The introduction of advances in molecular genetics, immunology, analytical biochemistry, morphology and other fundamental disciplines led to the identification of a whole class of new diseases — the so-called diseases of cellular organelles. Thanks to the study of the structure and functions of subcellular formations in various human pathologies, it became possible to identify mitochondrial diseases, lysosomal diseases, and peroxisomal diseases. However, while the study of the first two classes of diseases is progressing significantly, insufficient attention is being paid to the study of peroxisomal diseases.

OBJECTIVE

To study the role of gas chromatography-mass spectrometry (GC-MS) and molecular genetics methods in the diagnosis of peroxisomal diseases in children.

MATERIAL AND METHODS

The study included 35 pediatric patients with suspected peroxisomal disease and 369 control children. The study of long-chain fatty acids was carried out using the chromatography-mass spectrometry method using a gas chromatograph with an Agilent 7820/5977 mass spectrometer (Agilent Technologies, USA). A targeted molecular genetic analysis was also carried out in 5 patients with symptoms of Zellweger syndrome without increased plasma levels of very long-chain fatty acids using the Ion Torrent PGM System for Next-Generation Sequencing device (Life Technologies, Thermo Fisher Scientific). DNA samples were prepared using the Ion AmpliSeq Library Kit 2.0 reagent kit according to the manufacturer’s protocol.

RESULTS

Based on the results of the analysis of blood plasma samples from 35 children from the group with suspected peroxisomal diseases, based on characteristic clinical and laboratory data, 17 patients with various nosological forms of peroxisomal diseases were identified. Three patients had Zellweger syndrome; the concentration of hexacosanoic acid (C26:0) in these patients was 3.58 μmol/L, 9.22 μmol/L, and 12.8 μmol/L, which exceeds the upper limit of its reference interval by 3—10 times. In three cases, the diagnosis of X-linked adrenoleukodystrophy was confirmed. Only one of three patients had exceeded levels of C24:0 and C22:0 acids. In two patients, the diagnosis of styrene transport protein deficiency was confirmed; the concentrations of phytanic and pristanic acids were 18.7 μmol/L, 29.8 μmol/L and 15.3 μmol/L, 22.8 μmol/L, respectively. In five patients, the diagnosis of Refsum disease in the infantile form was confirmed. The concentration of the diagnostic marker phytanic acid was in the range of 525—1458 μmol/L, which is significantly higher than the upper limit of the reference interval. Two children aged 3 and 4.5 years were diagnosed with Refsum’s disease in the adult form; the concentration of phytanic acid was 622 µmol/l and 128 µmol/l, respectively, which is higher than the age norm, but not as significant as in patients with the infantile form of this disease. In two cases, the diagnosis of rhizomelic punctate chondrodysplasia type I was confirmed. Targeted sequencing of the PEX2, PEX10, PEX12, PEX16, and PEX11B genes using NGS in 5 patients with suspected Zellweger syndrome without an increase in the level of very long-chain fatty acids revealed missense in 2 children mutation W223X.

CONCLUSIONS

Our study shows the importance of chromatographic and molecular genetic diagnosis of peroxisomal diseases in children. It is recommended to use gas chromatography-mass spectrometry analysis of long-chain fatty acids as the most informative chromatographic method. For molecular genetic analysis, due to the genetic heterogeneity of this group of diseases, it is most informative to carry out whole exome sequencing using the NGS method, which will allow a more accurate nosological diagnosis.