The impact of age and number of mutations on the size of clonal hematopoiesis.

Clonal hematopoiesis (CH) represents the clonal expansion of hematopoietic stem cells and their progeny driven by somatic mutations. Accurate risk assessment of CH is critical for disease prevention and clinical decision-making. The size of CH has been showed to associate with higher disease risk, yet, factors influencing the size of CH are unknown. In addition, the characteristics of CH in long-lived individuals are not well documented. Here, we report an in-depth analysis of CH in longevous (≥90 y old) and common (60~89 y old) elderly groups. Utilizing targeted deep sequencing, we found that the development of CH is closely related to age and the expression of aging biomarkers. The longevous elderly group exhibited a significantly higher incidence of CH and significantly higher frequency of TET2 and ASXL1 mutations, suggesting that certain CH could be beneficial to prolong life. Intriguingly, the size of CH neither correlates significantly to age, in the range of 60 to 110 y old, nor to the expression of aging biomarkers. Instead, we identified a strong correlation between large CH size and the number of mutations per individual. These findings provide a risk assessment biomarker for CH and also suggest that the evolution of the CH is influenced by factor(s) in addition to age.

ETS1 acts as a regulator of human healthy aging via decreasing ribosomal activity.

Adaptation to reduced energy production during aging is a fundamental issue for maintaining healthspan or prolonging life span. Currently, however, the underlying mechanism in long-lived people remains poorly understood. Here, we analyzed transcriptomes of 185 long-lived individuals (LLIs) and 86 spouses of their children from two independent Chinese longevity cohorts and found that the ribosome pathway was significantly down-regulated in LLIs. We found that the down-regulation is likely controlled by ETS1 (ETS proto-oncogene 1), a transcription factor down-regulated in LLIs and positively coexpressed with most ribosomal protein genes (RPGs). Functional assays showed that ETS1 can bind to RPG promoters, while ETS1 knockdown reduces RPG expression and alleviates cellular senescence in human dermal fibroblast (HDF) and embryonic lung fibroblast (IMR-90) cells. As protein synthesis/turnover in ribosomes is an energy-intensive cellular process, the decline in ribosomal biogenesis governed by ETS1 in certain female LLIs may serve as an alternative mechanism to achieve energy-saving and healthy aging.

Transcriptome evidence reveals enhanced autophagy-lysosomal function in centenarians.

Centenarians (CENs) are excellent subjects to study the mechanisms of human longevity and healthy aging. Here, we analyzed the transcriptomes of 76 centenarians, 54 centenarian-children, and 41 spouses of centenarian-children by RNA sequencing and found that, among the significantly differentially expressed genes (SDEGs) exhibited by CENs, the autophagy-lysosomal pathway is significantly up-regulated. Overexpression of several genes from this pathway, CTSB, ATP6V0C, ATG4D, and WIPI1, could promote autophagy and delay senescence in cultured IMR-90 cells, while overexpression of the Drosophila homolog of WIPI1, Atg18a, extended the life span in transgenic flies. Interestingly, the enhanced autophagy-lysosomal activity could be partially passed on to their offspring, as manifested by their higher levels of both autophagy-encoding genes and serum beclin 1 (BECN1). In light of the normal age-related decline of autophagy-lysosomal functions, these findings provide a compelling explanation for achieving longevity in, at least, female CENs, given the gender bias in our collected samples, and suggest that the enhanced waste-cleaning activity via autophagy may serve as a conserved mechanism to prolong the life span from Drosophila to humans.

Familial longevity study reveals a significant association of mitochondrial DNA copy number between centenarians and their offspring.

Reduced mitochondrial function is an important cause of aging and age-related diseases. We previously revealed a relatively higher level of mitochondrial DNA (mtDNA) content in centenarians. However, it is still unknown whether such an mtDNA content pattern of centenarians could be passed on to their offspring and how it was regulated. To address these issues, we recruited 60 longevity families consisting of 206 family members (cohort 1) and explored their mtDNA copy number. The results showed that the first generation of the offspring (F1 offspring) had a higher level of mtDNA copy number than their spouses (p < 0.05) independent of a gender effect. In addition, we found a positive association of mtDNA copy number in centenarians with that in F1 offspring (r = 0.54, p = 0.0008) but not with that in F1 spouses. These results were replicated in another independent cohort consisting of 153 subjects (cohort 2). RNA sequencing analysis suggests that the single-stranded DNA-binding protein 4 was significantly associated with mtDNA copy number and was highly expressed in centenarians as well as F1 offspring versus the F1 spouses, thus likely regulates the mtDNA copy number in the long-lived family members. In conclusion, our results suggest that the pattern of high mtDNA copy number is likely inheritable, which may act as a favorable factor to familial longevity through assuring adequate energy supply.

