerance and biochemical findings of MADD^[30]. The second, a 51-year-old Dutch patient with a severe neuromuscular phenotype had initially presented aged three years with muscle weakness post- influenza infection. He subsequently developed impaired motor skills, progressive exercise intolerance in childhood, early-onset ataxia, myoclonus, dysarthria, and dysphagia^[31]. Muscle biopsy in both patients demonstrated ragged-red fibers, lipid storage, and decreased staining for succinate dehydrogenase (SDH, FAD-dependent mitochondrial respiratory chain Complex II) and COX (mitochondrial respiratory chain Complex IV). Complex II deficiency was revealed in cultured skin fibroblasts and muscle from the first and second patients, respectively^[30,31]. Dramatic improvements in exercise tolerance and endurance and biochemical abnormalities were reported in both patients following oral riboflavin supplementation^[30,31].

SLC25A32 has recently been revealed to be a novel regulator of cancer cell proliferation and mitochondrial FAD metabolism. SLC25A32 knock-down in sensitive tumor cells resulted in inhibition of the FAD-dependent Complex II, increased succinate levels, and reduced oxygen consumption rate^[32]. These findings corroborate evidence of decreased Complex II protein levels and OXPHOS activity, which is a marker for mitochondrial FAD in muscle of patients with severe neuromuscular phenotype and novel variants in SLC25A32^[30,31]. Reduction of mitochondrial FAD concentrations by inhibition of SLC25A32 is anti-proliferative in a subset of tumor cell lines and has potential clinical applications as a novel cancer target by increasing oxidative stress and reducing tumor growth^[32].

Flavin adenine dinucleotide synthase (FAD) synthase deficiency (OMIM # 255100)

The FLAD1 gene encodes FAD synthase (EC 2.7.7.2), which catalyzes the adenylation of FMN into the redox cofactor FAD. Human FADS has previously been shown to be a bifunctional enzyme with both FAD synthase and hydrolase activity^[33]. The enzyme contains an N-terminal molybdopterin binding (MPTb) domain, which has FAD hydrolase activity, and a C-terminal 3’-phosphoadenosine-5’-phosphosulfate (PAPS) reductase domain, which is sufficient to catalyze FAD synthesis and has thus been renamed the FADS domain^[33,34]. Multiple FADS isoforms generated due to alternative splicing of the encoding FLAD1 gene result in distribution within different subcellular localizations^[34-36].Two isoforms, the more abundant cytosolic (FADS2) and the mitochondrial (FADS1), have been described in detail^[33]. Human FADS1, FADS2, and FADS3 possess both catalytic domains and are bifunctional, while FADS4 and FADS5 present the sole mMPTb domain and are monofunctional enzymes^[37]. A novel cytosolic FADS6 isoform recently characterized contains only the PAPS domain with considerable FAD synthesis activity, which allows survival of affected patients with biallelic FLAD1 frameshift variants^[37].The FADS6 isoform has approximately 70% of the FAD-synthesizing capacity of human FADS2. Its catalytic efficiency (kcat/Km) is however higher due to a lower Km for FMN and ATP compared to FADS2^[38].

The recently described clinical spectrum for FAD synthase deficiency has ranged from neonatal-onset, a lethal disease with metabolic myopathy, cardiomyopathy, swallowing, speech difficulties, and respiratory insufficiency, to later-onset, a potentially treatable neuromuscular disorder with lipid storage myopathy, MADD-like metabolic aberrations, and combined respiratory-chain deficiency^[34]. To date, sixteen patients have been reported with age of onset ranging from birth to 44 years^[34,39-45]. Most patients (92%) have however presented in infancy, with five out of thirteen dying within the first twelve months of life. The first patient described by Taylor et al.^[39], a Turkish male infant of consanguineous parents, presented at age four months with respiratory insufficiency, lipid myopathy, decreased activities of mitochondrial respiratory chain Complexes I and IV, and early death at eight months of age. A homozygous 4-bp deletion (c.397_400delTTCT) in the FLAD1 gene was identified, resulting in a frameshift and premature termination (Phe134CysfsTer8). Functional studies were not performed; however, in silico predictions of the deleterious effect of the mutations supported a causal association.

