Mitochondrial fatty acid oxidation is a crucial metabolic pathway responsible for the degradation of fatty acids, serving as a significant energy source for the heart, skeletal muscle, and liver, especially during periods of reduced glucose supply, such as prolonged fasting, febrile illness, or muscular exertion [1]. Mitochondrial fatty acid oxidation disorders (FAODs) comprise a diverse group of genetic autosomal recessive diseases involving the transportation or oxidation of fatty acids. This includes processes like the uptake and activation of fatty acids, the carnitine cycle, the beta-oxidation cycle, and electron transfer [2]. Currently, over 15 distinct FAODs have been identified through enzymatic and molecular analyses [3]. Some notable FAODs include carnitine uptake defect, carnitine palmitoyl-CoA transferase (CPT) 1-2 deficiency, carnitine-acylcarnitine translocase deficiency, very long chain acyl-CoA dehydrogenase deficiency (VLCADD), long chain hydroxyacyl-CoA dehydrogenase deficiency or mitochondrial trifunctional protein deficiency (LCHADD/MTPD), medium-chain acyl-CoA dehydrogenase deficiency (MCADD), medium/short chain hydroxyacyl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency (SCADD), and multiple acyl-CoA dehydrogenase deficiency [4]. VLCADD demonstrates characteristic elevations in C14:1, C14, and C16 acylcarnitines, with clinical presentations varying by age from cardiac complications in infants to exercise-induced rhabdomyolysis in adults. LCHADD/MTPD manifests with cardiomyopathy, hepatopathy, and skeletal myopathy. A unique feature is retinopathy that develops in long-term survivors. The characteristic biochemical profile includes elevated C16-OH and C18:1-OH acylcarnitines, with increased 3-hydroxydicarboxylic acids in urine. MCADD, one of the more common FAODs, is biochemically characterized by elevated C6-C10 acylcarnitines, particularly C8, with patients typically experiencing fasting hypoglycemia and hepatomegaly. SCADD, although predominantly asymptomatic, presents with elevated C4 acylcarnitine and ethylmalonic acid, and can manifest as developmental delay and muscular weakness in symptomatic cases [5].

These defects exhibit a heterogeneous clinical presentation and varying ages of onset, determined by the specific enzyme involved, residual activity, and exposure to catabolic events throughout life [6]. FAODs can present with life-threatening symptoms; the most severe form manifests with hypertrophic cardiomyopathy, hepatic encephalopathy, or severe hypoketotic hypoglycemia. Conversely, a less severe, later-onset myopathic form is characterized by exercise-induced myopathy and rhabdomyolysis [7].

After the development of tandem mass spectrometry (TMS) for the analysis of acylcarnitines, the scope of disorders, including FAODs, detectable through newborn screening (NBS) has significantly expanded [8]. The incidence of FAODs exhibits variability based on geographical region and ethnic background, ranging from 1:3,300 in Turkey to 1:217,000 in Taiwan [9]. In South Korea, TMS NBS was implemented in 2002, and from 2006 to 2015, the prevalence of FAODs was identified as 1:13,205 newborns [10]. Early detection and treatment of FAODs through NBS play a crucial role in preventing acute, potentially life-threatening events. This proactive approach facilitates the avoidance of prolonged fasting and ensures prompt treatment for affected infants and children during episodes of acute illness [11]. Although large-scale studies comparing clinical outcomes of FAODs before and after the implementation of expanded NBS in Korea are lacking, this study aimed to investigate the clinical and genetic characteristics and outcomes of patients diagnosed through NBS or symptomatic presentation to unravel the complexities of FAODs based on our experience.

1. Patients

We conducted a retrospective analysis of patients with FAODs diagnosed and monitored from July 2010 to November 2022 at the Department of Medical Genetics, Pusan National University Children’s Hospital in Yangsan, South Korea. Approval for this study was obtained from the Institutional Review Board at Pusan National University Children’s Hospital (IRB number: 05-2023-030). The analysis focused on the clinical features of these patients, examining symptomatic presentations such as rhabdomyolysis, cardiomyopathy, and developmental delay. The requirement to obtain informed consent was waived.

