Erythrocyte Membrane

The erythrocyte fatty acid membrane profile analysis was carried out as previously described, using the erythrocyte membrane pellet obtained by standard methods [39] (link). For this study, selection of the erythrocyte fraction was made by modification of a literature procedure for the selection of aged erythrocytes (red blood cell age >3 months), with cells selected for high density and small diameter compared to the average erythrocyte population [40] .
One mL of whole blood was first centrifuged at 2000 g for 5 min to eliminate the plasma, and a second round of centrifugation was then carried out at 4000 g at 4°C for 5 min in order to yield a stratification by cell density. The bottom layer (2.5 mm from the bottom of tube) consisted of erythrocyte cells, which were evaluated for their diameter using a Scepter™ 2.0 Cell Counter (Merck Millipore, Milan, Italy) to characterize the cell selection from each blood sample. The results were also compared with the cell population obtained from standard density gradient separation [41] (link), [42] .
Briefly, lipids were extracted from erythrocyte membranes according to the method of Bligh and Dyer [43] . The phospholipid fraction was controlled by TLC as previously described [39] (link), then treated with KOH/MeOH solution (0.5 M) for 10 min at room temperature and under stirring [44] (link).
Fatty acid methyl esters (FAME) were extracted using n-hexane; the hexane phase was collected and dried with anhydrous Na₂SO₄. After filtration, the solvent was eliminated by evaporation using a rotating evaporator, and the thin white film of the FAME was subsequently dissolved in a small volume of n-hexane. Approximately 1 µL of this solution was injected into the GC. A Varian CP-3800 gas chromatograph, with a flame ionization detector and an Rtx-2330 column (90% biscyanopropyl-10% phenylcyanopropyl polysiloxane capillary column; 60 m, 0.25 mm i.d., 0.20 µm film thickness) was used for the analysis. Temperature was held at 165°C held for the initial 3 min, followed by an increase of 1°C/min up to 195°C, held for 40 min, followed by a second increase of 10°C/min up to 250°C, held for 5 min. The carrier gas was helium, held at a constant pressure of 29 psi. Methyl esters were identified by comparison with the retention times of commercially available standards or trans fatty acid references, obtained as described elsewhere [45] (link).

InCHIANTI GWAS: Inverse normal transformation was applied to plasma fatty acid concentrations to avoid inflated type I error due to non-normality [51] (link). The genotypes were coded 0, 1 and 2 reflecting the number of copies of an allele being tested (additive genetic model). For X-chromosome analysis, the average phenotype of males hemizygous for a particular allele was treated assumed to match the average phenotype of females homozygous for the same allele. Association analysis was conducted by fitting simple regression test using the fastAssoc option in MERLIN [52] (link). Narrow heritability reflects the ratio of the trait’s additive variance to the total variance [51] (link),[53] (link). In all the analyses, the models were adjusted for sex, age and age squared. The genomic control method was used to control for effects of population structure and cryptic relatedness [54] (link). An approximate genome-wide significance threshold of 1×10⁻⁷ (∼0.05/495343 SNPs) was used. For each fatty acid concentration, a second analysis included the most significant SNP from the first pass analysis as a covariate. Linkage disequilibrium coefficints within the region of interest were calculated using GOLD [55] (link).
For the other phenotypes (total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol and BMI), the traits were normalized either by natural log or square root transformation when necessary. Associations for each SNP were investigated using the general linear model (GLM) procedure in SAS.
GOLDN: Inverse normal transformation was applied to erythrocyte membrane fatty acid concentration to achieve approximate normality. For the additive model, genotype coding was based on the number of variant alleles at the polymorphic site. With no significant sex modification observed, men and women were analyzed together. We used the generalized estimating equation (GEE) linear regression with exchangeable correlation structure as implemented in the GENMOD procedure in SAS (Windows version 9.0, SAS Institute, Cary, NC) to adjust for correlated observations due to familial relationships. Potential confounding factors included study center, age, sex, BMI, smoking (never, former and current smoker), alcohol consumption (non-drinker and current drinker), physical activity, drugs for lowering cholesterol, diabetes and hypertension and hormones. A two-tailed P value of <0.05 was considered to be statistically significant.

An aliquot of the blood samples (250 μL) used for the HDFM measurements was then used for the separation of the blood cells, isolation of mature RBC membrane phospholipids and examination of fatty acid composition, applying previously published protocols^{7 (link)}. We evaluated the fatty acids of the mature erythrocyte membrane phospholipids as relative percentages (% rel) referred to a cluster of 12 fatty acids (10 cis and 2 trans fatty acids)² . The mean % rel ± standard deviation (sd) of the RBC membrane fatty acid cluster for healthy and ASD children with the decreased value of DHA in the ASD children (P = 0.0424) are reported in the Supplementary Table 4.

