| Backgrounds:PSEN1,PSEN2 and APP are the causative genes of autosomal dominant inheritance familial Alzheimer’s disease(ADAD).ADAD with pathogenic/likely pathogenic variants shared similar clinical features with sporadic Alzheimer’s disease(AD)and had its unique clinical characteristics.As ADAD with mutation was relatively rare,previous researches on Chinese ADAD were mostly based on case reports,lacking a systematic study on the mutations of PSEN1,PSEN2,and APP and the clnical features of ADAD in a large AD cohort.Besides,previous studies have shown that the peripheral immune system of AD patients,including innate immunity and adaptive immunity,had changed.Current evidence showed that APP is closely related to immunity.However,the characteristics of peripheral immune cells in AD patients with pathogenic mutations in APP are still unclear.Objectives:To study the rare variants of PSEN1,PSEN2 and APP and clarify the pathogenic mutation spectrum in a large AD cohort from China.Genotype-phenotype correlation analysis was performed in ADAD patients with pathogenic/likely pathogenic mutations.Besiedes,the single-cell sequencing method was used to study the changes of immune cell composition and transcriptome of peripheral blood immune cells in familial AD(FAD)patients and pre-symptomatic AD(pre AD)cases with pathogenic variant in APP systematically.Methods:The targeted capture and sequencing method was used to sequence the exon and exon-intron junction regions of PSEN1,PSEN2 and APP genes in 1610 AD patients from China,and the rare variants were verified by Sanger sequencing.The pathogenicity of rare variants was classfied according to the American College of Medical Genetics and Genomics(ACMG)guidelines in 2015.The distribution of pathogenic/likely pathogenic variants in Chinese AD patients and the clinical characteristics and influencing factors of Chinese ADAD patients were further explored.Besides,peripheral blood mononuclear cells(PBMCs)from three FAD patients with APP pathogenic mutation,three pre AD subjects with APP pathogenic mutation and three healthy controls were extracted for single-cell RNA sequencing and T cell receptor(TCR)sequencing.Then we study the proportion and transcriptomics changes of various periphery immune cells in FAD patients and pre AD subjects systematacially.Enrichment analysis and protein-protein interaction analysis of differentially expressed genes were carried out to search for the key genes of differentially expressed genes.Results:1.In this study,a total of 55 rare variants of PSEN1,PSEN2 and APP were detected in 86 AD patients,of which 25 variants were found in AD cases for the first time.29 variants were classified as pathogenic or likely pathogenic variants according to the ACMG guidelines,including22 PSEN1 variants,four PSEN2 variants,and 3 APP variants,which were detected in 40 unrelated AD patients.And nine variants(PSEN1:c.451G>A;p.V151 M,PSEN1:c.500＿502del;p.I168 del,PSEN1:c.507＿509del;p.S169 del,PSEN1:c.802C>T;p.L268 F,PSEN1:c.1139A>G;p.K380 R,PSEN1:c.1300G>A;p.A434 T,PSEN1:c.1369A>G;p.M457 V,PSEN2:c.715T>C;p.M239 T,and PSEN2:c.1073-2＿1073-1del AG)were first identified as likely pathogenic mutations for AD by our team.2.Among the 40 patients with a pathogenic/likely pathogenic variant,34 were early-onset FAD,one was late-onset FAD,four were early-onset sporadic AD(SAD),and one was late-onset SAD.Among them,22 early-onset FAD cases,one late-onset FAD case,and two early-onset SAD cases carried PSEN1 mutations;six early-onset FAD patients,one early-onset SAD patient,and one late-onset SAD patient carried PSEN2 mutations;six early-onset FAD patients and one early-onset SAD patient carried APP variants.3.The frequencies of PSEN1,PSEN2,and APP pathogenic/likely pathogenic variants were 1.55%,0.50%,and 0.43%,respectively,in the entire AD cohort,4.89%,1.28%,and 1.28%,respectively,in the FAD cohort,0.18%,0.18%,and 0.09%,respectively,in the SAD cohort,2.94%,0.86%,and 0.86%,respectively,in the early-onset AD cohort,0.13%,0.13%,and 0.00%,respectively,in the late-onset AD cohort,and8.73%,2.38%,and 2.38%,respectively,in the early-onset FAD cohort.The onset-age of patients with a pathogenic/likely pathogenic variant was significantly earlier than those without a pathogenic/likely pathogenic variant(p < 0.001).4.A total of 52 ADAD patients,including 40 AD patients with pathogenic/likely pathogenic variants and another 42 AD patients from eight AD families,formed the ADAD cohort.The initial symptoms included memory loss(50/52)and abnormal mental behavior(2/52),other