Dichotomous metabolic networks govern human ILC2 proliferation and function.

Group 2 innate lymphoid cells (ILC2s) represent innate homologs of type 2 helper T cells (TH2) that participate in immune defense and tissue homeostasis through production of type 2 cytokines. While T lymphocytes metabolically adapt to microenvironmental changes, knowledge of human ILC2 metabolism is limited, and its key regulators are unknown. Here, we show that circulating 'naive' ILC2s have an unexpected metabolic profile with a higher level of oxidative phosphorylation (OXPHOS) than natural killer (NK) cells. Accordingly, ILC2s are severely reduced in individuals with mitochondrial disease (MD) and impaired OXPHOS. Metabolomic and nutrient receptor analysis revealed ILC2 uptake of amino acids to sustain OXPHOS at steady state. Following activation with interleukin-33 (IL-33), ILC2s became highly proliferative, relying on glycolysis and mammalian target of rapamycin (mTOR) to produce IL-13 while continuing to fuel OXPHOS with amino acids to maintain cellular fitness and proliferation. Our results suggest that proliferation and function are metabolically uncoupled in human ILC2s, offering new strategies to target ILC2s in disease settings.

Origins of tissue and cell-type specificity in mitochondrial DNA (mtDNA) disease.

Mutations of mitochondrial (mt)DNA are a major cause of morbidity and mortality in humans, accounting for approximately two thirds of diagnosed mitochondrial disease. However, despite significant advances in technology since the discovery of the first disease-causing mtDNA mutations in 1988, the comprehensive diagnosis and treatment of mtDNA disease remains challenging. This is partly due to the highly variable clinical presentation linked to tissue-specific vulnerability that determines which organs are affected. Organ involvement can vary between different mtDNA mutations, and also between patients carrying the same disease-causing variant. The clinical features frequently overlap with other non-mitochondrial diseases, both rare and common, adding to the diagnostic challenge. Building on previous findings, recent technological advances have cast further light on the mechanisms which underpin the organ vulnerability in mtDNA diseases, but our understanding is far from complete. In this review we explore the origins, current knowledge, and future directions of research in this area.

Tracer-based lipidomics enables the discovery of disease-specific candidate biomarkers in mitochondrial ß-oxidation disorders.

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore the presence of disease-specific markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease-specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease-specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

HiFi long-read amplicon sequencing for full-spectrum variants of human mtDNA.

BACKGROUND: Mitochondrial diseases (MDs) can be caused by single nucleotide variants (SNVs) and structural variants (SVs) in the mitochondrial genome (mtDNA). Presently, identifying deletions in small to medium-sized fragments and accurately detecting low-percentage variants remains challenging due to the limitations of next-generation sequencing (NGS). METHODS: In this study, we integrated targeted long-range polymerase chain reaction (LR-PCR) and PacBio HiFi sequencing to analyze 34 participants, including 28 patients and 6 controls. Of these, 17 samples were subjected to both targeted LR-PCR and to compare the mtDNA variant detection efficacy. RESULTS: Among the 28 patients tested by long-read sequencing (LRS), 2 patients were found positive for the m.3243 A > G hotspot variant, and 20 patients exhibited single or multiple deletion variants with a proportion exceeding 4%. Comparison between the results of LRS and NGS revealed that both methods exhibited similar efficacy in detecting SNVs exceeding 5%. However, LRS outperformed NGS in detecting SNVs with a ratio below 5%. As for SVs, LRS identified single or multiple deletions in 13 out of 17 cases, whereas NGS only detected single deletions in 8 cases. Furthermore, deletions identified by LRS were validated by Sanger sequencing and quantified in single muscle fibers using real-time PCR. Notably, LRS also effectively and accurately identified secondary mtDNA deletions in idiopathic inflammatory myopathies (IIMs). CONCLUSIONS: LRS outperforms NGS in detecting various types of SNVs and SVs in mtDNA, including those with low frequencies. Our research is a significant advancement in medical comprehension and will provide profound insights into genetics.

