Association of hyperhomocysteinemia with genetic variants in key enzymes of homocysteine metabolism and methotrexate toxicity in rheumatoid arthritis patients

Objectives

The study investigated the association between plasma homocysteine, folate and vitamin B12 with 5,10 methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), thymidylate synthase (TYMS 2R → 3R) and methionine synthase (MTR A2756G) polymorphisms and methotrexate (MTX) treatment and toxicity in Tunisian Rheumatoid arthritis (RA) patients.

Methods

A total of 185 patients with RA were included. Homocysteine (Hcy) was assessed by fluorescence polarization immunoassay, and folate and vitamin B12 were measured by chemiluminescence immunoassays. The genetic polymorphisms were analyzed by PCR or PCR-RFLP. Hyperhomocysteinemia (HHC) was considered for Hcy?>?15 µmol/L.

Results

MTHFR C677T polymorphism was associated with HHC in RA patients (multi-adjusted OR, 95% CI 2.18, [1.07–4.57]; p?=?0.031). No association was detected with the remaining polymorphisms. Plasma Hcy, folate, and vitamin B12 did not differ according to each polymorphism, or with MTX treatment or toxicity. However, HHC was more prevalent in patients with than those without MTX toxicity (32.7 vs. 16.7%; p?=?0.035).

Conclusions

The MTHFR 677TT genotype is an independent risk factor for HHC in Tunisians RA patients. HHC could be a useful marker of MTX toxicity in RA patients.

     

Immune tumoral microenvironment in gliomas: focus on CD3+ T cells,Vδ1+ T cells,and microglia/macrophages

Gliomas are histologically defined as low-grade gliomas (LGG) and high-grade gliomas (HGG). The most common type of HGG is the glioblastoma (GBM). We aimed to determine the immunological characteristics of CD3 T-cells, Vδ1 T-cells, and microglia/macrophages infiltrating brain gliomas. We collected 24 formalin-fixed paraffin-embedded samples issued from 19 cases of GBM and 5 cases of LGG. An immunohistochemical analysis was performed using anti-CD3, anti-Vδ1, and anti-iba-1 antibodies. Labelling indexes (LI) of CD3 and Vδ1 were evaluated quantitatively, and other CD3, Vδ1, and iba-1 staining characteristics were evaluated qualitatively. The median age of patients was 49 years in GBM and 52 years in LGG. The sex ratio was 1.4 and GBM predominated in males (p?=?0.05). In GBM, the medians of CD3-LI and Vδ1-LI were 30 and 3.5 respectively. CD3-LI inversely correlated with survival in GBM cases (r?=????0.543; p?=?0.016). CD3 staining intensity correlated with CD3-LI (p?<?0.0001) and with the survival in GBM cases (p?=?0.003). Compared to LGG, the CD3-LI, the intensity of intra-tumoral Vδ1 staining, and the amount of iba-1 were higher in GBM (p?=?0.042; p?=?0.014; and p?=?0.001 respectively). The iba-1 organization was more amoeboid in older patients and more branched in younger patients (p?=?0.028) and tended to be more amoeboid in cases with high iba-1 amount (p?=?0.09). Our results suggest that a high level of CD3-LI and a strong intra-tumoral infiltration of Vδ1 T-cells as well as a high involvement of TAM can be considered potential markers of poor prognosis of GBM. However, this requires further studies on more balanced GBM-LGG sample, including an expanded panel of biomarkers.

     

Skeletal clinical characteristics of osteogenesis imperfecta caused by haploinsufficiency mutations in COL1A1

COL1A1 haploinsufficiency mutations lead to the mildest form of osteogenesis imperfecta (OI), OI type I. The skeletal clinical characteristics resulting from such mutations have not been characterized in detail. In this study we assessed 86 patients (36 male, 50 female; mean age 13.3 years; range, 0.6 to 54 years) with COL1A1 haploinsufficiency mutations, of whom 70 were aged 21 years or less (“pediatric” patients). Birth history was positive for fracture or long‐bone deformity in 12% of patients. The average rate of long‐bone fracture (femur, tibia/fibula, humerus, radius/ulna) in pediatric patients was 0.62 fractures per year, one‐half of which affected the tibia/fibula. Long‐bone fracture rate was negatively associated with age and lumbar spine areal bone mineral density. Vertebral compression fractures were observed in 71% of the 58 pediatric patients who had lateral spine radiographs. The median number of vertebral fractures was higher for females (median 4; range, 0 to 14) than for males (median 1; range, 0 to 8) (p = 0.03). Lumbar spine areal bone mineral density was negatively associated with the severity of vertebral compression fractures, as reflected in the spine deformity index. Scoliosis was present in about 30% of pediatric patients but the Cobb angle was <30 degrees in all cases. The average final height Z‐score was –1.1, representing a deficit of 8 to 10 cm compared to the general population. In summary, OI patients with COL1A1 haploinsufficiency mutations have high rates of significant skeletal involvement. Systematic follow‐up of growing patients with COL1A1 haploinsufficiency mutations including radiographic screening for vertebral compression fractures and scoliosis is warranted.     

