Age-related bone loss is associated with FGF21 but not IGFBP1 in healthy adults.

What is the central question of this study? Fibroblast growth factor 21 (FGF21) plays important therapeutic roles in metabolic diseases but is associated with bone loss, through insulin-like growth factor binding protein 1 (IGFBP1), in animals. However, the effect of the FGF21-IGFBP1 axis on age-related bone loss has not been explored in humans. What is the main finding and its importance? Using 'genetically linked' parent and child family pairs, we show that the FGF21 concentration, but not the IGFBP1 concentration, is higher in older than in younger adults. Our results suggest that age-associated decline in bone mineral density is associated with FGF21 and increased bone turnover but not likely to involve IGFBP1 in healthy humans. ABSTRACT: Bone fragility increases with age. The fibroblast growth factor 21 (FGF21)-insulin-like growth factor binding protein 1 (IGFBP1) axis regulates bone loss in animals. However, the role of FGF21 in mediating age-associated bone fragility in humans remains unknown. The purpose of this study was to explore the FGF21-regulatory axis in bone turnover and the age-related decline in bone mineral density (BMD). Twenty 'genetically linked' family (parent and child) pairs were recruited. Younger adults were 22-39 years old and older adults 60-71 years old. The BMD and serum concentrations of FGF21, IGFBP1, receptor activator of nuclear factor-κB ligand (RANKL), tartrate-resistant acid phosphatase 5b (TRAP5b) and bone-specific alkaline phosphatase (BAP) were measured. Older adults had 10-18% lower BMD at the hip and spine (P < 0.008) and a twofold higher FGF21 concentration (P < 0.001). The IGFBP1 concentration was similar in younger and older adults (P = 0.961). The RANKL concentration was 44% lower (P = 0.006), whereas TRAP5b and BAP concentrations were 36 and 31% higher (P = 0.01 and P = 0.004), respectively, in older adults than in younger adults. Adjusting for sex did not affect these results. The FGF21 concentration was negatively correlated with BMD at the spine (r = -0.460, P = 0.003), but not with the IGFBP1 concentration (r = -0.144, P = 0.374). The IGFBP1 concentration was not correlated with BMD at the hip or spine (all P > 0.05). In humans, FGF21 might be involved in the age-associated decline in BMD, especially at the spine, through increased bone turnover. IGFBP1 is unlikely to be the downstream effector of FGF21 in driving the age-associated decline in BMD and in RANKL-associated osteoclast differentiation.

Complex Copy Number Variation of AMY1 does not Associate with Obesity in two East Asian Cohorts.

The human amylase gene locus at chromosome 1p21.1 is structurally complex. This region contains two pancreatic amylase genes, AMY2B, AMY2A, and a salivary gene AMY1. The AMY1 gene harbors extensive copy number variation (CNV), and recent studies have implicated this variation in adaptation to starch-rich diets and in association to obesity for European and Asian populations. In this study, we showed that by combining quantitative PCR and digital PCR, coupled with careful experimental design and calibration, we can improve the resolution of genotyping CNV with high copy numbers (CNs). In two East Asian populations of Chinese and Malay ethnicity studied, we observed a unique non-normal distribution of AMY1 diploid CN genotypes with even:odd CNs ratio of 4.5 (3.3-4.7), and an association between the common AMY2A CN = 2 genotype and odd CNs of AMY1, that could be explained by the underlying haplotypic structure. In two further case-control cohorts (n = 932 and 145, for Chinese and Malays, respectively), we did not observe the previously reported association between AMY1 and obesity or body mass index. Improved methods for accurately genotyping multiallelic CNV loci and understanding the haplotype complexity at the AMY1 locus are necessary for population genetics and association studies.

Genome-wide association study identifies ZFHX1B as a susceptibility locus for severe myopia.