A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical application.

BACKGROUND: Sequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing). RESULTS: By using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases. CONCLUSIONS: Pime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes.

Mitochondrial phosphate-carrier deficiency mimicking infantile-onset Pompe disease.

The mitochondrial phosphate carrier is critical for adenosine triphosphate synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. Variants in the SLC25A3 gene coding mitochondrial phosphate carrier lead to failure in inorganic phosphate transport across mitochondria. The critical dependence on mitochondria as an energy source is especially evident in tissues with high-energy demands such as the heart, muscle; defects in the mitochondrial energy production machinery underlie a wide range of primary mitochondrial disorders that present with cardiac and muscle diseases. The characteristic clinical picture of a prominent early-onset hypertrophic cardiomyopathy and lactic acidosis may be an indication for analysis of the SLC25A3 gene. Here, described a patient with suspicion of infantile Pompe disease due to involvement of heart and muscle and high-level of plasma creatinine kinase but finally diagnosed mitochondrial phosphate-carrier deficiency.

Adult presentation of a rare mitochondrial tRNA Val gene mutation-an expanding clinical phenotype.

BACKGROUND AND PURPOSE: Late-onset mitochondrial disorders are diagnostically challenging with significant heterogeneity in disease presentation. A case is reported of a 67-year-old gentleman who presented with a 3-month history of seizures, recurrent encephalopathy, ataxia and weight loss, preceded by recent initiation of haemodialysis for end-stage chronic kidney disease. METHODS: Extensive work-up including serological, cerebrospinal fluid, magnetic resonance imaging and electroencephalography was performed. Whole exome sequencing and muscle biopsy confirmed the diagnosis. RESULTS: Magnetic resonance imaging brain demonstrated a single non-enhancing T2 fluid attenuated inversion recovery hyperintense cortical/subcortical signal change in the right temporal lobe and cerebellar atrophy. Given the subacute presentation of uncertain aetiology, he was empirically treated for autoimmune/paraneoplastic encephalitis. Despite radiological resolution of the cortical abnormality 2 weeks later, there was no clinical improvement. Further collateral history unveiled a mildly ataxic gait and longstanding hearing loss suggestive of a genetic cause. Whole exome sequencing revealed a likely pathogenic, heteroplasmic mitochondrial DNA variant in the MT-TV gene, m.1659T>C, present at higher levels of heteroplasmy in muscle (91%) compared to other mitotic tissues. A high fat/protein diet and multivitamins including co-enzyme Q10 were commenced. Treatment of the nutritional deficiency and avoidance of intermittent fasting due to unreliable oral intake secondary to encephalopathy probably contributed to the clinical improvement seen over the ensuing few months, with resolution of his encephalopathy and return to his baseline gait and weight. CONCLUSION: An adult case is reported with an acute neurological presentation mimicking encephalitis, caused by a heteroplasmic m.1659T>C MT-TV variant, previously reported once in a child who displayed a different clinical phenotype.

A diagnostic framework to identify vestibular involvement in multi-sensory neurological disease.

BACKGROUND AND PURPOSE: Identifying vestibular causes of dizziness and unsteadiness in multi-sensory neurological disease can be challenging, with problems typically attributed to central or peripheral nerve involvement. Acknowledging vestibular dysfunction as part of the presentation provides an opportunity to access targeted vestibular rehabilitation, for which extensive evidence exists. A diagnostic framework was developed and validated to detect vestibular dysfunction, benign paroxysmal positional vertigo or vestibular migraine. The specificity and sensitivity of the diagnostic framework was tested in patients with primary mitochondrial disease. METHODS: Adults with a confirmed diagnosis of primary mitochondrial disease were consented, between September 2020 and February 2022. Participants with and without dizziness or unsteadiness underwent remote physiotherapy assessment and had in-person detailed neuro-otological assessment. The six framework question responses were compared against objective neuro-otological assessment or medical notes. The output was binary, with sensitivity and specificity calculated. RESULTS: Seventy-four adults completed the study: age range 20-81 years (mean 48 years, ±SD 15.05 years); ratio 2:1 female to male. The framework identified a vestibular diagnosis in 35 participants, with seven having two diagnoses. The framework was able to identify vestibular diagnoses in adults with primary mitochondrial disease, with a moderate (40-59) to very high (90-100) sensitivity and positive predictive value, and moderate to high (60-74) to very high (90-100) specificity and negative predictive value. CONCLUSIONS: Overall, the clinical framework identified common vestibular diagnoses with a moderate to very high specificity and sensitivity. This presents an opportunity for patients to access effective treatment in a timely manner, to reduce falls and improve quality of life.

