Genes associated with antibody-dependent cell activation are overexpressed in renal biopsies from patients with antibody-mediated rejection.

INTRODUCTION: Antibody-mediated rejection (ABMR) is dependent on complement activating donor-specific anti-HLA antibodies (DSA). This is commonly detected by C4d deposition in allografts. However, recent data define a C4d negative ABMR phenotype suggesting a role for complement-independent DSA injury, antibody-dependent cellular cytotoxicity (ADCC). METHODS: Here, we established an in vitro ADCC model that identified human ADCC-activated genes using microarray analysis. We subsequently interrogated renal allograft biopsies from patients with ABMR and controls for mRNA expression of the ADCC-activated gene set. RESULTS: We identified 13 ADCC-activated genes. Six gene expression assays including 8 of the 13 genes (CCL3, CCL4/CCL4L1/CCL4L2, CD160, IFNG, NR4A3 and XCL1/XCL2) were analyzed in 127 kidney biopsies obtained from HLA-sensitized (HS), non-HS patients and control individuals. Most ADCC-activated genes showed significantly higher expression in the transplant samples compared to the controls (p<0.0005). The gene expression levels were significantly higher in HS and non-HS transplant patients who developed ABMR compared to those who did not (p=0.04-0.002). There was no difference in the gene expression levels between C4d positive and negative ABMR (p=0.26-0.99). Samples from high PRA (>80%) or positive DSA patients showed higher gene expression levels for the ADCC-activated genes compared to low PRA (<80%) and negative DSA patients (p=0.04-0.001). CONCLUSION: ADCC pathways are active in transplant patients with ABMR, and likely mediate allograft injury, providing a potential mechanism for C4d negative ABMR.

Significant reduction of ATP production in PHA-activated CD4+ cells in 1-day-old blood from transplant patients.

BACKGROUND: Global immunosuppression can be measured by assessing adenosine triphospate (ATP) levels in mitogen-stimulated CD4+ T cells. METHODS: We investigated the effect of storage time on ATP levels in 234 blood samples from 18 healthy individuals and 152 transplant patients. The difference between day 0 (<13 hours post-blood draw) and day 1 (24-37 hours) measurements was analyzed and compared with various factors; a subset of samples was also analyzed in 6-hour intervals. RESULTS: The ATP levels were significantly lower on day 1 compared with that on day 0 in healthy individuals (279±159 vs 414±159 ng/mL, P<0.001) and patients (356±209 vs 455±221 ng/mL, P<0.0001). Of the 18 healthy individuals, 17 showed ATP reduction, whereas 192 (89%) of 216 patients did so on day 1 (24.8±24.1%). In the time course analysis, ATP levels decreased with the blood storage time in healthy and patient samples, and the reduction began as early as 7 hours post-blood draw. The reduction rate was significantly higher in patient samples with low day 0 ATP levels compared with samples with moderate or high levels (44.7±31.3% vs 23.2±23.6% or 18.7±15.7%; P<0.001). The reduction rate in patients treated with alemtuzumab induction was slightly higher than that in daclizumab-treated patients (28.8±24.6% vs 21.3±21.3%, P=0.09). CD4+ cell number did not change within 24 hours post-blood draw, but CD4 expression decreased 2.0±2.8% (P<0.05). CONCLUSIONS: The ATP levels are significantly lower in 1-day-old blood compared with fresh blood, suggesting that fresh blood should be used for assessing the T cell immune function to obtain the most accurate results.

Further evidence for the role of ENPP1 in obesity: association with morbid obesity in Finns.