CETP polymorphisms confer genetic contribution to centenarians of Hainan, south of China.

OBJECTIVE: In this paper, we will discuss if the CETP polymorphism contributes to the centenarians in Hainan island. METHODS: We tested the TaqIB and I405V polymorphisms of CETP gene among 276 centenarians and 301 matched healthy individuals by AS-PCR and analyzed the data with SPSS software package (Version 19.0). RESULTS: Our data indicated that allele B1 and V have the significant differences between centenarians and healthy control groups with P < 0.001. Further analysis implied that genotypes B1B1 (P < 0.001, OR = 0.148, 95% CI = 0.095-0.230) and VV (P < 0.001 and OR = 0.353, 95% CI = 0.237-0.525) were significantly different between centenarians and matched controls. The combination of B and V, such as B1B1-II (P < 0.001, OR = 0.128, 95% CI = 0.049-0.329), B1B1-IV (P < 0.001, OR = 0.115, 95% CI = 0.056-0.237), B1B2-VV (P < 0.05, OR = 0.534, 95% CI = 0.310-0.920), and B2B2-VV (P < 0.001, OR = 0.198, 95% CI = 0.086-0.453) have significant differences between centenarians and matched healthy individuals from Hainan. CONCLUSION: Our results implied that allele B1B1 and VV, as well as the combination B1B1-II, B1B1-IV, B1B2-VV and B2B2-VV may contribute to the longevity in centenarians of Hainan, south of China.

Mitochondrial DNA plays an equal role in influencing female and male longevity in centenarians.

The mitochondrion is a double membrane-bound organelle which plays important functional roles in aging and many other complex phenotypes. Transmission of the mitochondrial genome in the matrilineal line causes the evolutionary selection sieve only in females. Theoretically, beneficial or neutral variations are more likely to accumulate and be retained in the female mitochondrial genome during evolution, which may be an initial trigger of gender dimorphism in aging. The asymmetry of evolutionary processes between gender could lead to males and females aging in different ways. If so, gender specific variation loads could be an evolutionary result of maternal heritage of mitochondrial genomes, especially in centenarians who live to an extreme age and are considered as good models for healthy aging. Here, we tested whether the mitochondrial variation loads were associated with altered aging patterns by investigating the mtDNA haplogroup distribution and genetic diversity between female and male centenarians. We found no evidence of differences in aging patterns between genders in centenarians. Our results indicate that the evolutionary consequence of gender dimorphism in mitochondrial genomes is not a factor in the altered aging patterns in human, and that mitochondrial DNA contributes equally to longevity in males and females.

Improved lipids, diastolic pressure and kidney function are potential contributors to familial longevity: a study on 60 Chinese centenarian families.

Centenarians are a good healthy aging model. Interestingly, centenarians' offspring are prone to achieve longevity. Here we recruited 60 longevity families and investigated the blood biochemical indexes of family members to seek candidate factors associated with familial longevity. First, associations of blood indexes with age were tested. Second, associations of blood parameters in centenarians (CEN) with their first generation of offspring (F1) and F1 spouses (F1SP) were analyzed. Third, genes involved in regulating target factors were investigated. We found that total cholesterol (TC) and triglyceride (TG) increased with age (20-80 years), but decreased in CEN. Similarly, blood urea nitrogen (BUN) and blood creatinine (BCr) increased with age (20-80 years), but were maintained on a plateau in CEN. Importantly, we first revealed dual changes in blood pressure, i.e., decreased diastolic blood pressure but increased systolic blood pressure in CEN, which associated with altered CST3 expression. Genetic analysis revealed a significant association of blood uric acid (BUA) and BCr in CEN with F1 but not with F1SP, suggesting they may be heritable traits. Taken together, our results suggest serum lipids, kidney function and especially diastolic pressure rather than systolic pressure were improved in CEN or their offspring, suggesting these factors may play an important role in familial longevity.

Mitochondrial DNA content contributes to healthy aging in Chinese: a study from nonagenarians and centenarians.

Mitochondrial DNA (mtDNA) content plays an important role in energy production and sustaining normal physiological function. A decline in the mtDNA content and subsequent dysfunction cause various senile diseases, with decreasing mtDNA content observed in the elderly individuals with age-related diseases. In contrast, the oldest old individuals, for example, centenarians, have a delayed or reduced prevalence of these diseases, suggesting centenarians may have a different pattern of the mtDNA content, enabling them to keep normal mitochondrial functions to help delay or escape senile diseases. To test this hypothesis, a total of 961 subjects, consisting of 424 longevity subjects and 537 younger control subjects from Hainan and Sichuan provinces of China, were recruited for this study. The mtDNA content was found to be inversely associated with age among the age of group 40-70 years. Surprisingly, no reduction of mtDNA content was observed in nonagenarians and centenarians; instead, these oldest old showed a significant increase than the elderly people aged between 50 and 70 years. The results suggest the higher mtDNA content may convey a beneficial effect to the longevity of people through assuring sufficient energy supply.