Cascade testing in mitochondrial diseases: a cross-sectional retrospective study.

BACKGROUND: Cascade testing can offer improved surveillance and timely introduction of clinical management for the at-risk biological relatives. Data on cascade testing and costs in mitochondrial diseases are lacking. To address this gap, we performed a cross-sectional retrospective study to provide a framework for cascade testing in mitochondrial diseases, to estimate the eligibility versus real-time uptake of cascade testing and to evaluate the cost of the genetic diagnosis of index cases and the cost of predictive cascade testing. METHODS: Data was collected through retrospective chart review. The variant inheritance pattern guided the identification of eligible first-degree relatives: (i) Males with mitochondrial DNA (mtDNA) single nucleotide variants (SNVs) - siblings and mothers. (ii) Females with mtDNA SNVs - siblings, mothers and offspring. (iii) Autosomal Dominant (AD) nuclear DNA (nDNA) variants - siblings, offspring and both parents. (iv) Autosomal Recessive (AR) nDNA variants - siblings. RESULTS: We recruited 99 participants from the Adult Mitochondrial Disease Clinic in Sydney. The uptake of cascade testing was 55.2% in the mtDNA group, 55.8% in the AD nDNA group and 0% in AR nDNA group. Of the relatives in mtDNA group who underwent cascade testing, 65.4% were symptomatic, 20.5% were oligosymptomatic and 14.1% were asymptomatic. The mean cost of cascade testing for eligible first-degree relatives (mtDNA group: $694.7; AD nDNA group: $899.1) was lower than the corresponding index case (mtDNA group: $4578.4; AD nDNA group: $5715.1) (p < 0.001). CONCLUSION: The demand for cascade testing in mitochondrial diseases varies according to the genotype and inheritance pattern. The real-time uptake of cascade testing can be influenced by multiple factors. Early diagnosis of at-risk biological relatives of index cases through cascade testing, confirms the diagnosis in those who are symptomatic and facilitates implementation of surveillance strategies and clinical care at an early stage of the disease.

Atypical presentation of Pearson syndrome in an infant with suspected myelodysplastic syndrome.

BACKGROUND: Anemia exhibits complex causation mechanisms and genetic heterogeneity. Some cases result in poor outcomes with multisystemic dysfunction, including renal tubulopathy. Early diagnosis is crucial to improve management. CASE-DIAGNOSIS/TREATMENT: A 21-month-old female patient was admitted with severe anemia. Persistent neutropenia and dysplastic signs suggested myelodysplastic syndrome, but targeted gene panel results were negative. After multiple transfusions, spontaneous hematologic recovery was observed. At 4 years old, she presented failure to thrive, renal Fanconi syndrome, and severe metabolic acidosis. Differential diagnosis included Pearson syndrome (PS), a life-threatening condition associated with mitochondrial DNA (mtDNA), featuring anemia and pancreatic insufficiency. Further analysis revealed a ~ 7.5 kb mtDNA deletion. Until the age of 5, supportive care has been provided, without pancreatic insufficiency. CONCLUSIONS: This PS case highlights the importance of genetic testing, even in the absence of typical features. Understanding the nature of mitochondrial disorders enables treatment tailoring and counseling about the prognosis.

The Importance of Mitochondrial Disease Testing in Young Adults With New Onset Sensorineural Hearing Loss.