Severe myopia (defined as spherical equivalent < -6.0 D) is a predominant problem in Asian countries, resulting in substantial morbidity. We performed a meta-analysis of four genome-wide association studies (GWAS), all of East Asian descent totaling 1603 cases and 3427 controls. Two single nucleotide polymorphisms (SNPs) (rs13382811 from ZFHX1B [encoding for ZEB2] and rs6469937 from SNTB1) showed highly suggestive evidence of association with disease (P < 1 × 10(-7)) and were brought forward for replication analysis in a further 1241 severe myopia cases and 3559 controls from a further three independent sample collections. Significant evidence of replication was observed, and both SNP markers surpassed the formal threshold for genome-wide significance upon meta-analysis of both discovery and replication stages (P = 5.79 × 10(-10), per-allele odds ratio (OR) = 1.26 for rs13382811 and P = 2.01 × 10(-9), per-allele OR = 0.79 for rs6469937). The observation at SNTB1 is confirmatory of a very recent GWAS on severe myopia. Both genes were expressed in the human retina, sclera, as well as the retinal pigmented epithelium. In an experimental mouse model for myopia, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for Zfhx1b and Sntb1. These new data advance our understanding of the molecular pathogenesis of severe myopia.

Detection of hemi/homozygotes through heteroduplex formation in high-resolution melting analysis.

Heteroduplex formation, required for the complete detection of hemi/homozygotes using high-resolution melting analysis, can be induced either by pre-PCR mixing of genomic DNAs or by post-PCR mixing of PCR products from unknown and reference samples. This study investigates the effects of both methods using two single nucleotide polymorphisms in X-linked DMD gene. The results show that both methods resulted in the same effect when mixing samples with the same gene copy number. Mixing samples with different gene copy numbers has not been previously explored and we show that post-PCR mixing is insensitive to gene copy number differences as compared to pre-PCR mixing.

Fibroblast Growth Factor 21 Mediates the Associations between Exercise, Aging, and Glucose Regulation.

INTRODUCTION: Aging increases the prevalence of glucose intolerance, but exercise improves glucose homeostasis. The fibroblast growth factor 21 (FGF21)-adiponectin axis helps regulate glucose metabolism. However, the role of FGF21 in mediating glucose metabolism with aging and exercise remains unknown. PURPOSE: This study examined whether FGF21 responses to a glucose challenge are associated with habitual exercise, aging and glucose regulation. METHODS: Eighty age- and sex-matched healthy individuals were assigned to young sedentary and active (≤36 yr, n = 20 each group) and older sedentary and active (≥45 yr, n = 20 each group) groups. Fasted and postprandial blood glucose concentration and plasma concentration of insulin, FGF21, and adiponectin were determined during an oral glucose tolerance test (OGTT). RESULTS: During the OGTT, glucose concentrations were 9% higher (P = 0.008) and FGF21 concentrations were 58% higher (P = 0.014) in the older than the younger group, independent of activity status. Active participants had 40% lower insulin concentration and 53% lower FGF21 concentration than sedentary participants, independent of age (all P < 0.001). Adiponectin concentration during the OGTT did not differ by age (P = 0.448) or activity status (P = 0.611). Within the younger group, postprandial glucose, insulin and FGF21 concentrations during the OGTT were lower in active than in sedentary participants. In the older group, only postprandial insulin and FGF21 concentrations were lower in active participants. CONCLUSIONS: FGF21, but not adiponectin, response during the OGTT is higher in older than younger adults and lower in active than sedentary individuals. Exercise-associated reduction in OGTT glucose concentrations was observed in younger but not older adults.

Pulse Wave Velocity Is Associated with Increased Plasma oxLDL in Ageing but Not with FGF21 and Habitual Exercise.

