2 articles
Mitochondrial diseases present heterogeneous clinical features that overlap with numerous genetic disorders, making early diagnostic stratification essential. This study aimed to evaluate the performance of a stepwise molecular diagnostic algorithm integrating High-Resolution Melting qPCR screening and targeted sequencing in individuals suspected of mitochondrial pathology based on a Nijmegen Mitochondrial Disease Score (NMDS) ≥3.
The analysis included 240 patients with clinical suspicion of mitochondrial disease and an NMDS ≥3, all evaluated through a standardized clinical, biochemical, and instrumental assessment. Molecular testing followed a tiered workflow: initial qPCR-HRM screening for seven common mtDNA mutations, followed by targeted Sanger sequencing of mitochondrial genes, including POLG hotspot regions, in patients meeting predefined clinical and NMDS thresholds. For individuals subsequently identified with non-mitochondrial etiologies, next-generation sequencing approaches were performed in accredited external laboratories. Statistical evaluation relied on descriptive statistical methods and non-parametric comparative analyses.
Molecular analysis confirmed mitochondrial involvement in 37 patients (15.4%) and identified non-mitochondrial genetic disorders in 44 patients (18.3%), while 159 individuals (66.3%) remained without a definitive molecular diagnosis. Patients with mitochondrial involvement showed higher frequencies of severe neuromuscular dysfunction, developmental regression, ophthalmic manifestations including ophthalmoplegia, and cardiovascular involvement. By contrast, neurodevelopmental and behavioral impairments and dysmorphic features were more prevalent in non-mitochondrial and undiagnosed patients. Biochemically, elevated plasma lactate and hyperalaninemia were significantly more common among individuals with mitochondrial involvement. Neuroimaging findings in this group were characterized by cerebral and cerebellar atrophy and basal ganglia abnormalities. Consistently, NMDS values were markedly higher in patients with mitochondrial involvement, and their integration as threshold-based decision points within the stepwise diagnostic algorithm substantially enhanced diagnostic stratification, enabling more precise differentiation between mitochondrial involvement and alternative genetic etiologies.
The structured algorithm integrating NMDS-based selection, qPCR-HRM screening, and targeted sequencing demonstrated effective stratification of patients with suspected mitochondrial disease, achieving a combined diagnostic rate of 33.7%. These findings support the utility of this tiered approach in distinguishing mitochondrial from non-mitochondrial genetic conditions and in optimizing molecular diagnostic workflows.
Inborn errors of metabolism (IEM) make up a large group of disorders caused by an inherited defect of proteins that have enzymatic, carrier, receptor or structural roles. The cumulative prevalence of IEM in different populations is around 1:500 – 800 newborns, despite the fact that some of these disorders are extremely rare when taken individually. Early recognition and intervention are essential to avoiding disastrous consequences associated with IEM. The phenotype of IEM patients is very heterogeneous and only in combination with specialized metabolic test it can lead to a correct diagnosis. The aim of the study was defined as evaluation of importance of plasma amino acid profile in the diagnosis of IEM.
Plasma amino acids quantification have been performed by high performance liquid chromatography on Shimadzu LC-20 with post column derivatization with OPA in 15 patients aged from 0 to 13 years old, selected through medical genetic counseling, based on inclusion and exclusion criteria. Inform consent has been signed by parents after receiving all necessary information regarding the study. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) on dried blood spots and Nuclear Magnetic Resonance Spectroscopy (H1-NMR) on urine has been done as complementary tests.
The first line investigations showed acid-base imbalance (33,3%), hypoglycemia (46,6%), high lactate level (46,6%) and high ammonia level (20%). Plasma amino acid concentrations were abnormal in 4 patients (27%). Increased glycine (544 μmol/L, reference values 70,72 – 256,36 μmol/L), along with elevated glycine in cerebrospinal fluid (CSF) / plasma ratio (0,147, normal < 0.02) was detected in a patient with seizures, coma, and respiratory arrest indicating non-ketotic hyperglycinemia. High level of phenylalanine (Phe 1568µmol/L, reference values 26.52 – 221 µmol/L) has been identified in a patient suspected for Phenylketonuria after neonatal screening results (Phe > 3mg/dL). Also the ratio Phe / Tyr around 17 was specifically appreciated as for classical form of PKU. High alanine concentration (> 450 µmol/L) has been observed in two patients presenting severe metabolic acidosis and high lactate level, suggesting for a metabolic error with mitochondrial involvement. The results from extended newborn screening and NMR spectroscopy narrowed the spectrum of suspected diseases, facilitating the diagnosis. Molecular genetic tests are required for the confirmation of disease in all cases.
Quantitative amino acids analysis is an important tool for the diagnosis of “intoxication type” of IEMs and nutritional monitoring of individuals with already established diagnosis.