2. Biochemical and genetic testing

The biochemical findings (including plasma acylcarnitines), molecular analyses, and the progression of each patient’s condition were examined retrospectively. Any patient identified through NBS tests with abnormal results was subsequently referred to our department. Dried blood spot samples were collected at the hospital where the patients were born, and TMS analysis was conducted at commercial biochemical laboratories. Newborns with abnormal initial screening results underwent a repeat TMS. If the second screening results still exceeded the cutoff value, molecular genetic analyses were performed, and the diagnosis of FAOD was confirmed upon receiving a positive confirmation test. Acylcarnitine levels were measured using Liquid Chromatography TMS, as previously described. Genomic DNA was isolated from peripheral blood leukocytes. The polymerase chain reaction was conducted for all coding exons and exon-intron boundaries of acyl-CoA dehydrogenase very long chain (ACADVL) for VLCADD, hydroxy acyl-CoA dehydrogenase trifunctional multi-enzyme complex subunit alpha (HADHA) and beta (HADHB) for LCHADD/MTPD, ACADM for MCADD, ACADS for SCADD, and SLC22A5 for primary carnitine deficiency.

1. Clinical characteristics of patients with FAODs

FAODs were identified in 26 patients during the study period, comprising VLCADD (3 patients), LCHAD/MTPD (3 patients), MCADD (4 patients), SCADD (14 patients), and primary carnitine deficiency (2 patients) (Table 1, Fig. 1). Clinical characteristics of each disease group revealed variations in the age at diagnosis, ranging from 15 days after birth to 10 years of age. In instances of clinical presentation, VLCAD and LCHAD were diagnosed based on symptoms such as cardiomyopathy and severe metabolic acidosis, while the others were diagnosed through NBS and familial tests (Table 2). Patients in the VLCADD and LCHAD/MTPD groups commonly exhibited symptoms like recurrent rhabdomyolysis and cardiomyopathy. In contrast, none of the patients with MCADD and primary carnitine deficiency displayed symptoms. For SCADD, 35.7% of patients presented with symptoms, while 64.3% showed no symptoms (Table 2). The clinical presentations showed that all three patients with VLCADD displayed features of cardiomyopathy and recurrent rhabdomyolysis (Table 2). LCHAD/MTPD were identified through manifestations of cardiomyopathy with severe lactic acidosis at 2-3 months after birth (patients 5 and 6) and recurrent rhabdomyolysis at 2 years (patient 4).

Patient 4, diagnosed with LCHAD/MTPD, experienced recurrent rhabdomyolysis, leading to repeated hospitalizations and discharges due to polyneuropathy. Notably, he had a family history involving the unfortunate death of his fraternal twin brother from cardiac arrest at the age of 15 months. Patient 5, also with LCHAD/MTPD, suffered from recurrent rhabdomyolysis starting at around 3 months and later experienced repeated metabolic crises and cardiomyopathy. The patient succumbed to severe heart failure at around 11 months. No patients were identified with MCADD or primary carnitine deficiency based on symptomatic presentation during the study period.

In contrast to other FAODs groups, the clinical manifestations of patients with SCADD varied widely, ranging from asymptomatic cases to symptomatic presentations with cyclic vomiting, metabolic acidosis, and recurrent diarrhea. Among the 14 patients, five exhibited clinical symptoms such as lethargy, hypotonia, fatigue, and motor delay. The height standard deviation score (SDS) of each disease group revealed variations ranging from –1.87 to 1.74, and weight SDS ranged from –1.85 to 1.95 among different types of FAODs (Table 2).

2. Biochemical findings of patients with FAODs

Biochemical findings revealed that plasma levels of C14:1 and C14:2 were elevated in VLCADD, while C14OH, C16OH, and C18:1OH levels were elevated in LCHAD/MTPD. Elevated levels of C6 and C8 were observed in MCADD, and elevated levels of C2 and C4 were noted in SCADD. Notably, the C0 level was markedly decreased in primary carnitine deficiency (Table 3).