A further 15 patients from 13 unrelated families affected by MADD and/or multiple respiratory-chain deficiency and lipid storage myopathy were diagnosed with FLAD1 deficiency. Apart from one patient, all were identified by whole-exome sequencing or through candidate gene panels^[3]. Characteristic clinical manifestations included early infantile onset hypotonia, swallowing, speech difficulties, respiratory insufficiencies, and a fatal course (seven died within the first year of life and one at age 16 years). Cardiorespiratory collapse was the cause of death for a neonate who presented at 32 h of age and died at three days. One patient died of multiorgan failure at seven months due to recurrent episodes of sudden cardiac arrest necessitating a pacemaker. Another infant with cardiomyopathy and recurrent supraventricular tachycardias who required implantable cardioverter defibrillator placement showed a dramatic response to riboflavin supplementation and was still alive at 22 years. Interestingly, the acute metabolic decompensations observed in the neonatal forms of MADD caused by ETFA, ETFB, and ETFDH defects, including hypoglycemia, metabolic acidosis, and hyperammonemia have not been reported in early-onset FADS deficiency^[41]. The adult onset presentations at 20 and 44 years included exercise intolerance and progressive muscle weakness, with gait difficulties, bilateral foot drop, and arm weakness in the older patient. These individuals were alive at 44 and 56 years, respectively^[34,40].

Metabolic findings in FADS deficiency are typical of riboflavin deficiency or MADD, with increased acylcarnitines (C4-C14 even chain acylcarnitines; C5 and C5-DC; and C10:1, C14:1, C16:1, C18:1, and C18:2) and urinary organic acids (increased urinary excretion of ethylmalonic, adipic, suberic, glutaric and methylsuccinic acids, hexanoylglycine, and tiglylglycine)^[3]. Characteristic muscle pathology with pronounced lipid storage and global decrease of COX and/or SDH histochemical staining has been reported^[39]. Multiple respiratory chain enzyme deficiencies involving Complexes I-III were described in skeletal muscle biopsies in seven of eight cases tested^[34,39,41].

A possible genotype-phenotype correlation has been proposed with homozygous carriers of frameshift variants affecting the MPTb domain displaying a more severe clinical course than that observed in individuals harboring single amino acid changes in the FADS domain that are potentially more responsive to riboflavin therapy^[34].

Riboflavin supplementation resulted in clinical improvements in nine of ten patients treated^{[34,39-42,44,45]}. All five children who presented in infancy and treated with riboflavin were alive, two at 8 years, one at 22 years, one at 15 months^[44]), and one at 2 years 5 months^[45]. Treatment with riboflavin in two patients carrying homozygous c.401_404delTTCT mutations in FLAD1 at age three months resulted in milder improvements in spontaneous activity, muscle tone, vomiting, and alertness; however, it failed to prevent disease progression and demise by six and five months, respectively^[41]. The same variant had previously been reported in an untreated Turkish infant with multiple respiratory chain complex deficiencies who died at six months of age^[34]. It has been proposed that the partial response to riboflavin supplementation and residual FADS activity described in patients with biallelic frameshift mutations in exon 2 of FLAD1 may be attributed to the existence of a FADS isoform that lacks exon 2, but has an intact and functional FADS domain^[34]. However, the doses of riboflavin used in these patients may have been inadequate in ensuring long-term survival^[41].

A trial of riboflavin therapy should be offered to all FADS-deficient individuals while awaiting mutational analysis. The benefits of early diagnosis and treatment is exemplified by the fatal outcome of the untreated brother of an older sister who responded to riboflavin^[34].

Riboflavin kinase deficiency (OMIM # 613010)

RFK (EC 2.7.1.26) is a ubiquitous rate-limiting enzyme that catalyzes the first step in flavocoenzyme biosynthesis by phosphorylating riboflavin to form FMN, an obligatory step in riboflavin metabolism. FMN is subsequently adenylated by FAD synthetase to generate FAD^[46]. Embryonic lethality demonstrated before Day 7.5 of gestation in complete knockout mice was potentially attributable to secondary mitochondrial dysfunction from the effect on flavocoenzyme deficiencies on the electron transport chain, which is critically involved in energy generation^[47]. To date, there have been no reported human phenotypes of RFK deficiency. It is possible that defects in RFK may be incompatible with life due to its critical role noted above. Hypomorphic RFK mutations may result in clinical phenotypes that are not that dissimilar to patients with FLAD1 mutations^[3].

Acyl-CoA dehydrogenase-9 deficiency (ACAD9) (OMIM #611126)

Acyl-CoA dehydrogenase 9 (ACAD9), a mitochondrial protein, was initially demonstrated to catalyze the initial rate-limiting step in the beta-oxidation of long-chain fatty acids^[48]. It was subsequently proposed to be a novel assembly factor crucial for oxidative phosphorylation Complex I biogenesis, independent of its role in fatty acid oxidation^[49]. More recently, human ACAD9 was showed to be a bifunctional enzyme involving primarily oxidative phosphorylation Complex I biogenesis, with an additional moonlighting function in fatty acid oxidation (FAO)^[50].