CDK13-related disorder: Report of a series of 18 previously unpublished individuals and description of an epigenetic signature

PurposeRare genetic variants in CDK13 are responsible for CDK13-related disorder (CDK13-RD), with main clinical features being developmental delay or intellectual disability, facial features, behavioral problems, congenital heart defect, and seizures. In this paper, we report 18 novel individuals with CDK13-RD and provide characterization of genome-wide DNA methylation.MethodsWe obtained clinical phenotype and neuropsychological data for 18 and 10 individuals, respectively, and compared this series with the literature. We also compared peripheral blood DNA methylation profiles in individuals with CDK13-RD, controls, and other neurodevelopmental disorders episignatures. Finally, we developed a support vector machine–based classifier distinguishing CDK13-RD and non–CDK13-RD samples.ResultsWe reported health and developmental parameters, clinical data, and neuropsychological profile of individuals with CDK13-RD. Genome-wide differential methylation analysis revealed a global hypomethylated profile in individuals with CDK13-RD in a highly sensitive and specific model that could aid in reclassifying variants of uncertain significance.ConclusionWe describe the novel features such as anxiety disorder, cryptorchidism, and disrupted sleep in CDK13-RD. We define a CDK13-RD DNA methylation episignature as a diagnostic tool and a defining functional feature of the evolving clinical presentation of this disorder. We also show overlap of the CDK13 DNA methylation profile in an individual with a functionally and clinically related CCNK-related disorder.     

Association of serum levels of aggrecan ARGS,NITEGE fragments and radiologic knee osteoarthritis in Tunisian patients

ObjectivesProteolytic degradation of aggrecan is a hallmark of the pathology of osteoarthritis. The aim of this study was to develop enzyme-linked immunosorbent assay (ELISA) to quantify the serum levels of specific aggrecan fragments generated by aggrecanases-mediated cleavage. We investigated the relationships between these two aggrecan degradations fragments and urinary CTX-II levels.MethodsThe competitive ELISAs employ a polyclonal antibody raised against the aggrecan fragments containing two neoepitopes NITEGE³⁷³ and ³⁷⁴ARGSVI. We measured serum levels of ARGSV and NITEGE in 125 women with knee osteoarthritis (mean ± SD age of 53.6 ± 7.6 years, mean ± SD disease duration of 3.6 ± 3.8 years), and 57 women age-matched controls.ResultsAggrecan neoepitopes assays showed an intra- and inter-assay imprecision (CV) lower than 20% for both tests and good linearity. Median serum ARGSVI (by 18%; P = 0.002), and NITEGE (36.4%; P < 0.001) levels were significantly decreased in patients with knee osteoarthritis compared with controls. Minimal joint space width was negatively correlated with ARGSVI (r = –0.368, P = 0.04) and NITEGE (r = –0.274, P = 0.038) in knee osteoarthritis patients. Median urinary CTX-II levels were significantly increased by 39.5% (P = 0.001) in knee OA patients compared with controls.ConclusionMarkers of degradation aggrecan were analyzed for the first time in an African osteoarthritis population. These markers can be used to monitor aggrecanase activity in human joint disease. Their combination with CTX-II can improve clinical investigation of patients with osteoarthritis patients.     

Dissociation of the Role of Infralimbic Cortex in Learning and Consolidation of Extinction of Recent and Remote Aversion Memory

Medial prefrontal circuits have been reported to undergo a major reorganization over time and gradually take a more important role for remote emotional memories such as contextual fear memory or food aversion memory. The medial prefrontal cortex, and specifically its ventral subregion, the infralimbic cortex (IL), was also reported to be critical for recent memory extinction of contextual fear conditioning and conditioned odor aversion. However, its exact role in the extinction of remotely acquired information is still not clear. Using postretrieval blockade of protein synthesis or inactivation of the IL, we showed that the IL is similarly required for extinction consolidation of recent and remote fear memory. However, in odor aversion memory, the IL was only involved in extinction consolidation of recent, but not remote, memory. In contrast, only remote retrieval of aversion memory induced c-Fos activation in the IL and preretrieval inactivation of the IL with lidocaine impaired subsequent extinction of remote but not recent memory, indicating IL is necessary for extinction learning of remote aversion memory. In contrast to the effects in odor aversion, our data show that the involvement of the IL in the consolidation of fear extinction does not depend on the memory age. More importantly, our data indicate that the IL is implicated in the extinction of fear and nonfear-based associations and suggest dissociation in the engagement of the IL in the learning and consolidation of food aversion extinction over time.     

Ergosterol purified from medicinal mushroom Amauroderma rude inhibits cancer growth in vitro and in vivo by up-regulating multiple tumor suppressors

We have previously screened thirteen medicinal mushrooms for their potential anti-cancer activities in eleven different cell lines and found that the extract of Amauroderma rude exerted the highest capacity in inducing cancer cell death. The current study aimed to purify molecules mediating the anti-cancer cell activity. The extract of Amauroderma rude was subject to fractionation, silica gel chromatography, and HPLC. We purified a compound and identified it as ergosterol by EI-MS and NMR, which was expressed at the highest level in Amauroderma rude compared with other medicinal mushrooms tested. We found that ergosterol induced cancer cell death, which was time and concentration dependent. In the in vivo experiment, normal mice were injected with murine cancer cell line B16 that is very aggressive and caused mouse death severely. We found that treatment with ergosterol prolonged mouse survival. We found that ergosterol-mediated suppression of breast cancer cell viability occurred through apoptosis and that ergosterol up-regulated expression of the tumor suppressor Foxo3. In addition, the Foxo3 down-stream signaling molecules Fas, FasL, BimL, and BimS were up-regulated leading to apoptosis in human breast cancer cells MDA-MB-231. Our results suggest that ergosterol is the main anti-cancer ingredient in Amauroderma rude, which activated the apoptotic signal pathway. Ergosterol may serve as a potential lead for cancer therapy.