sex genotyping  (Transnetyx)

Journal: bioRxiv

Article Title: The 677C>T variant in methylenetetrahydrofolate reductase causes morphological and functional cerebrovascular deficits in mice

doi: 10.1101/2021.12.16.472805

Figure Lengend Snippet: ( A ) MTHFR enzymatic activity in the liver. Activity is significantly reduced in both males and females carrying one (CT) or two (TT) copies of the variant. (n=6/genotype except for TT males: n=4; P=0.0002 One-way ANOVA, Tukey post-hoc). ( B ) Liver enzymatic activity after the sample is heated is shown as a ratio of percent activity at 46 degrees Celsius/percent activity at 37 degrees Celsius. Activity is significantly reduced in male CT and TT mice. (n=3/genotype; P=0.0003, One-way ANOVA, Tukey post-hoc). ( C ) Western blots were performed on control (CC) male and female mice and determined that MTHFR protein expression does not differ by sex (left) using actin as a standard (n=6/sex; unpaired t-test). The ratio of the phosphorylated MTHFR isoform p70 to the non-phosphorylated isoform is also not significantly different between male and female controls (right; n=6/genotype; unpaired t-test). ( D ) Within males, total MTHFR does not significantly differ based on genotype (left; n=6/genotype except for TT males: n=4; One-way ANOVA, Tukey post-hoc). However, the ratio of phosphorylated to non-phosphorylated isoforms is reduced in CT and TT males (center; n=6/genotype except for TT males: n=4; P=0.0017, One-way ANOVA, Tukey post-hoc). MTHFR enzymatic activity correlates with total MTHFR protein (right; n=6/genotype except for TT males: n=4; correlation with activity, P=0.0127 Pearson r=0.6250). ( E ) In females, total MTHFR protein also does not significantly differ based on genotype (left; n=6/genotype; One-way ANOVA, Tukey post-hoc). The ratio of phosphorylated to non-phosphorylated isoforms is reduced in TT females (center; n=6/genotype; P=0.0119, One-way ANOVA, Tukey post-hoc). MTHFR enzymatic activity correlated with total MTHFR protein (right; n=6/genotype; correlation with activity, P= 0.0004 Pearson r=0.7471).

Article Snippet: Crude protein extract was prepared from 80 – 100 mg of frozen liver, ( n =6/sex/genotype, except TT males; n =4) and whole half brain ( n =6/genotype) in extraction buffer (50 mM potassium phosphate pH 7.2, 50 mM sucrose, 0.3 mM EDTA containing protease and phosphatase inhibitors (Pierce Protease Inhibitor Tablet and Halt Phosphatase Inhibitor Cocktail, ThermoScientific catalog numbers A32955 and 78420)) using a bead mill with steel beads (2 x 2 min at 20 Hz; TissueLyser, Qiagen).

Techniques: Activity Assay, Variant Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: The 677C>T variant in methylenetetrahydrofolate reductase causes morphological and functional cerebrovascular deficits in mice

doi: 10.1101/2021.12.16.472805

Figure Lengend Snippet: ( A ) Numerous cell types in the brain express Mthfr . Data obtained from the Brain RNA-seq database. ( B ) Single cell sequencing showing expression of Mthfr in cerebrovascular and perivascular cells. Data obtained from the Single-Cell RNA-seq Gene Expression Data. AC= astrocytes, N= neurons, OPC= oligodendrocyte precursor cells, NFO= newly formed oligodendrocytes, MO= myelinating oligodendrocytes, MG= microglia, MAC= macrophages, EC= endothelial cells, PC= pericytes, vSMC= venous smooth muscle cells, aaSMC= arteriolar smooth muscle cells, aSMC= arterial smooth muscle cells, FB1= fibroblast-like type 1, FB2= fibroblastlike type 2, OL= oligodendrocytes, EC1= endothelial cell type 1, EC2= endothelial cell type 2, EC3= endothelial cell type 3, vEC= venous endothelial cells, capilEC= capillary endothelial cells, aEC= arterial endothelial cells. ( C ) MTHFR enzymatic activity measured in brain tissue. Both males and females show significantly reduced activity based on genotype (n=4-6/sex/genotype; P=<0.0001, One-way ANOVA, Tukey post-hoc). ( D ) In brains of female mice, total MTHFR is significantly reduced in both the CT and TT groups (left; n=6/genotype; P=0.0002, One-way ANOVA). Enzymatic activity is correlated with MTHFR protein expression (right; correlation with activity, P=0.0001 Pearson r=0.7896). ( E ) Brain-derived metabolite levels: THF= tetrahydrofolic acid, DHF= dihydrofolic acid, 5-MTHF= 5-methyltetrahydrofolate, SAM= S-Adenosyl methionine, SAH= S-Adenosylhomocysteine. DHF (P= <0.0001; 95% CI {0.09381 to 0.1738}), 5-MTHF (P= <0.0001; 95% CI {-0.1831 to −0.07584}), SAM (P= <0.0001; 95% CI {-17.52 to −13.76}), SAH (P= <0.0001; 95% CI {6.445 to 8.966}) methionine (P= <0.0001; 95% CI {270.3 to 369.9}) and choline (P= <0.0001 95% CI {1107 to 1324}) demonstrate significant sex-differences, while THF (P= 0.0007; 95% CI {-0.2728 to 0.2883}), 5-MTHF (P= 0.0075; 95% CI {-0.1831 to −0.07584}), cystathionine (P= 0.0031 95%CI {1.798 to 25.69}) and betaine (P= 0.0086 95% CI {-7.633 to 3.749}) show genotype-specific differences (n=4-6/sex/genotype; Two-way ANOVA, Tukey post-hoc).