Major clinical presentations of ACAD9 deficiency include biventricular hypertrophic cardiomyopathy (85%), muscle weakness (75%), exercise intolerance (72%) and lactic acidosis^[51]. Two subgroups of ACAD9 deficient patients have been described, including early-onset, often lethal cardiac involvement presenting in infancy. Those surviving from this group performed more poorly than later-onset presentations. Interestingly, developmental delay and severe intellectual disability were only seen with early disease onset^[51]. ACAD9 is the only long-chain ACAD enzyme expressed in the brain and is especially abundant during fetal life^[49,52]. Hence, it has been proposed that the neurological symptoms present in the most severely affected ACAD9-deficient patients could be attributed to a deficiency in brain FAO. To date, no patients with two ACAD9 null mutations have been described, suggesting that the complete absence of the protein is lethal. Embryonic lethality was also proposed for the failure to generate homozygous knockout ACAD9 mice^[49].

Riboflavin supplementation resulted in alleviation of symptoms in 65% patients, and, most notably, improved survival was observed when commenced within the first year of life^[51]. Riboflavin responsiveness may be related to its essential function as a precursor of FAD cofactor for ACAD enzyme activity and stability. It also increases ACAD9 protein levels and rescues Complex I assembly, while also functioning as a chemical chaperone by improving folding of ACAD9 mutant proteins^[49]. Schiff et al.^[49] (2015) suggested that similar specific interventions used for VLCAD defects including avoidance of fasting, medium-chain triglycerides, or triheptanoin anaplerotic therapy could be beneficial in terms of long-term outcomes in patients.

FAD-dependent OXidoREDuctase deficiency (FOXRED1) (OMIM #618241)

FOXRED1 has been proposed to be a dual function protein. It plays a key role as an assembly factor for Complex I biogenesis, and, secondly, due to its oxidoreductase activity, it is hypothesized to participate in glycine metabolism which modulates glutathione biosynthesis, an antioxidant protecting the cells from ROS^[53-55]. Co-immunoprecipitation experiments suggest that FOXRED1 facilitates Complex I assembly by associating with the 370-kDa subcomplex and two other FAD-dependent Complex I assembly factors, ACAD9 and probably AIFM1^[55].

FOXRED1 mutations have been recognized as a cause of Complex I deficiency. To date, eight patients from six families have been described with variable clinical spectrum and severity^[55-60]. Age of onset of clinical manifestations ranged from birth to early infancy with one prenatal onset of oligohydramnios, severe intrauterine growth retardation, and periventricular cysts^[60]. The clinical manifestations included Leigh syndrome and infantile-onset encephalomyopathy with epilepsy, mild to severe psychomotor retardation, and hypotonia. Non-neurological features included congenital lactic acidosis, cardiomyopathy, hepatomegaly, kyphoscoliosis, optic atrophy, roving eye movements, strabismus, and distal renal tubular acidosis^[55-58,60]. Brain neuroimaging (MRI) findings ranged from normal to delayed myelination and features consistent with Leigh syndrome^[55-57]. Enzymatic studies supported Complex I deficiency in all patients. Additionally, a milder decrease in Complex II was observed in one patient^[59]. The majority of mutations identified were missense. Riboflavin supplementation was reported in only two patients^[57,60], of whom one had prenatal onset and died at three months of age^[60]. Outcome was available in 6/8 patients: 4/6 are alive with median age of 17 years (age range: 10-22 years) and another died at eight years. In contrast to patients with other nuclear encoded Complex I defects who typically have a severe clinical presentation and associated early death, it has been suggested that pathogenic variants in the FOXRED1 gene result in partial loss of function and are probably hypomorphic due to the longer survival of patients^[61].

COQ6 deficiency (OMIM #614650)

Human Coenzyme Q10 monooxygenase 6 (COQ6) is a flavoprotein involved in the biosynthesis of Coenzyme Q10 which operates as a redox carrier by transferring electrons from respiratory chain Complexes I and II to Complex III. Coenzyme Q10 is also a potent antioxidant^[62] and a cofactor of many mitochondrial dehydrogenases. It is required for pyrimidine nucleoside biosynthesis and has been implicated in the inhibition of apoptosis by preventing the collapse of the inner mitochondrial membrane^[63]. Studies using human cell line lacking functional COQ6 showed impaired COQ synthesis, severe ATP deficiency, and increased production of reactive oxygen species^[64].