OBJECTIVES: Sensorineural hearing loss (SNHL) occurs commonly as part of mitochondriopathies and varies in severity and onset. In this study, we characterized hearing with specific consideration for hearing loss as a potential early indicator of mitochondrial disease (MD). We hypothesize that genetic testing at the earliest detection of SNHL may lead to an earlier MD diagnosis. DESIGN: We reviewed the clinical and audiometric data of 49 patients undergoing genetic testing for MD. RESULTS: One-third of individuals with molecularly confirmed MD presented with SNHL. On average, patients had hearing loss at least 10 years before genetic testing. The collective audiometric profile includes mild to moderate SNHL at lower frequencies and moderate SNHL at 2 kHz and higher frequencies. CONCLUSIONS: This study suggests that screening for SNHL could be an early indicator of MD. We propose that the audiometric profile for those with a MD diagnosis may have clinical triage utility.

Genotype and Phenotype Characteristics of 58 Cases of Mitochondrial Epilepsy with Nuclear DNA Mutations in Children.

OBJECTIVE: Identify the genotype and clinical characteristics of mitochondrial epilepsy caused by nDNA mutations in Chinese children and explore the treatment and prognosis of the condition. STUDY DESIGN: This is a retrospective cohort study conducted at a single center, including patients diagnosed with an established nDNA mutation-associated primary mitochondrial disease between October 2012 and March 2023 who also met the practical clinical definition of epilepsy published by the ILAE in 2014. RESULTS: Of the 58 patients identified, 74.1% had an onset before the age of 1 year and 63.8% had seizures as their initial symptom. Developmental and epileptic encephalopathy (DEE) (31%) are the most common phenotypes. The most frequently observed MRI abnormalities include abnormal signal asymmetry in the bilateral basal ganglia and/or brainstem (34.7%), as well as brain atrophy, myelin sheath dysplasia, and corpus callosum dysplasia (32.7%). Of the 40 patients followed, seizure treatment was effective in 18 of the cases, while it was ineffective in 22. The mitochondrial DNA depletion syndrome (MDS) was found to be more difficult to control seizures than other phenotypes (P < 0.05). Additionally, the MDS was associated with a significantly higher mortality rate compared to alternative phenotypes (P < 0.05). CONCLUSIONS: The onset of mitochondrial epilepsy due to nDNA mutations is early and seizures are the most common initial symptom. DEE is the most common phenotype. Characteristic MRI abnormalities in the brain may be helpful in the diagnosis of primary mitochondrial disease. People with MDS typically face challenges in seizure control and have a poor prognosis.

Strategic validation of variants of uncertain significance in ECHS1 genetic testing.

BACKGROUND: Enoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in the ECHS1 gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem. METHODS: Here, we constructed an assay system to verify VUS function for ECHS1 gene. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis. RESULTS: The functional validation of VUS identified novel variants causing loss of ECHS1 function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system. CONCLUSIONS: In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.

Mitochondrial Biomarkers in the Omics Era: A Clinical-Pathophysiological Perspective.

Mitochondrial diseases (MDs) affect 4300 individuals, with different ages of presentation and manifestation in any organ. How defects in mitochondria can cause such a diverse range of human diseases remains poorly understood. In recent years, several published research articles regarding the metabolic and protein profiles of these neurogenetic disorders have helped shed light on the pathogenetic mechanisms. By investigating different pathways in MDs, often with the aim of identifying disease biomarkers, it is possible to identify molecular processes underlying the disease. In this perspective, omics technologies such as proteomics and metabolomics considered in this review, can support unresolved mitochondrial questions, helping to improve outcomes for patients.

Laboratory testing for mitochondrial diseases: biomarkers for diagnosis and follow-up.

