Using metabolic abnormalities of carriers in the neonatal period to evaluate the pathogenicity of variants of uncertain significance in methylmalonic acidemia.

Objective: To accurately verify the pathogenicity of variants of uncertain significance (VUS) in MUT and MMACHC genes through mass spectrometry and silico analysis. Methods: This multicenter retrospective study included 35 participating units (ClinicalTrials.gov ID: NCT06183138). A total of 3,071 newborns (within 7 days of birth) were sorted into carrying pathogenic/likely pathogenic (P/LP) variants and carrying VUS, non-variant groups. Differences in metabolites among the groups were calculated using statistical analyses. Changes in conservatism, free energy, and interaction force of MMUT and MMACHC variants were analyzed using silico analysis. Results: The percentage of those carrying VUS cases was 68.15% (659/967). In the MMUT gene variant, we found that C3, C3/C2, and C3/C0 levels in those carrying the P/LP variant group were higher than those in the non-variant group (p < 0.000). The conservative scores of those carrying the P/LP variant group were >7. C3, C3/C0, and C3/C2 values of newborns carrying VUS (c.1159A>C and c.1286A>G) were significantly higher than those of the non-variant group and the remaining VUS newborns (p < 0.005). The conservative scores of c.1159A>C and c.1286A>G calculated by ConSurf analysis were 9 and 7, respectively. Unfortunately, three MMA patients with c.1159A>C died during the neonatal period; their C3, C3/C0, C3/C2, and MMA levels were significantly higher than those of the controls. Conclusion: Common variants of methylmalonic acidemia in the study population were categorized as VUS. In the neonatal period, the metabolic biomarkers of those carrying the P/LP variant group of the MUT gene were significantly higher than those in the non-variant group. If the metabolic biomarkers of those carrying VUS are also significantly increased, combined with silico analysis the VUS may be elevated to a likely pathogenic variant. The results also suggest that mass spectrometry and silico analysis may be feasible screening methods for verifying the pathogenicity of VUS in other inherited metabolic diseases.

Neuroprotective effect of 1,25­dihydroxyvitamin D3 against hyperoxia­induced brain injury in premature rats.

Studies have shown that vitamin D plays a crucial role in brain development, brain metabolism and neuroprotection. There is little evidence for the neuroprotective effect of 1, 25­dihydroxyvitamin D3 (1,25­(OH)2D3) on various brain injury models. The aim of this study was to investigate the neuroprotection effect of 1,25­(OH)2D3 against hyperoxia­induced brain injury in premature rats. Sprague­Dawley rats were exposed to 95% oxygen or room air for 24 h and treated with 1,25­(OH)2D3 or normal saline for 14 consecutive days. The histopathological changes of optic chiasma tissue were observed by hematoxylin­eosin staining. Immunohistochemistry, qRT­PCR, and western blot were performed to detect the expression of integrin­ß1 and yes­associated protein (YAP) in the organization of the optic chiasm. Histopathological sections of optic chiasma showed visible optic nerve swelling, expanded nerve fiber space, uneven staining, obvious oligodendrocyte proliferation and disordered cell arrangement accompanied by inflammatory cell infiltration and exudation after 7 days and 14 days of hyperoxia exposure. The hyperoxia group treated with 1,25­(OH)2D3 were showed improvement of brain injury with reduced inflammatory exudation, uniform nerve fiber staining and less obvious oligodendrocyte proliferation. Immunohistochemical staining, qRT­PCR and western blot indicated that 1,25­(OH)2D3 treatment upregulated the expression of integrin­ß1 and YAP in the hyperoxia group on day 7. However, the expression of YAP was significantly increased compared with control group and treatment with 1,25­(OH)2D3 reduced the expression of YAP in the hyperoxic group on day 14. 1,25­(OH)2D3 may regulate the expression of integrin­ß1 and YAP to alleviate hyperoxia­induced brain injury in premature rats.