The rat blood plasma was obtained by centrifugation of whole blood and immediately used to determine biochemical markers of the functional state of the liver (the activity of alanine aminotransferase and aspartate aminotransferase, bilirubin concentration, and the thymol test) and lipid concentrations^{2 (link)}. ALT and AST activities were determined by Reitman-Fresnel method, total and direct bilirubin were determined by Endraschik method and thymol test was performed using thymol reagent checking the test kits (R&D enterprise Felicity-Diagnostics, Ukraine).
We determined the concentration of the following compounds in the blood plasma (mg%): phospholipids, cholesterol (CHOL), cholesterol esters (ECHOL), free fatty acids, triglycerides. Lipids were divided by the method of thin-layer chromatography⁴⁹ . Chromatographic separation of lipid components of plasma was carried out on “Silufol” plates. After treatment with an aqueous solution of phosphomolybdic acid, a quantitative assessment of the color intensity of each fraction was performed using a densitometer DO-1 M (“Shimadzu”, Japan, λ 620 nm)^{50 (link)}.
Blood cell mass was used to obtain erythrocyte plasma membrane preparations by the slightly modified Dodge’s method. Plasma membrane preparations were used to determine the ATPase activities of the primary active ion transport systems (total Mg²⁺, Na⁺, K⁺-ATPase, basal Mg²⁺-ATPase and Na⁺, K⁺-ATPase). The protein concentration in the preparations of the erythrocyte plasma membranes (PM) was determined by Lowry’s method^{51 (link)}. Total Mg²⁺, Na⁺, K⁺-ATPase activity was determined in the fraction of erythrocyte PMs in the standard incubation medium (in mM): 1 ATP, 3 MgCl₂, 125 NaCl, 25 KCl, 1 EGTA, 20 Hepes-Tris-buffer (pH 7.4), 1 NaN₃ (inhibitor of mitochondria ATPase), 0.1 µm thapsigargin (the selective inhibitor of Ca²⁺,Mg²⁺-ATPase of endoplasmatic reticulum) and 0.1% digitonin (the factor of PM perforation), at 37 °C. The Mg²⁺-ATPase activity was determined by the presence of a selective inhibitor Na⁺,K⁺-ATPase ouabain (1 mM) in the incubation medium. The Na⁺, K⁺-ATP activity was calculated as the difference between the total Mg²⁺, Na⁺, K⁺-ATPase and the ouabain-insensitive Mg²⁺-ATPase activity^{52 (link),53 (link)}.
This paper presents a statistical analysis of the experimental data obtained in the study and processed by the variation statistics methods using the Origin Pro 8 software. The samples were checked to belong to normally distributed general populations according to the Shapiro–Wilk criterion. The dispersion analysis was used to determine reliable differences between the mean values of samplings, and the post-test comparison was made using the Tukey test. In all cases, the results were reliable on the condition of the probability value p under 5% (p < 0.05). The obtained results were presented as the arithmetic mean ± standard error of the mean value, and the n value was determined by the total in the number of experiments.

Tightly synchronised 3D7 schizonts expressing endogenously GFP-tagged GAPM2 (glideosome-associated protein with multiple membrane spans 2^{56 (link),57 (link)}) were isolated from a 10–20 mL culture (5–10% parasitaemia) using 60% Percoll ^{55 (link)}, washed twice in pre-warmed RPMI, and treated with 1 µM of the PKG-inhibitor compound 2 (provided by Dr. Mike Blackman, The Francis Crick Institute, UK)^{58 (link),59 (link)}, and incubated for 6 h. Segmented schizonts were washed once and then resuspended in pre-warmed RPMI without Albumax or phenol red but with the addition of 1 µM E64 (Sigma) to allow the parasitophorous vacuole to rupture but prevent rupture of the red blood cell membrane. This helped us identify very late stage schizonts in the SEM. Cells were kept warm and vitrified within an hour.

From April 2015 to June 2021, 20 Japanese families with suspected DHSt were enrolled in this study. This study was performed in accordance with the principles of the Declaration of Helsinki and approved by the ethics committee of the institution. After obtaining written informed consent, blood samples were collected from all patients. In addition, we collected detailed clinical information from the attending doctors, including family histories, clinical courses, and physical findings.
In most patients, when possible, we first performed additional red cell membrane functional examinations, including the acidified glycerol hemolysis time (AGLT) test, flow-cytometric osmotic fragility (FCM-OF) test, and eosin-5’-maleimide (EMA) binding test with a negative direct antiglobulin test as per previously reported methods^{10 (link)}. DHSt was suspected when clinical findings such as hemolytic anemia with stomatocytosis and hemochromatosis not due to transfusion, positive family history, and past history of perinatal edema were observed, and laboratory tests revealed elevated MCV, increased % residual red cells (%RRC) in the FCM-OF test, and normal or increased EMA binding.
Genomic DNA was extracted from the patient’s peripheral blood using a QIAamp DNA extraction kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). The Haloplex HS target enrichment system (Agilent Technologies, Santa Clara, CA, USA) was used for TCS. Using SureDesign (https://earray.chem.agilent.com/suredesign/home.htm), the target panel was designed to include all coding exons and intron‒exon boundaries of the 74 possible candidate genes^{10 (link)}. Massive parallel sequencing was performed using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Raw data were aligned to the human genome sequence GRCh37/hg19. The generated FASTQ files were imported into SureCall v3.5 (Agilent Technologies) for variant calling. Analysis following the filtering of the obtained variants was described previously^{10 (link)}. The obtained variants were filtered according to the following strategy: (1) variant frequencies were below 1% in 1000G_EAS and ALL (1000 Genomes), HGDV, and dbSNP; (2) synonymous variants were excluded (nonsynonymous variants, variants associated with frameshift, insertion/deletion variants, and variants in splicing donor/acceptor sites were included); (3) variants with allele frequencies less than 30% of the total read depth were excluded; and (4) the CADD_phred was higher than 20 if obtained. Variant information obtained using wANNOVAR (http://wannovar.wglab.org/) was used for curation. Integrative Genomics Viewer (https://software.broadinstitute.org/software/igv/) was used for visual evaluation. All variants were evaluated using the guidelines proposed by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP)^{11 (link)}.
The existence of the identified variants in the probands of the enrolled patients was confirmed using conventional PCR-Sanger sequencing. Genotyping for αLELY (low expression allele of SPTA1), UGT1A1, and Memphis I and II (SLC4A1) was also performed using conventional PCR-Sanger sequencing for all patients^{12 (link)–15 (link)}.