Article Snippet: Crude protein extract was prepared from 80 – 100 mg of frozen liver, ( n =6/sex/genotype, except TT males; n =4) and whole half brain ( n =6/genotype) in extraction buffer (50 mM potassium phosphate pH 7.2, 50 mM sucrose, 0.3 mM EDTA containing protease and phosphatase inhibitors (Pierce Protease Inhibitor Tablet and Halt Phosphatase Inhibitor Cocktail, ThermoScientific catalog numbers A32955 and 78420)) using a bead mill with steel beads (2 x 2 min at 20 Hz; TissueLyser, Qiagen).

Techniques: RNA Sequencing Assay, Sequencing, Expressing, Activity Assay, Derivative Assay

Journal: bioRxiv

Article Title: The 677C>T variant in methylenetetrahydrofolate reductase causes morphological and functional cerebrovascular deficits in mice

doi: 10.1101/2021.12.16.472805

Figure Lengend Snippet: ( A ) Representative images are shown from mice at 6 and 18 months of age. From right to left, the anatomical location of the image as determined by the Allen Mouse Brain Atlas, CT images showing clear locations of identifiable brain structures, PET images showing changes in blood perfusion via tracer concentration, the overlay of the PET and CT, and finally autorad demonstrating radioactive decay on the far right (n=6/sex/genotype/age). ( B ) 13 brain regions were determined by principal component analysis to explain 80% of the variance in blood flow within all mice. Data is presented as brain regions normalized to the cerebellum.

Article Snippet: Crude protein extract was prepared from 80 – 100 mg of frozen liver, ( n =6/sex/genotype, except TT males; n =4) and whole half brain ( n =6/genotype) in extraction buffer (50 mM potassium phosphate pH 7.2, 50 mM sucrose, 0.3 mM EDTA containing protease and phosphatase inhibitors (Pierce Protease Inhibitor Tablet and Halt Phosphatase Inhibitor Cocktail, ThermoScientific catalog numbers A32955 and 78420)) using a bead mill with steel beads (2 x 2 min at 20 Hz; TissueLyser, Qiagen).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: The 677C>T variant in methylenetetrahydrofolate reductase causes morphological and functional cerebrovascular deficits in mice

doi: 10.1101/2021.12.16.472805

Figure Lengend Snippet: ( A ) From right to left, representative confocal images of immunolabeled Collagen IV-expressing vascular basement membrane, GFAP-expressing astrocytes, and their respective Imaris renderings in 6 month male and female mice from each genotype. ( B ) Vascular density was assessed in 3 regions of the cortex at 6 and 18 months of age. The volume of ColIV per image was calculated using Imaris software and the sum of the volume/image was used for analysis. 6 month old males and females show significantly reduced vascular volume in the frontal cortex of TT or both CT and TT mice, respectively (n=6/sex/genotype/age; P=<0.0001 for genotype, 95% CI {-36337 to 89790} Two-way ANOVA, Tukey post-hoc). ( C ) Astrocyte expression of GFAP was assessed in the same set of images using the same protocol for Imaris analysis. In 6 month old males, there was a significant increase in GFAP-expressing astrocytes in both the visual cortex and the frontal cortex (n=5-6/sex/genotype/age; visual cortex P=0.0026 95% CI {-73800-220053}, frontal cortex P=0.0012 95% CI {-98179 to 48259}, Two-way ANOVA, Tukey post-hoc).

Article Snippet: Crude protein extract was prepared from 80 – 100 mg of frozen liver, ( n =6/sex/genotype, except TT males; n =4) and whole half brain ( n =6/genotype) in extraction buffer (50 mM potassium phosphate pH 7.2, 50 mM sucrose, 0.3 mM EDTA containing protease and phosphatase inhibitors (Pierce Protease Inhibitor Tablet and Halt Phosphatase Inhibitor Cocktail, ThermoScientific catalog numbers A32955 and 78420)) using a bead mill with steel beads (2 x 2 min at 20 Hz; TissueLyser, Qiagen).

Techniques: Immunolabeling, Expressing, Software