Fibroblast growth factor 21 (FGF21) and adiponectin increase the expression of genes involved in antioxidant pathways, but their roles in mediating oxidative stress and arterial stiffness with ageing and habitual exercise remain unknown. We explored the role of the FGF21-adiponectin axis in mediating oxidative stress and arterial stiffness with ageing and habitual exercise. Eighty age- and sex-matched healthy individuals were assigned to younger sedentary or active (18-36 years old, n = 20 each) and older sedentary or active (45-80 years old, n = 20 each) groups. Arterial stiffness was measured indirectly using pulse wave velocity (PWV). Fasted plasma concentrations of FGF21, adiponectin and oxidized low-density lipoprotein (oxLDL) were measured. PWV was 0.2-fold higher and oxLDL concentration was 25.6% higher (both p < 0.001) in older than younger adults, despite no difference in FGF21 concentration (p = 0.097) between age groups. PWV (p = 0.09) and oxLDL concentration (p = 0.275) did not differ between activity groups but FGF21 concentration was 9% lower in active than sedentary individuals (p = 0.011). Adiponectin concentration did not differ by age (p = 0.642) or exercise habits (p = 0.821). In conclusion, age, but not habitual exercise, was associated with higher oxidative stress and arterial stiffness. FGF21 and adiponectin did not differ between younger and older adults, meaning that it is unlikely that they mediate oxidative stress and arterial stiffness in healthy adults.

Airway microbiome composition correlates with lung function and arterial stiffness in an age-dependent manner.

OBJECTIVE: To investigate age-associated changes in airway microbiome composition and their relationships with lung function and arterial stiffness among genetically matched young and elderly pairs. METHODS: Twenty-four genetically linked family pairs comprised of younger (≤40 years) and older (≥60 years) healthy participants were recruited (Total n = 48). Lung function and arterial stiffness (carotid-femoral pulse wave velocity (PWV) and augmentation index (AIx)) were assessed. Sputum samples were collected for targeted 16S rRNA gene amplicon sequencing and correlations between microbiome composition, lung function and arterial stiffness were investigated. RESULTS: Elderly participants exhibited reductions in lung function (FEV1 (p<0.001), FVC (p<0.001) and percentage FEV1/FVC (p = 0.003)) and a 1.3-3.9-fold increase in arterial stiffness (p<0.001) relative to genetically related younger adults. Elderly adults had a higher relative abundance of Firmicutes (p = 0.035) and lower relative abundance of Proteobacteria (p = 0.014), including specific genera Haemophilus (p = 0.024) and Lautropia (p = 0.020) which were enriched in the younger adults. Alpha diversity was comparable between young and elderly pairs (p>0.05) but was inversely associated with lung function (FEV1%Predicted and FVC %Predicted) in the young (p = 0.006 and p = 0.003) though not the elderly (p = 0.481 and p = 0.696). Conversely, alpha diversity was negatively associated with PWV in the elderly (p = 0.01) but not the young (p = 0.569). Specifically, phylum Firmicutes including the genus Gemella were correlated with lung function (FVC %Predicted) in the young group (p = 0.047 and p = 0.040), while Fusobacteria and Leptotrichia were associated with arterial stiffness (PWV) in the elderly (both p = 0.004). CONCLUSION: Ageing is associated with increased Firmicutes and decreased Proteobacteria representation in the airway microbiome among a healthy Asian cohort. The diversity and composition of the airway microbiome is independently associated with lung function and arterial stiffness in the young and elderly groups respectively. This suggests differential microbial associations with these phenotypes at specific stages of life with potential prognostic implications.

Linear analogues of human beta-defensin 3: concepts for design of antimicrobial peptides with reduced cytotoxicity to mammalian cells.

A series of engineered linear analogues [coded as F6, W6, Y6, A6, S6 and C(Acm)6] were modeled, designed, synthesized and structurally characterized by mass spectra, circular dichroism, hydrophobicity analysis and molecular modeling. We have screened antimicrobial activity, hemolysis to rabbit erythrocytes, and cytotoxicity to human conjunctival epithelial cells. No significant hemolytic effect was observed for hBD3 or from five of the six analogues [F6, Y6, A6, S6 and C(Acm)6] over the range of 3-100 microg mL(-1). The six linear analogues have reduced cytotoxicity to human conjunctival epithelial cells over the range of 6-100 microg mL(-1) compared to hBD3. By tuning the overall hydrophobicity of linear hBD3 analogues, reduced cytotoxicity and hemolysis were obtained while preserving the antimicrobial properties. The decreased cytotoxicity of the linear analogues is suggested to be structurally related to the removal of disulfide bridges, and the flexible structure of the linear forms, which seem to be associated with loss of secondary structure. These results suggest a new approach for guiding the design of new linear analogues of defensin peptides with strong antibiotic properties and reduced cytotoxicity to mammalian cells.