3. Molecular characteristics of patients with FAODs

Germline mutations of the genes responsible for each FAOD were identified in 100% of alleles: 6 out of 6 alleles in VLCADD, 6 out of 6 alleles in LCHAD/MTPD, 8 out of 8 alleles in MCADD, 28 out of 28 alleles in SCADD, and 4 out of 4 alleles in primary carnitine deficiency (Table 3). In MCADD, 2 out of 8 mutations were novel: c.[843A>T] (p.Arg28Ser) and c.[1189T>A] (p.Tyr397Asn). Additionally, patients with SCADD had five novel variants: c.[1130C>T] (p.Pro377Leu), c.[277C>A] (p.Leu93Ile), c.[682G>A] (p.Glu228Lys), c.[700C>T] (p.Arg234Trp), and c.[431C>T] (p.Thr144lle) (Table 3). Fig. 2 illustrates the structure of the ACADS gene and highlights the locations of frequent mutations in SCADD patients. Three pathogenic variants and five novel variants were identified, with a predominant concentration in exon 2 and exon 9. Other variants were evenly distributed between exons 3 and 10. The schematic representation distinguishes four pathogenic variants (depicted in black) and 5 novel variants (depicted in red) of the ACADS gene. Among these variants, the variants of symptomatic SCADD patients were as follows: c.277C>A, c.682G>A, c.1031A>G c.700C>T, c.431C>T, and c.164C>T.

In the present study, we analyzed clinical features, biochemical, and genetic basis from 26 patients (23 families) with FAODs. The 16 patients were diagnosed with FAODs through NBS or familial tests, while 10 patients were diagnosed based on symptomatic presentations. NBS emerges as a crucial tool for early detection of inborn metabolic disorders, unveiling a broader spectrum of FAODs compared to cases identified through symptomatic presentations. These results align with previous reports on the efficacy of NBS programs [12]. The predominant defects identified in our cohort were SCADD (54%), followed by MCADD (15%), VLCADD (15%), and LCHADD/MTPD (11.5%). Notably, 100% of patients with VLCADD and 33% of those with LCHADD/MTPD were identified symptomatically, while none of the patients with MCADD, SCADD, and primary carnitine deficiency were diagnosed symptomatically; instead, they were identified through positive NBS or familial tests. In essence, VLCADD or LCHADD/MTPD emerge as the most commonly symptomatic FAODs, whereas all FAOD patients with MCADD, SCADD, and primary carnitine deficiency were identified through NBS, emphasizing the rarity of symptomatic presentations for these disorders.

Compared with previous Korean studies, Kang et al. [13] compared clinical outcomes between patients with long-chain FAODs identified through NBS and those diagnosed clinically. Among 14 patients identified through NBS, only three patients developed symptoms (rhabdomyolysis or cardiomyopathy), while 10 patients remained asymptomatic with normal development. In contrast, among eight clinically diagnosed patients, most presented with severe manifestations. Furthermore, two patients with LCHADD/MTPD deficiencies died from cardiomyopathy during the neonatal period, and one CPT-1 deficient patient developed developmental disability.

Most long-chain FAODs patients developed recurrent rhabdomyolysis and hypertrophic cardiomyopathy, irrespective of pre-symptomatic management. The variations in outcomes among FAODs patients are believed to be linked to disease characteristics and the pathogenic effects of mutations. In VLCADD or LCHADD/MTPD, patients identified through NBS exhibit a broad spectrum of severity. Some patients develop symptoms even before NBS results are available, while others may remain asymptomatic throughout the follow-up period. Notably, all patients with VLCADD or LCHADD/MTPD experienced recurrent rhabdomyolysis or severe cardiomyopathy. Specifically, cardiac arrest resulting from severe cardiomyopathy was observed in one patient with LCHADD/MTPD. These distinctions in clinical presentation are likely influenced by the phenotype-genotype correlation in patients with VLCADD or LCHADD/MTPD. Additionally, it is believed that residual enzyme activity correlates with clinical presentations, providing further insights for improved patient classification. In a particular study, patients with a residual activity less than 10% tend to exhibit clinical symptoms, while those with a residual activity exceeding 20% might never manifest severe symptoms, presenting only a mild biochemical phenotype [12]. Notably, for patients with a residual activity falling between 10 and 20%, there remains insufficient evidence regarding the impact of catabolic stress during inter-current illness as a potential trigger for severe symptoms [14]. The levels of residual activity may serve as indicators of a late-onset phenotype, particularly with a predominant muscular expression. However, further research is warranted to enhance our understanding of this association.

