| Objective(s): Autism is a highly hereditary and heterogeneous neurodevelopmental disorder with over 1000 related genes.Its principal clinical symptoms include delayed language development,poor social skills,restricted interests,repeated stereotyped behaviors,and a variety of severely harmful behaviors.Currently,there is no other effective treatment besides behavioral intervention.Epidemiology studies have suggested that advanced paternal age is an independent risk factor for autism,but the exact mechanism is unknown.The accumulation of de novo mutations in the germline is a widely recognized view,but it can only explain some of the risk mechanisms in aging fathers with autism.In this study,we conducted a whole exon sequence(WES)of genomic DNA from sperm and peripheral blood of healthy men of different ages.Through paired sample comparisons,we analyzed the patterns and characteristics of changes in sperm de novo mutations and inherited mutations with age,and explored the sperm-originating mechanism of autism in offspring caused by aged fathers.Methods: Twenty healthy pre-pregnancy-aged(> 40 years)and young(< 30years)men’s semen and peripheral blood were sampled,and the semen was centrifuged using a non-continuous density gradient to remove non-sperm cell components.Genomic DNA was extracted separately for WES,and results of single nucleotide polymorphisms(SNPs),insertions and deletions(Indels),structural variations(SVs),fusion genes(FGs),and copy number variations(CNVs)were obtained for two types of cells.By comparing sequencing data,these mutations were divided into sperm-only mutations(sperm de novo mutations),blood-only mutations,and shared mutations(inherited mutations).The differences in semen parameters between the two groups were compared,and their potential association with sperm de novo mutations was analyzed.We analyzed the distribution of mutations in functional regions of the genome and characteristics of de novo mutations and inheritance mutations in different types of sperm with age,as well as the mutation frequency distribution of mosaic single nucleotide variants(SNVs)in two groups of populations.To investigate the change pattern and characteristics of autism-related genes SVs with age,we examined the mutation trend of sperm de novo and inherited autism-related genes with age and compared the trend of mutation rate of different disease-related genes with age,including autism-related genes,neurodevelopmental genes(NDG),and genome-wide genes.The epigenetic causes of positive selection pressure on autism gene mutations were analyzed.According to our previous sperm DNA methylation group sequencing results,autism genes were divided into genes related to differential methylation regions(DMR)and non-DMR-related genes,and the mutation rates of the two types of genes were compared.Results:(1)Basic characteristics and sperm parameters were compared between the two groups,in addition to the influence on mutation: The average age difference between the aged men(43.10 ± 2.02 years)and the young men(25.00 ± 1.89years)was approximately 18.10 ± 0.13 years(P < 0.001);the average marriage age of aged men was higher than that of young men(33.43 ± 6.50 years vs.23.90 ± 2.02 years,P < 0.001).In the two groups,there were no significant differences in sperm density,activity rate,progressive rate,normal morphology rate,and acrosin activity.In comparison to the young group,the aged group’s DNA fragmentation index(DFI)of sperm was significantly higher(18.80% ± 4.47% vs.12.22% ± 2.33%,P = 0.001).There was no significant correlation between the changes in main semen parameters within the normal range and the number of de novo gene mutations in sperm(P > 0.05),while inheritance mutations were not correlated with the age of men at the time of sample collection(P > 0.05)but were positively correlated with the age of marriage(P<0.05).(2)Distribution of mutations in functional regions of the genome and characteristics of mutation distribution: deletion mutations(55.44%)made up the largest portion of sperm genome in various types of FGs,followed by insertion mutations(26.49%),and the remainder made up a relatively small portion.SVs also showed deletions(60.67%),followed by insertion mutations(33.18%),and a relatively small portion of the rest.SVs are mainly distributed in introns(60.97%),exons(16.17%),and inter gene regions(7.20%),with less distribution in other locations.Among the two groups,the de novo mutation characteristics of sperm,FGs and SVs deletion mutations in aged men were significantly higher than those in young men(P <0.05);only the SVs of aged males on introns were significantly higher than those of young males(P < 0.05).(3)The characteristics of de novo mutations in different types of sperm with age:both FGs and SVs demonstrated an increased tendency with advancing age,with an average increase of 0.8 and 1.8 mutations per year,respectively.The frequency of de novo pathogenic single nucleotide mutations(SNVs)in mosaic form in aged men was higher than that in young men.(4)Patterns and characteristics of changes in autism-related genes SVs with age:sperm de novo mutations showed a significant increasing trend with age(P < 0.05);inheritance mutations showed an upward trend with the increase in marriage age,but there was no statistical significance(P > 0.05);the mutation rate of NDG was significantly higher than that of whole genome genes,and the mutation rate of autismrelated genes was significantly higher than that of NDG;in addition,the mutation rate of DMR-related autism genes is significantly higher than that of total autism genes and non-DMR-related autism genes.Conclusion(s): The whole genome and de novo mutations in the sperm origin of autism-related genes accumulate with age,while the accumulation rate of autismrelated genes is faster than the whole genome,which may be attributed to the susceptibility to DNA methylation mutations in autism-related genes.Inheritance mutations were not associated with the age of the male at the time of sample collection but were associated with the age of marriage.Our results,while further clarifying the cumulative mechanism of age and sperm-originating de novo mutations,provide direct evidence for another possible mechanism of the age effect in fathers with autism: while males with autism susceptibility mutations do not show the typical clinical symptoms,they impact the marriage or reproductive age,postpone the reproductive age,and pass the genes to their children,increasing the risk of autism in their offspring.Our research provides innovative research ideas and diagnostic and therapeutic evidence for autism in aged paternal offspring and other paternal sperm-originating offspring diseases.In the future,it will be necessary to further expand the sample size and enrich the characteristics of subjects so as to further study the correlation and mechanism of the occurrence of diseases in aged fathers and offspring. |
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