Two models of experimental myopia in the mouse.

PURPOSE: The purpose of this study was to test the response of the mouse eye to two methods for the induction of experimental myopia. METHODS: Growth patterns of eyes were determined by axial length measurements from birth to adult in eyes of both sexes of normal mice examined on post-natal day 1 to 6 months and at 1 year. For the induction of experimental myopia, Balb/cJ mice were prepared with either unilateral lid suture or by a -10D spectacle lens placed over one eye at post-natal day 10. Other mice received a plano lens as a control for lens wear. Refraction was carried out at post-natal days of 28, 42 and 56 in lid suture and spectacle lens wear group by streak retinoscopy. Axial length was measured by a combination of video image photography, digital caliper, or Optical Low Coherence Interferometry (OLCI). Corroborative optical modeling of the mouse eye was carried out using ZEMAX ray tracing software. RESULTS: Axial length (AL) increased linearly between post-natal day 1 to day 56, plateauing at about 140 days. After 18 days of unilateral lid suture initiated 10 days after birth, the AL of experimental eyes was 3.032+/-0.003 mm, while AL in contra-lateral control eyes was 2.981+/-0.005 mm (mean+/-sem, p<0.05, n=40), after 32 days, the AL of experimental eyes was 3.290+/-0.004 mm, and the AL of control eyes was 3.104+/-0.002 mm (p<0.001, n=60). After 46 days of lid closure AL of experimental eyes was 3.592+/-0.003 mm, while AL of control eyes was 3.363+/-0.003 mm (p<0.001, n=80). Spectacle lens wear of 46 days duration increased AL in experimental eyes to 3.721+/-0.002 mm, while AL in control eyes was 3.354+/-0.003 mm (p<0.001, n=100). Refraction and ray tracing analysis substantiated the dimensional changes to be consistent with increased AL. CONCLUSIONS: Two procedures to induce experimental myopia, initiated at eye opening, produced significant myopic shifts corresponding to increases in axial lengths after 32 and 46 days of lid suture and after 46 days wearing a -10D spectacle lens.

Polymorphism studies of four miniSTR loci for three ethnic populations in Singapore.

MiniSTR loci has demonstrated to be an effective approach to recover genetic information from degraded sample, due to the improved PCR efficiency of their reduced PCR amplicon sizes. This study constructed a partial miniSGM panel and investigated the performance of four miniSTR loci, D2S1338, D16S539, D18S51 and FGA, in three ethnic populations residing in Singapore. The suitability of the miniSTR primers for Singapore populations was assessed for loci D16S539, D18S51 and FGA.

Presence of Burkholderia pseudomallei in Soil and Paddy Rice Water in a Rice Field in Northeast Thailand, but Not in Air and Rainwater.

Environmental Burkholderia pseudomallei has been postulated to be aerosolized during ploughing and heavy rain, and could result in inhalational melioidosis. Here, we determined the presence of B. pseudomallei in soil, paddy field water (PFW), air, and rainwater samples in a single rice paddy field in Ubon Ratchathani, northeast Thailand. In 2012, we collected 100 soil samples during the dry season, 10 PFW samples during the monsoon season, 77 air samples during ploughing (N = 31) and heavy rains (N = 46), and 60 rainwater samples during 12 rain events. We found that 32 soil samples (32%), six PFW samples (60%), and none of the air and rainwater samples were culture positive for B. pseudomallei. Other soil bacteria were isolated from air and rainwater samples. Mean quantitative count of B. pseudomallei estimated from two culture-positive PFW samples was 200 colony forming units/mL. Our findings suggest that the risk of melioidosis acquisition by inhalation in Thailand might be low.