In 2011, Heeringa et al.^[65] reported a novel cause for steroid-resistant nephrotic syndrome associated with sensorineural hearing loss due to variants in the COQ6 gene. Twenty-three patients in 16 families with similar clinical manifestations and two mutations in the COQ6 gene have been identified to date^[65-71]. Extra-renal manifestations such as ataxia, seizures, muscle weakness, white matter changes, and optic atrophy were less common^[65,68]. The age of onset of the renal disease varied from 2 months to 6.4 years but was most common in infancy and early childhood. The course was progressive with median age of onset of end stage renal disease between 13 months and 19 months^[65,68]. The most common histopathologic finding was focal segmental glomerulosclerosis. Abnormal mitochondrial proliferation in the podocytes was also noted on electron microscopy^[68].

Coenzyme Q10 supplementation alone at 5-30 mg/kg/day was reported in a few patients. Significant improvement in proteinuria with normal renal function was noted on follow-up of these patients^{[66,70,72-74]}) However, hearing loss had not improved after Coenzyme Q10 supplementation^[66,72]. Hence, early supplementation of Coenzyme Q10 seemed to help avoid development of massive proteinuria and consequently chronic renal failure^[72].

GFER deficiency (OMIM #613076)

Human Growth Factor ERV-1-like (GFER) (also known as augmenter of liver regeneration 1, ALR1) is one of the sulfhydryl oxidases with a FAD-binding domain, which together with disulfide carrier Mia40 forms part of the disulfide relay system (DRS) and constitutes the mitochondrial import and assembly (MIA) pathway. The MIA pathway plays an important role in the biogenesis of many mitochondrial intramembrane space (IMS) proteins^[75]. GFER has two distinct isoforms: the long isoform (205 amino acids, 23 kD), which is mainly located in the IMS of the mitochondria, and the shorter isoform (125 amino acids, 15 kD), which is present in the nucleus. Mia40 functions as an import receptor of cysteine-rich substrates in the IMS and promotes oxidative folding of these proteins. It is re-oxidized by GFER in a disulfide-transfer reaction with electrons transferred to cytochrome c, thus connecting the DRS to the electron-transport chain of the mitochondria and establishing its role in Complex IV activity^[76].

In 2009, Di Fonzo et al.^[77] reported homozygous missense mutations (c.581 G > A p.R194H) in the GFER gene in three siblings from a consanguineous Moroccan family. Subsequent functional studies using analogous mutation (R182H) in yeast ERV1 showed altered mitochondrial morphology, mitochondrial instability, reduced cyt C oxidase activity, weaker FAD binding, decreased thermal protein stability, and altered protein folding^[77,78]. Daithanka et al.^[79] (2010) also characterized the human GFER/ALR protein and demonstrated that the R194H led to marked loss of protein stability by an increased rate of FAD dissociation and enhanced proteolysis susceptibility with only minimal effect on enzymatic activity.

There are eight patients in four families reported with mutations in the GFER gene^[77,80,81], to date. The age range of the patients was 3 to 21 years with one deceased at 21 years of age. The predominant features were neuromuscular including hypotonia and psychomotor retardation of variable severity, muscle hypotrophy, progressive muscle weakness requiring ventilatory support, and, additionally, cataracts and lactic acidosis. Other clinical features included movement disorder, dysautonomia, cachexia and orthopedic problems^[81], hearing loss^[77], and adrenal insufficiency^[80]. MRI of the brain showed normal findings (4/6), cerebellar and moderate cortical atrophy (1/6), and thin corpus callosum (1/6). Histopathologic muscle findings included abnormal mitochondrial morphology, ragged red fibers, COX negative, and atrophic fibers. Enzymatic testing revealed isolated Complex IV deficiency or combined deficiencies of Complexes I-IV. The missense mutation (c.581 G > A; p.R194H) was identified in six out of the eight patients.

Riboflavin supplementation was not reported in these patients. Experiments have indicated that the FAD cofactor is detached from Erv1 R182H during the catalytic reaction and inactivated the protein^[78]. The functional defect was shown to recover by adding extra FAD^[78]. Hence, riboflavin supplementation in patients with GFER mutations may potentially have clinical benefit, but this is speculative.