Correlation between the Xba I polymorphism of apoB gene and serum lipid profiles in Li ethnic group.

OBJECTIVE: To study correlation between the Xba I polymorphism of apoB gene and plasma lipid profiles in Li ethnic group. METHODS: Total 151 cases of healthy Li people were recruited randomly by cluster sampling and 200 Han people were recruited as control; blood was drawn to analyze Xba I polymorphism distribution of apoB gene and serum lipid levels. RESULTS: There were lower serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in serum of Li people; while, high density lipoprotein cholesterol (HDL-C), X-/X+ genotype and X+ allele frequencies exhibited higher levels than Han people. Interestingly, HDL-C level was reduced, while LDL-C level was enhanced in subjects carrying heterozygous (X-/X+) genotype compared to homozygous (X-/X-) genotype. Additionally, there were no difference in serum level of triglyceride, TC, apoprotein A (apo A) and apoprotein B (apo B) between Li and Han people, the same results were showed between X-/X+ and X-/X- genotype carriers. CONCLUSIONS: Xba I polymorphism of apoB gene is correlated to the profiles of serum lipid level, X-/X+ genotype carriers are phenotyped with higher LDL-C level and lower level of HDL-C in Li ethnic group.

Absence of A673T variant in APP gene indicates an alternative protective mechanism contributing to longevity in Chinese individuals.

A673T, a rare variant in the amyloid-ß precursor protein gene, shows a protective potential against Alzheimer's disease (AD) and age-related cognitive decline in an Icelandic population. Although A673T was observed independently in a Finnish population, this variant was absent in 8721 Asian subjects. The conflicting observations suggest that the contribution of A673T may be confined to Europeans and Americans rather than Asians. Nevertheless, A673T confers a protective function against AD and thus may be observed only in longevity subjects; thus it is not surprising to see its absence when the general populations or the patient cohorts were considered. To test whether the A673T contributes to the Chinese population, 1237 healthy longevity subjects (mean age 96.9 years) and 1404 matched younger controls (mean age 44.2 years) were genotyped for the variant. Our study failed to observe this variant in either the longevity subjects or the controls. Given the previous observation from Asians, our results suggest that the A673T variant is not involved in longevity in Chinese individuals; some other protective mechanisms may contribute to a lower incidence of AD in Chinese nonagenerians and centenarians.

[Identification of a novel large deletion of factor subunit A mRNA associated with hereditary factor deficiency].

OBJECTIVE: To analyze the expressed mRNA of the factor subunit A (FA) in monocyte in a hereditary factor (F) deficiency family. METHODS: The F A mRNA of the proband and the other family members was analyzed by RT-PCR, semi-quantitative RT-PCR, cloning and sequencing. The three dimensional structure of the protein was predicted by SWISS-MODEL and viewed by RASMIOL. RESULTS: (1) A large in frame deletion from codons 11 to 279, spanning from exon 2 to 7 of F A (DelCD11-279), was identified in the proband at mRNA level and a truncated protein is predicted composed of 464 amino acids. Compared with the normal and the other families, the proband showed lower level of F A mRNA in RT-PCR. (2) SWISS-MODEL analysis showed that the truncated protein lacked the ß-sandwich and a part of catalytic core, resulting in loss of the normal catalytic domains. CONCLUSION: DelCD11-279 of F A mRNA is associated with hereditary F deficiency. The reduced expressing level of F A gene is one of the causes resulting in F deficiency in the patients.

E2F1 enhances 8-chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells.