common symptoms were personality changes,speech disorders,mental behavior abnormalities,gait disorders,extrapyramidal symptoms,epilepsy,myoclonus,and cerebrovascular events.The incidence of myoclonus,epilepsy,and extrapyramidal symptoms was significantly lower than that in whites.5.The mean onset-ages of PSEN1,PSEN2 and APP were46.74±7.96 years,53.78±11.42 years and 48.56±5.83 years,respectively.No significant difference was found in the onset-age among the three genes(p > 0.05).The onset-age of ADAD patients with APOE ε4 allele were similar to those without APOE ε4 allele(p > 0.05).No statistical difference in the onset-age was observed between patients with PSEN1 mutation before codon 200 and patients with PSEN1 mutation after codon200(p > 0.05).6.A total of 88146 high-quality cells were identified from nine samples,which can be divided into eight cell types according to the marker genes of peripheral immune cells,including 51949 T cells,14261 monocytes,13209 natural killer(NK)cells,7082 B cells,915monocyte-derived dendritic cells(m DC),316 plasmacyte-derived dendritic cells,235 megakaryocytes,and 179 mast cells.Compared with the control group,the proportions of monocytes,B cells,and m DC in the FAD group were significantly different(p = 0.049,p< 0.001,and p =0.024,respectively),and the proportions of T cells,NK cells,and B cells in the pre AD group were significantly different(p < 0.001,p < 0.001,and p = 0.011,respectively).Besides,significant differences were observed in the proportions of T cells,monocytes,NK cells,and B cells between FAD group and pre AD group(p < 0.001,p = 0.002,p < 0.001,and p <0.001,respectively).7.Furthermore,T cells were divided into 10 subpopulations,monocytes were divided into three subpopulations,NK cells were divided into two subpopulations,and B cells were divided into four subpopulations according to the different marker genes.The proportions of four T cell subsets and one monocyte subset in the FAD group were significantly different from those in the control group(p < 0.05).There were statistical differences in the proportions of four T cell subsets and one B cell subset between the pre AD group and the control group(p <0.05).And significant differences were found in the proportions of three T cell subsets and two monocyte subsets between the pre AD group and the FAD group(p < 0.05).8.A total of 274 differentially expressed genes were identified among the T cells,monocytes,NK cells,B cells and m DC when comparing the FAD group to the control group;312 differentially expressed genes existed between the pre AD group and the control group and 136 differentially expressed genes existed between the FAD group and the pre AD group,most of the differentially expressed genes are unique to a certain cell type.Pathway enrichment analysis showed that differentially expressed genes were mainly enriched in infection-related pathways and autoimmune disease-related pathways,which were mainly driven by the HLA genes.9.Compared with the control group,the FAD and pre AD groups shared 167 differentially expressed genes with consistent trend.Protein-protein interaction analysis shows that HLA molecules,mitochondrial proteins encoded by mitochondrial genes,and S100 protein family may be the key molectures of differentially expressed genes.Conclusions:1.In the cohorts of AD,early-onset AD,FAD,and early-onset FAD,the frequencies of pathogenic/likely pathogenic variants in the AD causative genes were 2.48%,4.66%,7.45%,and 13.49%,respectively.Genetic testing should be recommended for early-onset AD patients and AD patients who have a clear positive family history.2.The most common causative gene in Chinese AD population was PSEN1,followed by PSEN2 and APP.3.Most of the Chinese ADAD cases with pathogenic/likely pathogenic variants started with episodic memory impairment,and some initially presented with psychological and behavioral abnormalities.4.Compared with the control group,various immune cells in the PBMCs were dysregulated in both the pre AD and FAD patients with APP pathogenic variants,including a consistent decrease in the proportion of B cells.5.Compared with healthy controls,the transcription of a variety of peripheral immune cells in the FAD patients and the pre AD subjects was changed.Most of the differentially expressed genes were cell-type specific,and the differentially expressed genes were mainly enriched in infection-related pathways and autoimmune diseases related pathways,which were mainly driven by the HLA genes.35 figures,38 tables,196 references... |