FDXR deficiency (OMIM #617717)

FDXR (Ferredoxin Reductase) has one FAD/NAD(P)-binding domain and two NAD(P)-binding domains. It is the sole ferredoxin reductase in humans and has important roles in iron-sulfur (Fe-S) cluster biogenesis and heme synthesis^[82]. Mitochondrial membrane associated FDXR reduces the ferredoxins, FDX1 and FDX2, and transfers electrons from NADPH to the mitochondrial cytochrome P450 system, thus initiating the mitochondrial electron transport chain reaction^[83]. Studies using patient derived fibroblasts showed loss of FDXR function led to decreased enzyme activity, reduced functions of Complexes I-III, significant increase in reactive oxygen species production, and mitochondrial iron overload^[84,85].

There are 27 patients in 19 families with mutations in the FDXR gene reported in the literature^[84-86]. A cohort of eight patients presented predominantly with auditory neuropathy and optic atrophy with onset in childhood or adolescence^[84]. There were no other clinical features except for mild language delay in one and retinitis pigmentosa, ophthalmoplegia, and lower limbs hypoallesthesia in another patient^[84]. However, clinical variability was present in another cohort of patients who presented with neurological features of early onset, i.e., infancy to childhood^[85]. Apart from visual problems, i.e., retinitis pigmentosa and/or optic atrophy, and hearing loss, this cohort of 13 unrelated patients also had other clinical features such as hypotonia, global developmental delay, axonal sensorimotor polyneuropathy, regression after febrile illness, ataxia, failure to thrive, progressive microcephaly, and encephalopathy^[85]. Respiratory chain enzyme activities, when reported, showed normal activities or combined deficiencies. Post-mortem findings of one FDXR patient showed neuronal loss and vacuoles in the cerebral cortex and increased biomarkers for gliosis, astrocyte activation, and neurodegeneration^[86]. These findings were also observed in a homozygous p. R389Q mouse model, suggesting that inflammation could be a major component of the pathology in the neurodegeneration of FDXR mutations^[86]

NDUFV1 deficiency (OMIM # 618225)

NADH: ubiquinone oxireductase flavoprotein 1 (NDUFV1) is a 51-kDa highly conserved nuclear encoded subunit comprising the electron input (N) functional module of Complex I^[87]). It contains NADH-, FMN-, and Fe-S-binding sites^[88].

Mutations in the NDUFV1 gene causing Complex I deficiency have been identified in 41 patients in 33 families^[56,88-110]. The median age of onset of clinical manifestations was 8.0 months (range: birth to 6 years). The most common clinical features were motor delay or neurodevelopmental regression usually precipitated by an illness, hypotonia, dystonia, seizures, spasticity, and cognitive impairment. Oculomotor impairment including ptosis, strabismus, and ophthalmoplegia was also frequently observed. Typical Leigh syndrome^[88,107]and late-onset Leigh syndrome have been reported^[95,103]. The clinical course was variable with some patients having slower progression of neurological manifestations^[90,91,103]. Brain MRI and MRS findings included features of typical Leigh syndrome, white matter changes, basal ganglia changes, elevated lactate peak, infantile striatal necrosis^[97], and cavitating leukoencephalopathy^[109]. Outcome was available in 34 patients; ten died at a median age of six months (range: three days to three years). The median age among surviving patients was seven years (range: 1-15 years) and severity of neurocognitive outcome varied from mild learning disability^[91,96] to significant impairment^[101,103]. Riboflavin supplementation was only reported in seven patients^{[94,97,98,101]} and was given with other supplements such as thiamine, Coenzyme Q10, creatine, and alpha lipoic.

NDUFV2 deficiency (OMIM #618229)

Human NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), a 24-kDa binuclear [2Fe-2S] cluster containing protein, is highly conserved and one of the nuclear encoded subunits of Complex I which is involved in electron transfer. It is also one of the subunits comprising the electron input (N) functional module of Complex I^[87].

Phenotypic variability is observed in patients with Complex I deficiency due to mutations in the NDUFV2 gene. Bénit et al.^[111] 2003 reported three affected siblings in a consanguineous family presenting with early onset hypertrophic cardiomyopathy, truncal hypotonia, feeding difficulties, growth retardation, and early death within the first year of life. Another patient also presented with hypertrophic cardiomyopathy and encephalopathy^[112]. However, a separate family with three affected siblings presented with Leigh syndrome without cardiac involvement^[113]. These patients reported by Cameron et al.^[113] had seizures by 10 months of age, progressive clinical course, and two died at 19 months and 10 years. One of these patients was started on riboflavin plus other supplements. One sibling is still alive at 32 years despite a vegetative state by three years of age. All these patients were homozygous for a 4-bp deletion in intron 2 (IVS2 + 5_ + 8delGTAA) of the NDUFV2 gene^[113]. Studies using human disease cell model showed that this deletion caused a significant reduction in the mitochondrial targeting ability of the NDUFV2 protein^[114].

