In this study, we aimed to assess clinical, laboratory and molecular features of newborns with clinical suspicion for systemic primary carnitine deficiency (CUD), medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). The implementation of newborn screening programs for fatty acid β-oxidation disorders (FAODs) has changed the natural course of these diseases, facilitating the initiation of preventive or therapeutic measures for affected newborns shortly after birth. This study included 94 newborns who were admitted between 2016 and 2023 because of biochemical signs of CUD, MCADD and VLCADD, and provided clinical, biochemical and genotypic data. Definitive molecular diagnosis confirmed that 16/94 newborns (17%) were true positives of the NBS, and 17 novel variants were detected in SLC22A5, ACADM and ACADVL genes. We assessed the clinical evolution of patients over time. This study expands the genotypic spectrum of SLC22A5, ACADM and ACADVL and highlights the role of genetics in identifying and correctly characterising FAODs.

Carnitine is essential for the import of long-chain fatty acids into mitochondria, where they are used for energy production. The carnitine transporter OCTN2 (novel organic cation transporter 2, SLC22A5) mediates carnitine uptake across the plasma membrane and as such facilitates fatty acid metabolism in most tissues. OCTN2 dysfunction causes systemic primary carnitine deficiency (SPCD), a potentially lethal disorder. Despite its importance in metabolism, the mechanism of high-affinity, sodium ion-dependent transport by OCTN2 is unclear. Here we report cryo-EM structures of human OCTN2 in three conformations: inward-facing ligand-free, occluded carnitine- and Na+-bound, and inward-facing ipratropium-bound. These structures define key interactions responsible for carnitine transport and identify an allosterically coupled Na+ binding site housed within an aqueous cavity, separate from the carnitine-binding site. Combined with electrophysiology data, we provide a framework for understanding variants associated with SPCD and insight into how OCTN2 functions as the primary human carnitine transporter.

Systemic primary carnitine deficiency (SPCD) is a rare congenital fatty acid metabolism disorder causing impaired β-oxidation and energy production, leading to hypoglycemia, metabolic encephalopathy, and sudden death. Early diagnosis and treatment, including L-carnitine supplementation and fasting avoidance, can improve prognosis. However, newborn screening (NBS) criteria differ by region, and standardized guidelines are lacking. This report presents a case of SPCD undetected by NBS, resulting in basal ganglia damage and dystonia due to metabolic decompensation. A 1-year-9-month-old girl with no abnormalities on NBS presented with impaired consciousness. She exhibited hypoketotic hypoglycemia, hyperammonemia, and myocardial hypertrophy. Suspecting a fatty acid metabolism disorder, L-carnitine and high-calorie infusion were initiated. Laboratory tests revealed markedly low serum total and free carnitine levels, and genetic analysis confirmed a homozygous SLC22A5 mutation. Brain MRI on day 7 revealed bilateral basal ganglia and substantia nigra abnormalities. The patient developed severe dystonia and respiratory failure, requiring ECMO management. L-DOPA was initiated on day 62, resulting in improvements in dystonia, swallowing, and motor function. By day 88, MRI showed resolution of basal ganglia abnormalities, though cerebral atrophy persisted. Basal ganglia damage is a rare but severe SPCD complication. L-DOPA may alleviate dystonia by acting on dopaminergic neurons in the substantia nigra. Early ketone measurement during emergencies is crucial for diagnosing fatty acid metabolism disorders. A standardized NBS protocol with a defined carnitine cutoff value is essential for early detection and prevention of SPCD complications. Primary carnitine deficiency (PCD) is a disorder of the carnitine cycle that results in defective fatty acid oxidation. If untreated, it encompasses a broad clinical spectrum including: (1) metabolic decompensation in infancy typically presenting between age three months and two years with episodes of hypoketotic hypoglycemia, poor feeding, irritability, lethargy, hepatomegaly, elevated liver transaminases, and hyperammonemia triggered by fasting or common illnesses such as upper respiratory tract infection or gastroenteritis; (2) childhood myopathy involving heart and skeletal muscle with onset between age two and four years; (3) pregnancy-related decreased stamina or exacerbation of cardiac arrhythmia; (4) fatigability in adulthood; and (5) absence of symptoms. The latter two categories often include mothers diagnosed with PCD after newborn screening has identified low carnitine levels in their infants. The diagnosis of PCD is established in a proband with consistent biochemical analyte findings and/or suggestive clinical and laboratory features by identification of biallelic pathogenic variants in SLC22A5 on molecular genetic testing. In individuals with suspected PCD and negative molecular testing, a carnitine transport assay using cultured skin fibroblasts may be available. Targeted therapy: Metabolic decompensation and skeletal and cardiac muscle function improve with 100-200 mg/kg/day oral levocarnitine if it is started before irreversible organ damage occurs. Supportive care: Routine treatment includes preventing hypoglycemia with frequent feeding and avoidance of prolonged fasting; notifying designated metabolic center in advance of scheduled surgical or medical procedures; hospitalization for intravenous glucose administration for individuals who are required to fast for a procedure or who cannot tolerate oral intake due to illness such as gastroenteritis; implementing transitional care plan prior to adulthood. Emergency outpatient treatment includes levocarnitine and carbohydrate supplementation, antipyretics for fever, and antiemetics for occasional vomiting. Acute inpatient treatment includes high-calorie fluids, insulin as needed, intravenous or oral levocarnitine (100-200 mg/kg/day), evaluation of muscle and liver involvement by measuring serum creatine kinase concentration and liver transaminases, and evaluation by cardiologist with EKG and echocardiogram for cardiomyopathy. Surveillance: Monitor plasma carnitine concentration frequently until levels reach normal range; once levels reach normal range, measure three times a year during infancy and early childhood, twice a year in older children, and annually in adults. Assess growth and development at each visit throughout childhood. Neuropsychological testing and quality of life assessment as needed. EKG and echocardiogram annually during childhood and less frequently in adulthood. Agents/circumstances to avoid: Fasting longer than age-appropriate periods; catabolic illness; inadequate calorie provision during other stressors. Evaluation of relatives at risk: Evaluation of all sibs of any age by molecular genetic testing if the SLC22A5 pathogenic variants in the family are known or measurement of plasma-free carnitine concentration to identify as early as possible those who would benefit from institution of treatment and preventive measures. Pregnancy management: Pregnant women with PCD require close monitoring of plasma carnitine levels and increased carnitine supplementation as needed to maintain plasma carnitine levels in the normal range. PCD is inherited in an autosomal recessive manner. If both parents are known to be heterozygous for an SLC22A5 pathogenic variant, each sib of an affected individual has at conception a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Once the SLC22A5 pathogenic variants have been identified in an affected family member, molecular genetic carrier testing for at-risk relatives and prenatal/preimplantation genetic testing are possible.

