Study Of Red Blood Cell Abnormity And Mechanism In Prader-Willi Syndrome

BackgroundPrader-Willi syndrome,abbreviated as PWS,is a rare disease caused by deletion or abnormal methylation of paternal fragment of q11.2-q13.PWS can be divided into deletion subtype,maternal uniparental diploid subtype and imprinting defect subtype.The genes involved included protein-coding genes CYFIP1,GABRA5,GABRB3,GABRG3,IMP3,MAGEL2,MKRN3,NDN,NIPA1,NIPA2,SNRPN,SNURF,TUBGCP5,small nucleolar RNA(snoRNA)and long non-coding RNA(lncRNA).Before the age of three,the main clinical manifestations are severe hypotonia,poor sucking reflex,neurodevelopmental delay and feeding disorders.After the age of three,the main clinical manifestations are behavioral disorders,especially gluttony and related morbid obesity,development delay,gonadal dysplasia,hypothyroidism and special face.However,the diagnosis of PWS depends on methylation-specific multiplex ligation probe amplification(MS-MLPA)technology,which brings difficulties to the diagnosis in primary hospitals.At present,the main treatment of PWS is the application of growth hormone and symptomatic treatment,but there is no fundamental treatment.Therefore,PWS needs to be further studied.Although current studies on PWS mainly focus on the nervous system,endocrine system and obesity,whether the blood system is affected has not been studied.Red blood cells(RBCs)are the most abundant cells in the human body.In addition to transporting oxygen,RBCs are also involved in the occurrence and development of obesity and nervous system diseases due to the abnormalities of RBCs’ metabolites such as NO and membrane lipid components;besides,RBCs are involved in the pathogenesis of cardiovascular and cerebrovascular diseases.Therefore,exploring whether red blood cells change in PWS and the mechanism of change will be beneficial to understand PWS further.Purpose1.To observe whether the phenotypes of RBCs in PWS change at different age stages;2.To explore which part of the cell structure causes the abnormity of RBCs in PWS;3.To explore whether the abnormity of RBCs in PWS is the secondary changes caused by morbid obesity or hormone abnormalities or directly caused by gene defects;4.To screen the target genes by gene library construction;5.To search for simple indicators to assist in the diagnosis of PWS.Methods(Technical route is shown in Figurel)1.Subjects enrollment:Subjects were divided into PWS patients,healthy controls and obese controls who visited Shandong Provincial Hospital from 2019 to 2021.Age,gender,height,weight,diagnosis and treatment information were recorded.Thyroid function,growth hormone,blood glucose,blood lipids,liver function and other abnormal indicators in PWS were detected by machines of the same model.All subjects had signed informed consent forms.2.Grouping:The subjects who met the inclusion and exclusion criteria were divided into ≥3 years old group and<3 years old group based on age,and the subjects in each age group were studied separately.PWS patients included in the study were divided into initial patients and patients receiving treatment.The patients were diagnosed by MSMLPA.The patients receiving treatment maintained the normal range of thyroid function and growth hormone,while the initial patients had not received any treatments.The age distribution of subjects in each group was consistent with normal distribution.(1)Treatment group:In the group of ≥3 years old,the control group was divided into healthy control group(n=16)and simple obese control group(n=16)because PWS patients(n=16)had morbid obesity.The subjects under three years old were divided into PWS group(n=10)and control group(n=16).(2)Initial patients(n=5).3.The phenotype changes of RBCs by routine blood test,blood smear and red blood cell osmotic fragility were observed by unpaired t test.4.Simple indicators that can be used to distinguish patients from controls would be screened.5.The changes of red blood cell structure would be observed:(1)Hemoglobin:crystal violet staining and electron microscopy were used to observe whether hemoglobin formed inclusion bodies in PWS patients’ RBCs,and high performance liquid chromatography(HPLC)was used to observe the proportion of globin chains;(2)Membrane skeleton:changes in membrane skeleton proteins were detected;(3)Membrane lipids:changes in lipid composition of erythrocyte membranes were detected by targeted lipidomics.6.Correlation analysis was conducted on red blood cell routine indexes,serum EPO,Fe,hemolysis indexes,obesity and hormones.The changes of red blood cells before and after hormone therapy in PWS patients with initial onset were observed.At the same time,unpaired t-test was used to compare the changes of red blood cells in different age groups,treatment groups,BMI groups,and gene subtypes of PWS patients,so as to further determine the causes of red blood cell changes.7.CRISPR-Cas9 technology or shRNA was used to construct gene libraries of PWS deletion fragments,and