The aim of this study was to investigate a series of single-nucleotide polymorphisms (SNPs) in the genes MC2R, MC3R, MC4R, MC5R, POMC, and ENPP1 for association with obesity. Twenty-five SNPs (2-7 SNPs/gene) were genotyped in 246 Finns with extreme obesity (BMI > or = 40 kg/m2) and in 481 lean subjects (BMI 20-25 kg/m2). Of the obese subjects, 23% had concomitant type 2 diabetes. SNPs and SNP haplotypes were tested for association with obesity and type 2 diabetes. Allele frequencies differed between obese and lean subjects for two SNPs in the ENPP1 gene, rs1800949 (P = 0.006) and rs943003 (P = 0.0009). These SNPs are part of a haplotype (rs1800949 C-rs943003 A), which was observed more frequently in lean subjects compared to obese subjects (P = 0.0007). Weaker associations were detected between the SNPs rs1541276 in the MC5R, rs1926065 in the MC3R genes and obesity (P = 0.04 and P = 0.03, respectively), and between SNPs rs2236700 in the MC5R, rs2118404 in the POMC, rs943003 in the ENPP1 genes and type 2 diabetes (P = 0.03, P = 0.02 and P = 0.02, respectively); these associations did not, however, remain significant after correction for multiple testing. In conclusion, a previously unexplored ENPP1 haplotype composed of SNPs rs1800949 and rs943003 showed suggestive evidence for association with adult-onset morbid obesity in Finns. In this study, we did not find association between the frequently studied ENPP1 K121Q variant, nor SNPs in the MCR or POMC genes and obesity or type 2 diabetes.

Unraveling the complex genetics of familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL) constitutes a substantial risk factor for atherosclerosis since it is observed in about 20% of coronary heart disease (CHD) patients under 60 years. FCHL, characterized by elevated levels of total cholesterol (TC) and triglycerides (TGs), or both, is also one of the most common familial hyperlipidemias with a prevalence of 1%-6% in Western populations. Numerous studies have been performed to identify genes contributing to FCHL. The recent linkage and association studies and their replications are beginning to elucidate the genetic variations underlying the susceptibility to FCHL. Three chromosomal regions on 1q21-23, 11p and 16q22-24.1 have been replicated in different study samples, offering targets for gene hunting. In addition, several candidate gene studies have replicated the influence of the lipoprotein lipase (LPL) gene and apolipoprotein A1/C3/A4/A5 (APOA1/C3/A4/A5) gene cluster in FCHL. Recently, the linked region on chromosome 1q21 was successfully fine-mapped and the upstream transcription factor 1 (USF1) gene identified as the underlying gene for FCHL. This finding has now been replicated in independent FCHL samples. However, the total number of variants, the risk related to each variant and their relative contributions to the disease susceptibility are not known yet.

Common hepatic nuclear factor-4alpha variants are associated with high serum lipid levels and the metabolic syndrome.

Hepatic nuclear factor-4alpha (HNF-4alpha), a transcription factor involved in the regulation of serum lipid and glucose levels, has recently been associated with type 2 diabetes. The HNF-4alpha gene (HNF4A) resides on chromosome 20q12-q13.1, which, in addition to type 2 diabetes, has also previously been linked to high triglycerides in Finnish familial combined hyperlipidemia (FCHL) families. FCHL, characterized by elevated levels of serum total cholesterol, triglycerides, or both, is a common dyslipidemia observed in up to 20% of patients with premature coronary heart disease. Considering the clear phenotypic overlap between type 2 diabetes and FCHL, both predisposing to high serum triglycerides and glucose intolerance, we tested this gene for association in dyslipidemic families originating from two distinct populations, Finnish and Mexican, and comprising 1,447 subjects. Our data show that common HNF4A variants and haplotypes are associated with elevated serum lipid levels and the metabolic syndrome (P = 0.008-0.04), as well as with elevated glucose parameters (P = 0.008-0.03), using family-based association analysis. Importantly, both Finnish and Mexican families shared two common lipid-associated HNF4A haplotypes (P = 0.005 for total cholesterol and 0.006 for triglycerides). In conclusion, we show for the first time that common HNF4A variants are associated with high serum lipid levels and the metabolic syndrome.

Cross-species analyses implicate Lipin 1 involvement in human glucose metabolism.