The E2F1 transcription factor is a well known regulator of cell proliferation and apoptosis, but its role in response to DNA damage is less clear. 8-Chloro-adenosine (8-Cl-Ado), a nucleoside analog, can inhibit proliferation in a variety of human tumor cells. However, it is still elusive how the agent acts on tumors. Here we show that A549 and H1299 cells formed DNA double-strand breaks after 8-Cl-Ado exposure, accompanied by E2F1 upregulation at protein level. Overexpressed wild-type (E2F1-wt) colocalized with double-strand break marker γ-H2AX and promoted G2/M arrest in 8-Cl-Ado-exposed A549 and H1299, while expressed S31A mutant of E2F1 (E2F1-mu) significantly reduced ability to accumulate at sites of DNA damage and G2/M arrest, suggesting that E2F1 is required for activating G2/M checkpoint pathway upon DNA damage. Transfection of either E2F1-wt or E2F1-mu plasmid promoted apoptosis in 8-Cl-Ado-exposed cells, indicating that 8-Cl-Ado may induce apoptosis in E2F1-dependent and E2F1-independent ways. These findings demonstrate that E2F1 plays a crucial role in 8-Cl-Ado-induced G2/M arrest but is dispensable for 8-Cl-Ado-induced apoptosis. These data also suggest that the mechanism of 8-Cl-Ado action is complicated.

Evidence of gene conversion in the evolutionary process of the codon 41/42 (-CTTT) mutation causing beta-thalassemia in southern China.

The 4-bp deletion (-CTTT) at codon 41/42 (CD41/42) of the human beta-globin gene represents one of the most common beta-thalassemia mutations in East Asia and Southeast Asia, which is historically afflicted with endemic malaria, thus hypothetically evolving under natural selection by malaria infection. To understand the evolutionary process of generating the beta(CD41/42) allele and its maintenance, including the effect of natural selection on the pattern of linkage disequilibrium (LD), we sequenced a 15.933-kb region spanning 20.693 kb of the beta-globin cluster surrounding the 4-bp deletion using a sample from a Chinese population consisting of 24 normal individuals and 16 heterozygotes for the deletion. Forty-nine polymorphic sites were found. Analysis of the data, using a variety of methods including formal population genetics analysis and visual approaches, suggests that the spread of the CD41/42 (-CTTT) deletion is most likely mediated by interallelic gene conversion, although independent deletions in different lineages are also possible. The neutrality test resulted in a significant positive Tajima's D for the beta-globin locus, which is consistent with the existence of balancing selection. This suggests that the 4-bp deletion that occurred at this locus may be an event that is subject to natural selection, due to malaria, which leads to the heterozygote advantage, spread widely with further help by conversion and migration. The evolutionary process of this mutant through gene conversion that could conceivably take place between the 4-bp deletion and the normal sequence in the respective region is discussed in detail.

[Molecular characterization of 71 cases of glucose-6-phosphate dehydrogenase deficiency in Hainan province].

OBJECTIVE: To molecularly analyze in Han and Li individuals of glucose-6-phosphate dehydrogenase deficiency in Hainan, China. METHODS: The amplification refractory mutation system (ARMS) was employed to detect G1376T, G1388A and A95G mutations. The coding regions and flanking intronic regions from the second to the thirteenth exons of G6PD gene was analyzed by DNA sequencing to characterize the gene mutations in samples without G1376T, G1388A and A95G mutations. RESULTS: Among 29 Han cases of G6PD deficiency, 11 had G1376T (37.9%), 2 G1388A (6.9%), 1 G1376T and G1388A (3.4%) and 1 G1376T and A95G (3.4%) were identified. Mutations of G1376T, G1388A, A95G and their complex accounted for 51.7% of G6PD deficiency in the Han individuals. Among 42 Li cases of G6PD deficiency, 25 had G1376T (59.5%), 6 G1388A (14.3%), 2 A95G (4.8%), 4 G1376T and G1388A (9.5%), 1 G1376T and A95G (2.4% )were identified. These mutations accounted for 90.5% of the Li individuals. Gene mutation of 18 cases (14 Han and 4 Li individuals) remained unknown. Sequencing results of the 18 samples indicated that one case had a single base of T deletion at nucleotide 636 or 637 in the 5th intron (IVS-5 636 or 637 T del) and two cases had C1311T with IVS-11 T93C mutation. CONCLUSION: G6PD G1376T and G1388A are the most common mutations in the populations of the Han and Li nationalities in Hainan. The IVS-5 636 or 637 T del mutation is first reported in Chinese, and the complex mutation of G1376T/A95G is first found in the Li nationality.

Cyp2c19 genotype and omeprazole hydroxylation phenotype in Chinese Li population.