Mu opioid receptor gene polymorphisms and heroin dependence in Asian populations.

The distribution of three polymorphisms of the mu opioid receptor gene (OPRM1) was investigated in four different Asian populations, and in heroin-dependent subjects deriving from three of these populations. For the A118G polymorphism, we found significant differences in allele and genotype frequencies between different ethnic groups and highly significant association with heroin dependence in Indians for both genotype distribution (p = 0.024) and allele frequency (p = 0.009). For the C17 T polymorphism, the minor allele was documented in Chinese and Malays for the first time. Molecular haplotyping revealed complete linkage dis-equilibrium between the A118G and C17 T polymorphisms. Linkage disequilibrium between the A118G and C1031G polymorphisms was found to be almost complete in all four ethnic groups.

Identification of a novel repetitive DNA element and its use as a molecular marker for strain typing and discrimination of ara- from ara+ Burkholderia pseudomallei isolates.

A novel 10-bp repeat (5'-CGACGCAGGC-3')34 was identified in a strain of Burkholderia pseudomallei, the first repetitive element found in this species. A pair of primers, based on the flanking sequences of the repetitive region, was used in PCR and DNA sequence analysis to determine the genomic structure and distribution of the repetitive element in 76 arabinose- (ara-) and 7 ara+ B. pseudomallei isolates. DNA fragments of 400-700 bp were amplified in all ara- isolates. Ara+ isolates were characterised by a uniform fragment of 402 bp. Nucleotide sequence analysis of these fragments revealed broad heterogeneity of the variable-number tandem repeats with 26 distinct alleles ranging between (5'-CGACGCAGGC-3')13 and (5'-CGACGCAGGC-3')45 identified in the ara- isolates. In contrast, a novel non-repetitive sequence was identified in each of the ara+ isolates. This was confirmed by Southern blot analysis. Such biotype-specific variable-number tandem repeats may be useful as genetic markers for rapid strain differentiation of ara- isolates.

Association between an intronic apolipoprotein E polymorphism and bone mineral density in Singaporean Chinese females.

INTRODUCTION: Apolipoprotein E (ApoE) is implicated in the pathogenesis of osteoporosis. OBJECTIVE: To investigate possible association of the non-classical APOE gene +113C/G (rs440446) intron 1 enhancer polymorphism with bone mineral density (BMD) in a homogeneous Chinese population in Singapore. METHODS: A total of 655 volunteers, males and females, aged between 31 and 72 years, from the public participated. BMD was measured using dual-energy X-ray absorptiometry and APOE +113C/G (rs440446) genotypes were determined by Sequenom MassARRAY system. To adjust for potential confounders, anthropometric, demographic, and lifestyle determinants were obtained, and serum lipids and E(2) were measured. RESULTS: The +113C/G (rs440446) polymorphism within the APOE gene was associated with BMD in Chinese Singaporean females only. Females with the heterozygous CG genotype were significantly associated with reduced total, lumbar spine, and femoral neck of hip BMD, after multilevel adjustment of confounders. The association was stronger in the spine than in the hip. When females were stratified according to WHO classification for osteoporosis, those with CG and GG genotypes had increased risk (OR 3.50 and 2.22, respectively) of developing osteopenia/osteoporosis in the lumbar spine. Serum lipids did not explain the influence of APOE +113 C/G (rs440446) on BMD. CONCLUSION: This study demonstrated an association between APOE +113C/G (rs440446) polymorphism with measures of BMD in Singaporean Chinese females.

Polymorphism studies of six miniSTR loci for three ethnic populations in Singapore.

MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimen, because of the improved PCR efficiency of their reduced PCR amplicon sizes. This study constructed two miniSTR panels comprising six miniSTR loci, including D2S1776, D3S4529, D6S474, D9S2157, D10S1435 and D12ATA63. The allele frequency distribution, forensic parameters and heterozygosity in three Singapore populations are reported in this paper. All six loci showed relatively high polymorphism with observed heterozygosity > 0.7. However, many of these six loci also demonstrated significant population differentiation for the three populations.

Molecular characterization of a polymorphic 3-Mb deletion at chromosome Yp11.2 containing the AMELY locus in Singapore and Malaysia populations.

Amelogenin paralogs on Chromosome X (AMELX) and Y (AMELY) are commonly used sexing markers. Interstitial deletion of Yp involving the AMELY locus has previously been reported. The combined frequency of the AMELY null allele in Singapore and Malaysia populations is 2.7%, 0.6% in Indian and Malay ethnic groups respectively. It is absent among 541 Chinese screened. The null allele in this study belongs to 3 Y haplogroups; J2e1 (85.7%), F* (9.5%) and D* (4.8%). Low and high-resolution STS mapping, followed by sequence analysis of breakpoint junction confirmed a large deletion of 3 to 3.7-Mb located at the Yp11.2 region. Both breakpoints were located in TSPY repeat arrays, suggesting a non-allelic homologous recombination (NAHR) mechanism of deletion. All regional null samples shared identical breakpoint sequences according to their haplogroup affiliation, providing molecular evidence of a common ancestry origin for each haplogroup, and at least 3 independent deletion events recurred in history. The estimated ages based on Y-SNP and STR analysis were approximately 13.5 +/- 3.1 kyears and approximately 0.9 +/- 0.9 kyears for the J2e1 and F* mutations, respectively. A novel polymorphism G > A at Y-GATA-H4 locus in complete linkage disequilibrium with J2e1 null mutations is a more recent event. This work re-emphasizes the need to include other sexing markers for gender determination in certain regional populations. The frequency difference among global populations suggests it constitutes another structural variation locus of human chromosome Y. The breakpoint sequences provide further information to a better understanding of the NAHR mechanism and DNA rearrangements due to higher order genomic architecture.

Real-time fluorescence-based PCR for detection of malaria parasites.

A fluorescence-based real-time 5' nuclease PCR capable of detecting all four human malaria parasites was developed to screen large numbers of samples during an outbreak to prevent further transmission of malaria. The effectiveness of antimalarial therapy for malaria patients can be monitored by determining the reduction of parasitemia by this method.

A novel susceptibility locus on chromosome 2 in the (New Zealand Black x New Zealand White)F1 hybrid mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is inherited as a complex polygenic trait. (New Zealand Black (NZB) x New Zealand White (NZW)) F(1) hybrid mice develop symptoms that remarkably resemble human SLE, but (NZB x PL/J)F(1) hybrids do not develop lupus. Our study was conducted using (NZW x PL/J)F(1) x NZB (BWP) mice to determine the effects of the PL/J and the NZW genome on disease. Forty-five percent of BWP female mice had significant proteinuria and 25% died before 12 mo of age compared with (NZB x NZW)F(1) mice in which >90% developed severe renal disease and died before 12 mo. The analysis of BWP mice revealed a novel locus (chi(2) = 25.0; p < 1 x 10(-6); log of likelihood = 6.6 for mortality) designated Wbw1 on chromosome 2, which apparently plays an important role in the development of the disease. We also observed that both H-2 class II (the u haplotype) and TNF-alpha (TNF(z) allele) appear to contribute to the disease. A suggestive linkage to proteinuria and death was found for an NZW allele (designated Wbw2) telomeric to the H-2 locus. The NZW allele that overlaps with the previously described locus Sle1c at the telomeric part of chromosome 1 was associated with antinuclear autoantibody production in the present study. Furthermore, the previously identified Sle and Lbw susceptibility loci were associated with an increased incidence of disease. Thus, multiple NZW alleles including the Wbw1 allele discovered in this study contribute to disease induction, in conjunction with the NZB genome, and the PL/J genome appears to be protective.