In a cohort of 37 children with cavitating leukoencephalopathies, three (3/37, 8.1%) were compound heterozygotes for pathogenic mutations in the NDUFV2 gene^[109]. The age of onset of symptoms was 4-46 months with stable/improved clinical course and frontal predominant and deep white matter patterns on brain MRI^[109].

Succinic dehydrogenase subunit A deficiency (OMIM #252011)

Succinic dehydrogenase subunit A (SDHA), a flavoprotein with FAD as cofactor, is a nuclear encoded and catalytic subunit of Complex II (succinate dehydrogenase) which oxidizes succinate to fumarate in the Kreb’s cycle and transfers electrons to ubiquinone in the mitochondrial electron transport chain^[115].

Mutations in SDHA gene result in both a mitochondrial disease causing Complex II deficiency and tumor susceptibility. A large proportion (47%) of germline mutations in SDHA has been reported in gastrointestinal stromal tumors^[116]. Other tumors such as paraganglioma/pheochromocytoma, pituitary adenoma, and renal carcinoma have also demonstrated SDHA germline mutations^[117-121].

Complex II deficiency due to mutations in SDHA presents with variable clinical presentations and age of onset from prenatal to adulthood. The first report of a nuclear gene mutation causing a mitochondrial respiratory chain deficiency in humans was identified in two sisters presenting with developmental regression at 10 months of age, pyramidal tract signs, leukodystrophy with early demise, Complex II deficiency, and homozygous mutations (R554W) in the SDHA gene^[122,123]. Further reports of patients with SDHA mutations have since been described^[124-131]. Leigh or Leigh-like syndrome was a common phenotype. Slower progression with survival up to 11 years of age has been observed^{[126,127,130]}, and, in one patient, there was no apparent cognitive impairment at 10 years of age^[130]. Isolated cardiomyopathy presenting from 32 weeks in utero to 10 years of age was associated with high mortality in two large Bedouin families with homozygous G555E mutations in the SDHA gene^[132].

Adult presentation has been reported in two sisters (62 and 56 years) with normal cognition, late-onset (mid-40s), and slowly progressive cerebellar ataxia, visual impairment, optic atrophy, proximal weakness, and partial Complex II deficiency in skeletal muscle^[133]. Riboflavin supplementation did not prevent neurological progression in these two patients. These patients were found to be heterozygous for a missense mutation (R408C) in a highly conserved region of the mature SDHA protein, suggesting an autosomal dominant inheritance pattern^[134]. Another family with three affected members who presented with optic atrophy, dilated cardiomyopathy, ataxia, and isolated Complex II deficiency in fibroblast were also found to be heterozygous for a R451C missense mutation. This family serves as the second report supporting an autosomal dominant inheritance^[135].

Multiple Acyl-CoA Dehydrogenase Deficiency (OMIM# 231680)

Multiple acyl-coenzyme A dehydrogenase deficiency, also known as glutaric aciduria Type II (GAII), is an autosomal recessive disorder that affects the oxidation of fatty acids, BCAA, lysine, tryptophan, and choline. MADD is caused by deficiency of one of the two electron-transfer flavoproteins which transfer electrons from acyl-CoA dehydrogenases to Coenzyme Q in the respiratory chain: Electron Transfer Flavoprotein (ETF), encoded by ETFA and ETFB genes, and electron-transfer flavoprotein dehydrogenase (ETFDH), encoded by ETFDH^[136]. Metabolic defects resulting from impaired beta-oxidation include decreased ATP biosynthesis, excessive lipid accumulation in various organs, and insufficient gluconeogenesis^[137].