The currently available biomarkers generally lack the specificity and sensitivity needed for the diagnosis and follow-up of patients with mitochondrial diseases (MDs). In this group of rare genetic disorders (mutations in approximately 350 genes associated with MDs), all clinical presentations, ages of disease onset and inheritance types are possible. Blood, urine, and cerebrospinal fluid surrogates are well-established biomarkers that are used in clinical practice to assess MD. One of the main challenges is validating specific and sensitive biomarkers for the diagnosis of disease and prediction of disease progression. Profiling of lactate, amino acids, organic acids, and acylcarnitine species is routinely conducted to assess MD patients. New biomarkers, including some proteins and circulating cell-free mitochondrial DNA, with increased diagnostic specificity have been identified in the last decade and have been proposed as potentially useful in the assessment of clinical outcomes. Despite these advances, even these new biomarkers are not sufficiently specific and sensitive to assess MD progression, and new biomarkers that indicate MD progression are urgently needed to monitor the success of novel therapeutic strategies. In this report, we review the mitochondrial biomarkers that are currently analyzed in clinical laboratories, new biomarkers, an overview of the most common laboratory diagnostic techniques, and future directions regarding targeted versus untargeted metabolomic and genomic approaches in the clinical laboratory setting. Brief descriptions of the current methodologies are also provided.

Mitochondrial DNA disorders: from pathogenic variants to preventing transmission.

Mitochondrial DNA (mtDNA) disorders are recognized as one of the most common causes of inherited metabolic disorders. The mitochondrial genome occurs in multiple copies resulting in both homoplasmic and heteroplasmic pathogenic mtDNA variants. A biochemical defect arises when the pathogenic variant level reaches a threshold, which differs between variants. Moreover, variants can segregate, clonally expand, or be lost from cellular populations resulting in a dynamic and tissue-specific mosaic pattern of oxidative deficiency. MtDNA is maternally inherited but transmission patterns of heteroplasmic pathogenic variants are complex. During oogenesis, a mitochondrial bottleneck results in offspring with widely differing variant levels to their mother, whilst highly deleterious variants, such as deletions, are not transmitted. Complemented by a complex interplay between mitochondrial and nuclear genomes, these peculiar genetics produce marked phenotypic variation, posing challenges to the diagnosis and clinical management of patients. Novel therapeutic compounds and several genetic therapies are currently under investigation, but proven disease-modifying therapies remain elusive. Women who carry pathogenic mtDNA variants require bespoke genetic counselling to determine their reproductive options. Recent advances in in vitro fertilization techniques, have greatly improved reproductive choices, but are not without their challenges. Since the first pathogenic mtDNA variants were identified over 30 years ago, there has been remarkable progress in our understanding of these diseases. However, many questions remain unanswered and future studies are required to investigate the mechanisms of disease progression and to identify new disease-specific therapeutic targets.

Mitochondrial Diseases: A Diagnostic Revolution.

Mitochondrial disorders have emerged as a common cause of inherited disease, but are traditionally viewed as being difficult to diagnose clinically, and even more difficult to comprehensively characterize at the molecular level. However, new sequencing approaches, particularly whole-genome sequencing (WGS), have dramatically changed the landscape. The combined analysis of nuclear and mitochondrial DNA (mtDNA) allows rapid diagnosis for the vast majority of patients, but new challenges have emerged. We review recent discoveries that will benefit patients and families, and highlight emerging questions that remain to be resolved.

Methodologies in Mitochondrial Testing: Diagnosing a Primary Mitochondrial Respiratory Chain Disorder.

BACKGROUND: Mitochondria are cytosolic organelles within most eukaryotic cells. Mitochondria generate the majority of cellular energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OxPhos). Pathogenic variants in mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) lead to defects in OxPhos and physiological malfunctions (Nat Rev Dis Primer 2016;2:16080.). Patients with primary mitochondrial disorders (PMD) experience heterogeneous symptoms, typically in multiple organ systems, depending on the tissues affected by mitochondrial dysfunction. Because of this heterogeneity, clinical diagnosis is challenging (Annu Rev Genomics Hum Genet 2017;18:257-75.). Laboratory diagnosis of mitochondrial disease depends on a multipronged analysis that can include biochemical, histopathologic, and genetic testing. Each of these modalities has complementary strengths and limitations in diagnostic utility. CONTENT: The primary focus of this review is on diagnosis and testing strategies for primary mitochondrial diseases. We review tissue samples utilized for testing, metabolic signatures, histologic findings, and molecular testing approaches. We conclude with future perspectives on mitochondrial testing. SUMMARY: This review offers an overview of the current biochemical, histologic, and genetic approaches available for mitochondrial testing. For each we review their diagnostic utility including complementary strengths and weaknesses. We identify gaps in current testing and possible future avenues for test development.

