Aim: The levels of urine metabolites, rheumatoid factor (RF) and inflammatory factors are altered in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. However, the level of them has not been quantitatively evaluated yet, as well as the correlation between the urine metabolites, RF and inflammatory factors. This research aims to investigate the urine metabolites and inflammatory factors from patients with OA and early rheumatoid arthritis (e-RA) to explore the relationship between the urine metabolites and RF or inflammatory factors. Methods: A total of 455 individuals were included in this study. Urine specimen was collected from 190 healthy volunteers, 26 osteoarthritis patients and 239 RA patients in which 37 subjects were diagnosed as early stage RA (e-RA). Metabolites in urine were extracted and analyzed with liquid chromatography-mass spectrometry (LC-MS) technique. Urine RF and inflammatory factors were measured with MSD V-Plex Proinflammatory Panel 1 Human Kit. Results: RF and nine of the inflammatory factors were significantly elevated in e-RA compared with OA and controls. Nine kinds of metabolites levels were found to positively correlated with urine RF level, two of which including 2-Methylnaphthalene (r= 0.636, p= 0.00195) and 3,4-Dihydroxy-L-phenylalanine (r= 0.524, p= 0.0149) were significantly elevated in e-RA group. Conclusion: Urine from e-RA patients exhibited different levels of metabolites, rheumatoid factor (RF) and inflammatory factors from patients without RA and OA. Nine metabolites showed significant positive correlation with RF level. Among these nine metabolites, 2-Methylnaphthalene (r= 0.636, p= 0.00195) and 3,4-Dihydroxy-L-phenylalanine (r= 0.524, p= 0.0149) elevated at early stage of RA, which could serve as a marker for arthritis screening and early diagnostic.

Methylmalonic acidemia with homocystinuria (MMA-cblC) is an autosomal recessive genetic disorder of organic acid metabolism. Shandong, a northern province of China, has a significantly high incidence of about 1/4,000, suggesting a high carrying rate among the local population. The current study established a PCR technique involving high-resolution melting (HRM) to screen for carriers based on hotspot mutation analysis to further develop a preventive strategy to reduce the local incidence of this rare disease. Whole-exome sequencing of 22 families with MMA-cblC and a comprehensive literature review were used to identify MMACHC hotspot mutations in Shandong Province. Subsequently, a PCR-HRM assay based on the selected mutations was established and optimized for large-scale hotspot mutation screening. The accuracy and efficiency of the screening technique was validated using samples from 69 individuals with MMA-cblC and 1,000 healthy volunteers. Six hotspot mutations in the MMACHC gene (c.609G>A, c.658_660delAAG, c.80A>G, c.217C>T, c.567dupT and c.482G>A), which account for 74% of the alleles associated with MMA-cblC, were used to establish a screening technique. The established PCR-HRM assay detected 88 MMACHC mutation alleles in a validation study with 100% accuracy. In the general population in Shandong, the carrying rate of 6 MMACHC hotspot mutations was 3.4%. In conclusion, the 6 hotspots identified cover the majority of the MMACHC mutation spectrum, and the Shandong population has a particularly high carrying rate of MMACHC mutations. The PCR-HRM assay is highly accurate, cost-effective, and easy to use, making it an ideal choice for mass carrier screening.