The clinical phenotype is variable and has been classified into neonatal onset forms, with the most severely affected patients presenting with congenital anomalies (Type I) or without anomalies (Type II), and mild and/or later onset (Type III)^[138]. Severely affected patients present in the first few days of life with non-ketotic hypoglycemia, hyperammonemia, and metabolic acidosis accompanied by hypotonia, encephalopathy, hepatomegaly, cardiomyopathy, and poor prognosis. An odor of sweaty feet similar to that in isovaleric acidemia may be observed^[139]. Some patients have congenital anomalies (including large cystic kidneys, hypospadias, and neuronal migration defects that can be prenatally detected by fetal MRI and facial dysmorphism (low set ears, high forehead, and midfacial hypoplasia)^[139]. The most frequent clinical presentation is the milder myopathic Type III form, which manifests with fluctuating proximal and axial myopathy with exercise intolerance and occasionally respiratory insufficiency^[138] or rhabdomyolysis, although often with hepatomegaly, encephalopathy, and episodic lethargy, as well as vomiting and hypoglycemia often triggered by metabolic stress, episodes of which have been lethal in 5% of patients^[140]. Although most decompensations occur in childhood, severe metabolic crises have also been reported in adulthood^[138]. The majority of the 350 cases of late-onset MADD described in the literature carry mutations in the ETFDH gene (93%), while mutations in the ETFA (5%) and ETFB (2%) genes are less frequent^[138].

Laboratory parameters include increased creatine kinase and lactate levels, with low carnitine. Diagnostic confirmation is made by the increase of short-, medium-, and long-chain acyl-carnitines on acylcarnitine analysis and characteristic urinary organic acid pattern comprising elevated levels of glutaric, ethylmalonic, 3-hydroxyisovaleric, 2-hydroxyglutaric, 5-hydroxyhexanoic, and relevant ketonuria, particularly glycine conjugates of C4 and C5 acids^[137,138]. Muscle biopsies usually reveal lipid storage myopathy and secondary mitochondrial dysfunction with decreased Complexes I and II + III, attributable to deficiency of Coenzyme Q, which has been associated with increased ROS generation due to electron leak from misfolded variant ETFDH proteins and impaired Q10 binding affinity^[138].

A clear genotype-phenotype correlation has been reported with the heterogenous subtypes of MADD. Type I disease is usually associated with homozygosity for null mutations. Even minor amounts of residual ETF/ETFDH activity suffices to prevent embryonic development of congenital anomalies observed in Type II disease, whereas higher residual activity is found in the late-onset form/ Type III disease. The genotype-phenotype correlation within Type III patients is however poor, as exogenous stressors including febrile infections may modulate the residual activity^[138].

Riboflavin supplementation has successfully ameliorated clinical symptoms and metabolic abnormalities in almost all patients (98%) with late-onset MADD, the majority of whom have ETFDH mutations (93%)^[138]. High levels of FAD and FMN due to riboflavin supplementation promote folding and stability of flavoproteins, particularly at fever simulating temperatures (40 °C), permitting certain mutant flavoenzymes to reach a folding efficiency and stability that attenuates the enzyme deficiency^[7]. Combined treatment of riboflavin and Coenzyme Q10 has been advocated for use in riboflavin-responsive MADD based on studies which demonstrated improved mitochondrial dynamics with decreased ROS production and restoration of CoQ10 levels in patient fibroblasts co-treated with CoQ10 and riboflavin^[141].

Dihydrolipoamide dehydrogenase (E3) deficiency (OMIM# 246900)

The DLD gene encodes dihydrolipoamide dehydrogenase (also known as E3), a common flavoprotein shared by three mitochondrial α-ketoacid dehydrogenase multi-enzyme complexes: pyruvate dehydrogenase complex (PDHC), α-ketoglutarate dehydrogenase complex, and branched-chain α-keto acid dehydrogenase complex. It catalyzes the third stage of the reaction by re-oxidizing the reduced lipoyl moiety of E2 and generating lipoic acid and NADH^[142]. E3 is also a component of a fourth mitochondrial multienzyme complex, the glycine cleavage system, where it functions as the L protein, a housekeeping enzyme that, to date, has not appeared to impair the function of the system in vivo when associated with pathogenic variants of DLD^[143]. The metabolic derangements incorporate PDHC and TCA cycle defects: increased blood lactate and pyruvate, elevated plasma alanine, BCAA, presence of allo-isoleucine in plasma, and increased urinary lactic, pyruvic, 2-ketoglutaric, and branched-chain 2-hydroxy- and 2-ketoacid^[143,144].

The phenotypic spectrum of DLD deficiency includes: (1) classical E3 deficiency, which includes early-onset encephalopathy with progressive hypotonia, failure to thrive, hypoglycemia, ketoacidosis, and encephalopathy or Leigh-like encephalopathy; (2) a primarily hepatic presentation with Reye-like syndrome, wherein patients typically have normal intellect without residual neurologic deficits between acute metabolic episodes; and (3) a milder riboflavin responsive myopathic phenotype with exertional fatigue, intermittent elevation of blood lactate, ketoacidosis, creatine kinase, and mitochondrial proliferation^{[143,145,146]}. Riboflavin supplementation led to complete resolution of muscle weakness, improvement of metabolic abnormalities, partial restoration of the DLD protein, resolution of mitochondrial proliferation in muscle, and reduced ROS production in fibroblasts, supporting the evidence of a chaperone-like effect of riboflavin in promoting DLD protein stability and folding^[146].