Despite the benign clinical course observed in 3 patients with MCADD in the study, it is imperative to conduct a life-long follow-up evaluation for these patients, taking into account the potential development of late-onset metabolic episodes. As per the literature [14], enzyme activity below 10% is correlated with symptomatic manifestation in untreated subjects. Indeed, MCADD patients often require more emergency therapies and hospitalizations to prevent acute decompensation during febrile illness or challenging feeding, compared to other cases. It is noteworthy that patients with MCADD necessitate a life-long follow-up assessment due to the potential occurrence of late-onset metabolic episodes. Between 3 and 24 months of age, these patients may exhibit symptoms such as hypoketotic hypoglycemia, vomiting, lethargy, encephalopathy during febrile illness, or prolonged fasting. Additionally, sudden unexplained death may manifest as the initial presentation of MCADD [15,16].

All patients (n=14) with SCADD were identified solely through NBS or familial tests. Presently, there is a growing consensus to view SCADD as a benign biochemical phenotype rather than a disease. Consistent with previous reports, the majority of patients (n=9/14, 64.3%) in our study exhibited asymptomatic conditions with normal development and growth. However, among the 14 patients, five presented with neurological symptoms such as cyclic vomiting and hypotonia. Controversies persist regarding whether SCADD should be classified as a benign biochemical phenotype, a clinical disorder with incomplete penetrance, or a clinically relevant component of a multi-factorial or multi-genetic disorder [17]. In the present study, c.164C>T and c.1031A>G were the most common variants among patients with SCADD, which is consistent with previous reports from Chinese populations [18]. However, while Chinese studies reported all patients were asymptomatic regardless of genotype, our cohort demonstrated variable clinical manifestations ranging from asymptomatic to symptomatic presentations despite having similar genetic variants. This discrepancy in phenotypic expression among patients with identical mutations suggests that additional factors beyond ACADS genotype may influence the clinical presentation of SCADD. Studies on SCADD have reported highly variable clinical manifestations. While some studies have found that the majority of individuals identified through NBS remain asymptomatic, others have described a wide spectrum of symptoms including developmental delay, hypotonia, epilepsy, behavioral disorders, and myopathy [19]. The reported frequency of symptomatic cases also varies significantly across different studies, ranging from 20 to 60% of diagnosed individuals [18]. This variability in clinical presentation and symptom frequency suggests that additional genetic or environmental factors may influence the phenotypic expression of SCADD [19]. Further studies and long-term follow-up are essential to elucidate the clinical relevance of the SCADD phenotype.

This study presents the genetic landscape of mitochondrial long-chain FAODs in both pediatric and adult patients from a single center. However, there are several limitations to our study, including: 1) It is a retrospective cohort study previously on those diagnosed with mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects; 2) There was no standardized evaluation pathway for monitoring these patients, hindering the detailed and systematic assessment of outcomes. Despite these limitations, the insights derived from a substantial number of patients from a single center contribute valuable information to our understanding of mitochondrial long-chain fatty acid oxidation. Particularly, the identification of novel variants in MCADD (c.[843A>T], c.[1189T>A]) and SCADD (c.[1130C>T], c.[277C>A], c.[682G>A], c.[700C>T], c.[431C>T]) contributes to expanding the genetic spectrum of FAODs in Korean patients. However, the clinical significance of these novel variants requires further functional studies to determine their pathogenicity and potential impact on disease manifestation.

Additionally, NBS for FAODs revealed the relative frequency of each disease subtype and their general clinical characteristics. While NBS has proven effective in reducing mortality and morbidity associated with FAODs, the observed broad spectrum of disease severity across different diagnostic modes is partially explained by the respective genotypes of the patients. Careful, life-long observation of patients with FAODs is imperative for enhancing clinical outcomes and gaining a deeper understanding of the complexities associated with these disorders. Future large-scale, multicenter studies with standardized evaluation protocols and long-term follow-up are needed. In addition, pursuing efforts to follow outcomes in FAODs and elucidating possible underlying genetic and cellular mechanisms of FAODs, especially SCADD will be necessary to administer clinical management for physicians.

None.

This work was supported by a 2-Year Research Grant of Pusan National University.

Conception and design: CKC. Acquisition of data: MK. Analysis and interpretation of data: CKC, SY. Drafting the article: MK. Critical revision of the article: MK. Final approval of the version to be published: CKC.