cell models of gene knockout or knockdown were constructed.8.The changes of morphology,membrane skeleton,differentiation and oxidative stress of the cell model were observed,and the target genes were screened by monoclonal.Result1.Baseline characteristics of subjects 3 years olderThe baseline characteristics of subjects enrolled in this study were consistent with those reported in the literature.Compared with healthy controls,PWS patients showed significant obesity.There was no significant difference in thyroid hormone and growth hormone in PWS patients after symptomatic treatment compared with healthy and obese controls.2.PWS patients have microcytic anemia with iron deficiency and red blood cell morphological abnormalitiesIn groups of 3 years old or older,hemoglobin concentration(HGB),hematocrit(HCT),and mean red blood cell volume(MCV)were significantly lower in patients with PWS than in healthy and obese controls,although the red blood cell count(RBC)was not significantly altered.Consistent with this result,serum iron levels were decreased in the PWS group.In addition,erythropoietin(EPO)levels were higher than those in healthy controls,suggesting the presence of microcytic anemia associated with mild iron deficiency in PWS patients.Compared with healthy controls and obese controls,patients with PWS showed an increase in red blood cell distribution width(RDW),as well as an increase in bullseye shaped red blood cells and red blood cell fragments as indicated by Wright Giemsa staining of blood smears.Consistent with the above findings,erythrocyte osmotic fragility was increased,and serum heme,total bilirubin and indirect bilirubin were slightly increased,suggesting that erythrocytes in patients with PWS were more prone to hemolysis under osmotic pressure.These results suggested that erythrocyte instability increases in patients with PWS.Microcytic anemia and abnormal erythrocyte morphology were also present in the group less than three years old.3.RDW/HCT increased significantly in PWS groupRDW/HCT was higher in PWS group than in control group.The correlation matrix by Pearson Correlation analysis and receiver operating characteristic curve(ROC curve)suggested that RDW/HCT could be used as an auxiliary diagnostic index.4.PWS patients had membrane skeleton assembly disorders,membrane lipid abnormalities and slight increase of fetal hemoglobinNo inclusion bodies were observed in the blood smears of PWS patients by crystal violet staining.Electron microscopy showed that the internal structure of red blood cells was uniform.HPLC showed that the proportion of α and β globin peptide chains of red blood cells were normal,but the proportion of fetal hemoglobin(HbF)was slightly increased.Protein Coomassie brilliant blue staining and Western Blot results suggested that some erythrocyte skeleton proteins from PWS patients were reduced.Since Actin homeostasis plays an important role in membrane skeleton homeostasis,the ratio of Factin to G-actin was found to be decreased in erythrocytes of PWS patients,suggesting that Actin assembly disorder in erythrocytes of PWS patients may be the cause of membrane skeleton assembly disorder.Due to the tight connectivity of membrane lipids and membrane skeletons,OPLSDA analysis of targeted lipidomics of erythrocyte membranes showed that PWS patients were clearly differentiated from healthy and obese controls,with the most significant changes in the reduction of lipids related to membrane fluidity,such as polyunsaturated fatty acids.5.Erythrocyte changes in PWS patients are not related to obesity,subtype or age,but treatment can improve the phenotype of erythrocyte skeleton deletion and membrane lipidsErythrocyte stability,membrane skeleton proteins and membrane lipids did not differ among subtype groups,BMI groups or age groups.However,the erythrocyte membrane skeleton protein was partially improved in newly diagnosed patients before and after treatment,and the membrane lipid composition of untreated patients was markedly different from that of treated patients.However,even if the patients returned to normal with hormone treatment,there was still a clear difference from the control group.6.Gene deletion is the cause of red blood cell abnormalitiesCell models constructed using gene libraries showed that gene knockout or knockdown resulted in an increased proportion of abnormal cells and enhanced oxidative stress level,but did not affect the differentiation process of red blood cells.ConclusionOur study revealed that PWS patients had mild microcytic anemia associated with iron deficiency,as well as erythrocyte malformation and hemolysis tendencies,which were associated with abnormal assembly of erythrocyte membrane skeleton proteins and membrane lipid changes caused by abnormal assembly of F-actin.Increased oxidative stress due to genetic defects might be the mechanism.RDW/HCT could be used as an auxiliary diagnostic index,which provided a basis for the diagnosis of PWS in primary hospitals.