OCTN1 and OCTN2 are membrane transport proteins encoded by the SLC22A4 and SLC22A5 genes, respectively. Even though several transcripts have been predicted by bioinformatics for both genes, only one functional protein isoform has been described for each of them. Both proteins are ubiquitous, and depending on the physiopathological state of the cell, their expression is regulated by well-known transcription factors, although some aspects have been neglected. A plethora of missense variants with uncertain clinical significance are reported both in the dbSNP and the Catalogue of Somatic Mutations in Cancer (COSMIC) databases for both genes. Due to their involvement in human pathologies, such as inflammatory-based diseases (OCTN1/2), systemic primary carnitine deficiency (OCTN2), and drug disposition, it would be interesting to predict the impact of variants on human health from the perspective of precision medicine. Although the lack of a 3D structure for these two transport proteins hampers any speculation on the consequences of the polymorphisms, the already available 3D structures for other members of the SLC22 family may provide powerful tools to perform structure/function studies on WT and mutant proteins.

In this study, we evaluated the implementation of a second-tier genetic screening test using an amplicon-based next-generation sequencing (NGS) panel in our laboratory during the period of 1 September 2021 to 31 August 2022 for the newborn screening (NBS) of six conditions for inborn errors of metabolism: citrullinemia type II (MIM #605814), systemic primary carnitine deficiency (MIM #212140), glutaric acidemia type I (MIM #231670), beta-ketothiolase deficiency (#203750), holocarboxylase synthetase deficiency (MIM #253270) and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM # 246450). The custom-designed NGS panel can detect sequence variants in the relevant genes and also specifically screen for the presence of the hotspot variant IVS16ins3kb of SLC25A13 by the copy number variant calling algorithm. Genetic second-tier tests were performed for 1.8% of a total of 22,883 NBS samples. The false positive rate for these six conditions after the NGS second-tier test was only 0.017%, and two cases of citrullinemia type II would have been missed as false negatives if only biochemical first-tier testing was performed. The confirmed true positive cases were citrullinemia type II (n = 2) and systemic primary carnitine deficiency (n = 1). The false positives were later confirmed to be carrier of citrullinemia type II (n = 2), carrier of glutaric acidemia type I (n = 1) and carrier of systemic primary carnitine deficiency (n = 1). There were no false negatives reported. The incorporation of a second-tier genetic screening test by NGS greatly enhanced our program's performance with 5-working days turn-around time maintained as before. In addition, early genetic information is available at the time of recall to facilitate better clinical management and genetic counseling.