Clinical, radiological, biochemical and molecular characterization of a new case with multiple mitochondrial dysfunction syndrome due to IBA57: Lysine and tryptophan metabolites as potential biomarkers.

Iron­sulfur clusters (FeS) are one of the most primitive and ubiquitous cofactors used by various enzymes in multiple pathways. Biosynthesis of FeS is a complex multi-step process that is tightly regulated and requires multiple machineries. IBA57, along with ISCA1 and ISCA2, play a role in maturation of [4Fe-4S] clusters which are required for multiple mitochondrial enzymes including mitochondrial Complex I, Complex II, lipoic acid synthase, and aconitase. Pathogenic variants in IBA57 have been associated with multiple mitochondrial dysfunctions syndrome 3 (MMDS3) characterized by infantile to early childhood-onset psychomotor regression, optic atrophy and nonspecific dysmorphism. Here we report a female proband who had prenatal involvement including IUGR and microcephaly and developed subacute psychomotor regression at the age of 5 weeks in the setting of preceding viral infection. Brain imaging revealed cortical malformation with polymicrogyria and abnormal signal alteration in brainstem and spinal cord. Biochemical analysis revealed increased plasma glycine and hyperexcretion of multiple organic acids in urine, raising the concern for lipoic acid biosynthesis defects and mitochondrial FeS assembly defects. Molecular analysis subsequently detected compound heterozygous variants in IBA57, confirming the diagnosis of MMDS3. Although the number of MMDS3 patients are limited, certain degree of genotype-phenotype correlation has been observed. Unusual brain imaging in the proband highlights the need to include mitochondrial disorders as differential diagnoses of structural brain abnormalities. Lastly, in addition to previously known biomarkers including high blood lactate and plasma glycine levels, the increase of 2-hydroxyadipic and 2-ketoadipic acids in urine organic acid analysis, in the appropriate clinical context, should prompt an evaluation for the lipoic acid biosynthesis defects and mitochondrial FeS assembly defects.

MT-ATP6 mitochondrial disease identified by newborn screening reveals a distinct biochemical phenotype.

Although decreased citrulline is used as a newborn screening (NBS) marker to identify proximal urea cycle disorders (UCDs), it is also a feature of some mitochondrial diseases, including MT-ATP6 mitochondrial disease. Here we describe biochemical and clinical features of 11 children born to eight mothers from seven separate families who were identified with low citrulline by NBS (range 3-5 µM; screening cutoff >5) and ultimately diagnosed with MT-ATP6 mitochondrial disease. Follow-up testing revealed a pattern of hypocitrullinemia together with elevated propionyl-(C3) and 3-hydroxyisovaleryl-(C5-OH) acylcarnitines, and a homoplasmic pathogenic variant in MT-ATP6 in all cases. Single and multivariate analysis of NBS data from the 11 cases using Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) demonstrated citrulline <1st percentile, C3 > 50th percentile, and C5-OH >90th percentile when compared with reference data, as well as unequivocal separation from proximal UCD cases and false-positive low citrulline cases using dual scatter plots. Five of the eight mothers were symptomatic at the time of their child(ren)'s diagnosis, and all mothers and maternal grandmothers evaluated molecularly and biochemically had a homoplasmic pathogenic variant in MT-ATP6, low citrulline, elevated C3, and/or elevated C5-OH. All molecularly confirmed individuals (n = 17) with either no symptoms (n = 12), migraines (n = 1), or a neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) phenotype (n = 3) were found to have an A or U mitochondrial haplogroup, while one child with infantile-lethal Leigh syndrome had a B haplogroup.