Ethylmalonic aciduria secondary to ETHE1 dysfunction (OMIM# 602473)

Ethylmalonic encephalopathy (EE) is a rare, devastating, invariably fatal neurodegenerative disease caused by mutations in the ETHE1 gene, which encodes a mitochondrial sulfur dioxygenase critical in hydrogen sulfide (H₂S) detoxification^[147]. The characteristic clinical features are hydrogen sulfide mediated and include vasculopathy due to diffuse microvasculature injury responsible for multiple necrotic brain lesions, which lead to early-onset psychomotor regression, seizures, and global neurological impairment that subsequently evolve to severe psychomotor delay with spastic tetraparesis. Vascular lesions include diffuse and spontaneous relapsing petechial purpura, orthostatic acrocyanosis, hemorrhagic suffusions of mesothelial surfaces, and intestinal mucosa associated with chronic hemorrhagic diarrhea^[148].

Biochemical abnormalities resulting from accumulation of hydrogen sulfide and its derivative thiosulfate in crucial tissues including liver, brain, and colonic mucosa leading to inhibition of both short-chain acyl-CoA dehydrogenase with consequent elevation of ethylmalonate, C4- and C5-acylcarnitines predominantly in muscle and brain, and cytochrome c oxidase deficiency, blocking mitochondrial respiration and increasing lactic acid^[147,148]. Urinary thiosulfate is also markedly elevated^[149].

Current treatment modalities include the off-label use of common drugs such as N-acetylcysteine and metronidazole to lower the production and promote detoxification of toxic H2S. This combined exposure has produced some encouraging results with amelioration of some neurological abnormalities and marked attenuation or disappearance of the vascular lesions and diarrhea^[150], suggesting disease modification from the invariably fatal clinical course of EE^[151]. Therapeutic trial with riboflavin and/or Coenzyme Q10 resulted in decreased C4- and C5-acylcarnitines in one patient and mild improvements in chronic mucoid diarrhea and motor and cognitive functions in three patients^[152].

AIFM1 deficiency (OMIM#300816, OMIM#310490, OMIM#300614, OMIM#300232)

The X-linked AIFM1 gene encodes the apoptosis-inducing factor (AIF), a mitochondrial flavoprotein with a dual role, first as a FAD-dependent NADH oxidoreductase and second as a caspase-independent death effector which regulates apoptosis^[153,154]. AIFM1 mutations have been associated with wide spectrum of clinical phenotypes with X-linked recessive inheritance including a severe, early-onset mitochondrial encephalomyopathy with combined oxidative phosphorylation deficiency^[155], prenatal ventriculomegaly^[156], the Cowchock syndrome, an X-linked Charcot-Marie-Tooth disease (CMTX4) with axonal sensorimotor neuropathy, deafness and cognitive impairment^[157], auditory neuropathy spectrum disorder^[158], spondyloepimetaphyseal dysplasia with mental retardation^[159], and, more recently, cerebellar ataxia partially responsive to riboflavin^[160].

The AIFM1 protein is highly expressed in hairy cells and spiral ganglion within the inner ear, consistent with its crucial role in maintaining normal auditory function^[158]. Hence, the predominant phenotypes described with AIFM1 mutations are either mitochondrial encephalomyopathies or sensorineural hearing loss and auditory neuropathy as transmission from the inner ear to the auditory nerve and brain stem is distorted^[160]. Cerebellar ataxia, with or without vermian atrophy, was described only recently in relation to AIFM1 mutations, and has further widened the symptomatology^[160].

Supplementation with riboflavin was reported to temporarily slow the disease progression in a patient with the severe encephalomyopathy^[155]. A clear improvement of ataxia by 44% and 20% was observed in two patients, respectively, as measured by the ICARS score, an objective assessment that quantifies the magnitude of improvement in progressive ataxias, with a score of 5%-20% considered as substantial in providing a clinical impact. Prompt diagnosis through early recognition of constellation of symptoms including ataxia with sensorineural hearing loss especially auditory neuropathy would be important since the symptoms may be partially amenable to riboflavin treatment^[160].