Recent studies in the mouse have demonstrated that variations in lipin expression levels in adipose tissue have marked effects on adipose tissue mass and insulin sensitivity. In the mouse, lipin deficiency prevents normal adipose tissue development, resulting in lipodystrophy and insulin resistance, whereas excess lipin levels promote fat accumulation and insulin sensitivity. Here, we investigated the effects of genetic variation in lipin levels on glucose homeostasis across species by analyzing lipin transcript levels in human and mouse adipose tissues. A strong negative correlation was observed between lipin mRNA levels and fasting glucose and insulin levels, as well as an indicator of insulin resistance (HOMA-IR), in both mice and humans. We subsequently analyzed the allelic diversity of the LPIN1 gene in dyslipidemic Finnish families, as well as in a case-control sample of obese (n = 477) and lean (n = 821) individuals. Alleles were defined by genotyping seven single nucleotide polymorphisms (SNPs) of the critical DNA region over the LPIN1 gene. Intragenic SNPs and corresponding allelic haplotypes exhibited associations with serum insulin levels and body mass index (P = 0.002-0.04). Both the expression levels in adipose tissue across species and genetic data in human study samples highlight the importance of lipin in glucose homeostasis and imply that allelic variants of this gene have significance in human metabolic traits.

Locus for quantitative HDL-cholesterol on chromosome 10q in Finnish families with dyslipidemia.

Decreased HDL-cholesterol (HDL-C) and familial combined hyperlipidemia (FCHL) are the two most common familial dyslipidemias predisposing to premature coronary heart disease (CHD). These dyslipidemias share many phenotypic features, suggesting a partially overlapping molecular pathogenesis. This was supported by our previous pooled data analysis of the genome scans for low HDL-C and FCHL, which identified three shared chromosomal regions for a qualitative HDL-C trait on 8q23.1, 16q23.3, and 20q13.32. This study further investigates these regions as well as two other loci we identified earlier for premature CHD on 2q31 and Xq24 and a locus for high serum triglycerides (TGs) on 10q11. We analyzed 67 microsatellite markers in an extended study sample of 1,109 individuals from 92 low HDL-C or FCHL families using both qualitative and quantitative lipid phenotypes. These analyses provided evidence for linkage (a logarithm of odds score of 3.2) on 10q11 using a quantitative HDL-C trait. Importantly, this region, previously linked to TGs, body mass index, and obesity, provided evidence for association for quantitative TGs (P = 0.0006) and for a combined trait of HDL-C and TGs (P = 0.008) with marker D10S546. Suggestive evidence for linkage also emerged for HDL-C on 2q31 and for TGs on 20q13.32. Finnish families ascertained for dyslipidemias thus suggest that 10q11, 2q31, and 20q13.32 harbor loci for HDL-C and TGs.

The SLC6A14 gene shows evidence of association with obesity.

In our previous genome-wide scan of Finnish nuclear families, obesity was linked to chromosome Xq24. Here we analyzed this 15-Mb region by genotyping 9 microsatellite markers and 36 single nucleotide polymorphisms (SNPs) for 11 positional and functional candidate genes in an extended sample of 218 obese Finnish sibling pairs (sibpairs) (BMI > 30 kg/m2). Evidence of linkage emerged mainly from the obese male sibpairs, suggesting a gender-specific effect for the underlying gene. By constructing haplotypes among the obese male sibpairs, we restricted the region from 15 Mb to 4 Mb, between markers DXS8088 and DXS8067. Regional functional candidate genes were tested for association in an initial sample of 117 cases and 182 controls. Significant evidence was observed for association for an SNP in the 3'-untranslated region of the solute carrier family 6 member 14 (SLC6A14) gene (P = 0.0002) and for SNP haplotypes of the SLC6A14 gene (P = 0.0007-0.006). Furthermore, an independent replication study sample of 837 cases and 968 controls from Finland and Sweden also showed significant differences in allele frequencies between obese and non-obese individuals (P = 0.003). The SLC6A14 gene is an interesting novel candidate for obesity because it encodes an amino acid transporter, which potentially regulates tryptophan availability for serotonin synthesis and thus possibly affects appetite control.