Impact of measuring heteroplasmy of a pathogenic mitochondrial DNA variant at the single-cell level in individuals with mitochondrial disease.

Pathogenic mitochondrial DNA heteroplasmy has mainly been assessed with bulk sequencing in individuals with mitochondrial disease. However, the distribution of heteroplasmy at the single-cell level in skin fibroblasts obtained from individuals, together with detailed clinical and biochemical information, remains to be investigated. We used the mitochondrial DNA single-cell assay for the transposase-accessible chromatin sequencing method. Skin fibroblasts were obtained from six individuals with mitochondrial disease and pathogenic m.3243A>G variants of differing severity. Different distributions of heteroplasmy at the single-cell level were identified in skin fibroblasts from all six individuals. Four individuals with different outcomes showed similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity, while the distribution of single-cell heteroplasmy patterns differed among the individuals. This study showed different heteroplasmy distribution patterns at the single-cell level in individuals with the m.3243A>G variant, who had a similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity. Whether such different heteroplasmy distribution patterns explain the different clinical outcomes should be assessed further in future studies. Measuring heteroplasmy of pathogenic mitochondrial DNA variants at the single-cell level could be important in individuals with mitochondrial disease.

Severity estimation of very-long-chain acyl-CoA dehydrogenase deficiency via 13C-fatty acid loading test.

BACKGROUND: The clinical severity of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is difficult to predict using conventional diagnostic methods. METHODS: Peripheral blood mononuclear cells obtained from 14 VLCAD deficiency patients and 23 healthy adults were loaded with carbon-13-universally labeled (U-13C-) fatty acids. Differences in acylcarnitine ratios between the patients and healthy groups and correlations between acylcarnitine ratios and a newly established clinical severity score (CSS) in the patient group were statistically examined. RESULTS: There was a significant decrease in the 13C-C2/13C-C18 and 13C-C12/13C-C14 ratios in the U-13C-stearic acid loading test and in the 13C-C2/13C-C18:1 and 13C-C12:1/13C-C14:1 ratios in the U-13C-oleic acid loading test in the patient group. The values of each ratio were significantly correlated with the CSS, suggesting that they could predict disease severity. Additionally, patients with a higher 13C-C16/13C-C18 ratio than the 13C-C14/13C-C18 ratio in the U-13C-stearic acid loading test had a significantly higher CSS and were presumed to have more severe disease. CONCLUSIONS: Our data indicated that this method could be used to predict the clinical severity of VLCAD deficiency, and identify patients at a risk of severe disease. IMPACT: We established a novel method to predict the severity of VLCAD deficiency by performing a loading test with carbon-13-labeled fatty acids on peripheral blood mononuclear cells. The U-13C-oleic acid loading test was useful for comparing the patient group with the control group in terms of disease severity. The U-13C-stearic acid loading test was useful for identifying the more severely affected patients. These methods are relatively less invasive and enable rapid evaluation of the clinical severity.

Need for strict clinical management of patients with carnitine palmitoyltransferase II deficiency: Experience with two cases detected by expanded newborn screening.

In Japan, carnitine palmitoyltransferase II (CPTII) deficiency has been included as one of the primary target diseases in the expanded newborn mass screening program since 2018. However, many cases of the severe infantile hepatocardiomuscular form of CPTII deficiency showed severe neurodevelopmental delay or sudden death, which indicated that management of CPTII deficiency in the acute phase remains to be studied in detail. Herein, we discuss two cases diagnosed by newborn mass screening. Patient 1 was under strict clinical management from the neonatal period, with >20 admissions in 14 months, while Patient 2 was managed using a relatively relaxed approach, with only 2 admissions in the same period. Patient 1 showed normal development; however, Patient 2 expired at the age of 1 year 2 months. To develop strategies for preventing sudden deaths in patients with CPTII deficiency, this retrospective study focused on detailed clinical management practices and biochemical findings during the acute phase. We also investigated the correlation between conventional biomarkers (such as creatine kinase) and long-chain acylcarnitines. We propose that strict monitoring and immediate medical attention, even in case of slight fever or minor abdominal symptoms, can help prevent sudden death in patients with CPTII deficiency. Considering the higher morbidity rate of such patients, strict and acute management of CPTII deficiency cannot be overemphasized.

Use of cytosolic and myofibril markers in the detection of ongoing myocardial damage in patients with chronic heart failure.

PURPOSE: Measurement of serum levels of cytosolic and myofibril components of cardiac tissue could indicate ongoing myocardial damage in patients with chronic heart failure. METHODS: We correlated serum levels of a cytosolic marker (heart-type fatty acid-binding protein) and a myofibril marker (troponin T) with the severity of symptoms (based on the New York Heart Association [NYHA] class), neurohumoral derangement, and subsequent cardiac events in 56 patients with chronic heart failure. RESULTS: Mean (+/- SD) levels of heart-type fatty acid-binding protein were greater in patients with NYHA class III or IV heart failure (9.9 +/- 5.2 ng/mL) than in those with NYHA class II (4.9 +/- 1.9 ng/mL, P <0.0001). Detection of troponin T (> or =0.02 ng/mL) was also more common in patients with worse heart failure (81% [13/16] in class III or IV vs. 43% [17/40] in class II, P = 0.02). Significant correlations were found between heart-type fatty acid-binding protein levels and plasma levels of A-type natriuretic peptide (r = 0.45, P = 0.0004), B-type natriuretic peptide (r = 0.66, P <0.0001), and norepinephrine (r = 0.36, P = 0.006). Male sex (hazard ratio [HR] = 5.0; 95% confidence interval [CI]: 1.3 to 19), detectable troponin T levels (HR = 7.0; 95% CI: 1.1 to 44), heart-type fatty acid-binding protein (HR = 2.6 per 3.9-ng/mL increase; 95% CI: 1.1 to 6.5), and left ventricular ejection fraction (HR = 3.6 per 15% decrease; 95% CI: 1.2 to 11) were independently associated with subsequent cardiac events (8 deaths or 10 readmissions because of worsening heart failure). CONCLUSION: Heart-type fatty acid-binding protein and troponin T are markers of ongoing myocardial damage, and are associated with subsequent cardiac events in patients with chronic heart failure.