1. CYP2C19 is a polymorphism of cytochrome P450, which is responsible for the metabolism of many drugs. The genetic polymorphism shows interethnic variation and it has been demonstrated that the frequency of poor metabolizers (PM) and the distribution of alleles of CYP2C19 vary among Chinese ethnic nationalities. The aim of the present study was to investigate the incidence of CYP2C19 polymorphism in the Chinese Li population. 2. One hundred and sixty-five unrelated healthy Li subjects were identified with respect to CYP2C19 by genotype and phenotype analysis. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping. The plasma concentrations of omeprazole and 5-hydroxyomeprazole were assayed by reversed-phase high-performance liquid chromatography and the omeprazole hydroxylation index (HI) was determined. 3. The frequency distribution of omeprazole HI is bimodal and the antimode for HI was estimated to be 5.6. The prevalence of phenotypic PM in the Li population was 16.6% (13.7-19.5; 95% CI). Genotype analysis revealed that the frequencies of the CYP2C19*1, *2 and *3 alleles in the Li population were 0.617 (0.590-0.644; 95% CI), 0.353 (0.327-0.379; 95% CI) and 0.031 (0.021-0.041; 95% CI), respectively. The frequency of genotypic PM was 14.7% (11.9-17.5; 95% CI), which almost agreed with the frequency of phenotypic PM. Omeprazole HI was significantly different among the different genotype groups (P < 0.05). 4. The present study revealed that the incidence of the CYP2C19*1, *2 and *3 alleles in Chinese Li population is different to that in other ethnic populations of China. There was an obvious relationship between CYP2C19 genotype and omeprazole hydroxylation phenotype, and about 90% of phenotypic PM can be explained by the CYP2C19*2 and *3 alleles.

[Association of fibrinogen B beta -1420G/A, -993C/T and -854G/A gene polymorphism with coronary heart disease].

OBJECTIVE: To analyze the frequency of FGB gene -1420G/A, -993C/T and -854G/A polymorphisms, and their association with plasma fibrinogen levels in patients with coronary heart disease and in health adults. METHODS: The FGB gene -1420G/A, -993C/T and -854G/A polymorphisms were analyzed with restriction fragment length polymorphisms, polymerase chain reaction with allele-specific primer and nucleotide sequencing methods. Plasma fibrinogen levels were determined by turbidimetry. RESULTS: The frequencies of -1420A were 0.33 in patients with coronary heart disease and 0.26 in health adults. The frequencies of -1420A in coronary heart disease were apparently higher than that in health adults. There were no difference in frequencies of other two alleles. The logistic study suggested -1420G/A polymorphism was associated with coronary heart disease. There are significantly difference in plasma fibrinogen levels in two groups. Plasma fibrinogen levels were significantly increased in patients with coronary heart disease. CONCLUSION: This study suggests -1420G/A polymorphism may be associated with occurrence of coronary heart disease.

[A related analysis for alpha, beta fibrinogen gene haplotypes and nucleotide polymorphisms associated with the ischemic stroke in Hainan Han population].

OBJECTIVE: To analyze the association of that the polymorphisms and haplotypes of Taq I site in beta fibrinogen gene and the single nucleotide sites -455 G/A, -249 C/T, -148 C/T, +1689T/G, Bsm A I G/C, 448 G/A, Bcl I G/A, Hinf I A/C in beta-fibrinogen gene are linked up with the ischemic stroke(IS). METHODS: Turbidmetric assay was used to measure the plasma fibrinogen level of one hundred and sixty cases with ischemic stroke and one hundred and thirty healthy individuals from Hainanese Han population. The polymorphisms and genotypes were characterized by PCR-RFLP. Hardy-Weinberg equilibrium and statistical differences of allelic, genotype and haplotype frequencies were obtained by Chi-square test. Pairwise linkage disequilibrium was calculated and haplotypes of nine or four polymorphisms were estimated by the EH + program. RESULTS: There were highly significant differences in genotype frequencies and allelic frequencies of the polymorphisms -455 G/A, -148 C/T, 448 G/A, which happened between the IS group and control subjects (P< 0.01). However, the significant differences of the allelic frequencies in the other six polymorphisms were not found between the IS group and the control (P> 0.05). The odds ratio(OR) with the rare alleles of A -455, T -148 and A 448 is 2.46, 2.30 and 2.08 (95% confidence interval 1.153%-3.924%, 1.429%-3.694% and 1.298%-3.329%) respectively. No definite haplotype block was found by linkage disequilibrium analysis in the control group and the IS group. Association of haplotypes constructed from the nine polymorphisms with IS was not found. Among the haplotypes constructed from four polymorphisms including -455 G/A, -148 C/T, 448 G/A alleles, haplotype differences were found between the control group and the IS group. Haplotypes with G -455, C -148, G448 alleles appeared more frequently in control group(P< or = 0.01), whereas haplotypes with A -455, T -148, A 448 occurred more frequently in the IS group(P< 0.01). CONCLUSION: The results of multi-allele and haplotype analysis indicated that the polymorphisms -455 G/A, -148 C/T, 448 G/A in beta fibrinogen gene were the possible risk factors associated with the occurrence of ischemic stroke in Hainan Han population.