The impact of genetic variants on BMI increase during childhood versus adulthood.

BACKGROUND: Genetic variants that predispose individuals to obesity may have differing influences during childhood versus adulthood, and additive effects of such variants are likely to occur. Our ongoing studies to identify genetic determinants of obesity in American Indians have identified 67 single-nucleotide polymorphisms (SNPs) that reproducibly associate with maximum lifetime non-diabetic body mass index (BMI). This study aimed to identify when, during the lifetime, these variants have their greatest impact on BMI increase. SUBJECTS/METHODS: A total of 5906 Native Americans of predominantly Pima Indian heritage with repeated measures of BMI between the ages of 5 and 45 years were included in this study. The association between each SNP with the rates of BMI increase during childhood (5-19 years) and adulthood (20-45 years) were assessed separately. The significant SNPs were used to calculate a cumulative allelic risk score (ARS) for childhood and adulthood, respectively, to assess the additive effect of these variants within each period of life. RESULTS: The majority of these SNPs (36 of 67) were associated with rate of BMI increase during childhood (P-value range: 0.00004-0.05), whereas only nine SNPs were associated with rate of BMI change during adulthood (P-value range: 0.002-0.02). These 36 SNPs associated with childhood BMI gain likely had a cumulative effect as a higher childhood-ARS associated with rate of BMI change (ß=0.032 kg m(-2) per year per risk allele, 95% confidence interval: 0.027-0.036, P<0.0001), such that at age 19 years, individuals with the highest number of risk alleles had a BMI of 10.2 kg m(-2) greater than subjects with the lowest number of risk alleles. CONCLUSIONS: Overall, our data indicates that genetic polymorphisms associated with lifetime BMI may influence the rate of BMI increase during different periods in the life course. The majority of these polymorphisms have a larger impact on BMI during childhood, providing further evidence that prevention of obesity will need to begin early in life.

Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants.

BACKGROUND: In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. OBJECTIVE: The objective of this study is to compare BDNF concentrations of subjects with loss-of-function (LOF) and gain-of-function (GOF) MC4R variants with those of controls with common sequence MC4R. METHODS: Circulating BDNF was measured in two cohorts with known MC4R sequence: 148 subjects of Pima Indian heritage ((mean±s.d.): age, 15.7±6.5 years; body mass index z-scores (BMI-Z), 1.63±1.03) and 69 subjects of Hispanic heritage (10.8±3.6 years; BMI-Z, 1.57±1.07). MC4R variants were characterized in vitro by cell surface expression, receptor binding and cyclic AMP response after agonist administration. BDNF single-nucleotide polymorphisms (SNPs) rs12291186, rs6265 and rs7124442 were also genotyped. RESULTS: In the Pima cohort, no significant differences in serum BDNF was observed for 43 LOF subjects versus 65 LOF-matched controls (age, sex and BMI matched; P=0.29) or 20 GOF subjects versus 20 GOF-matched controls (P=0.40). Serum BDNF was significantly associated with genotype for BDNF rs12291186 (P=0.006) and rs6265 (P=0.009), but not rs7124442 (P=0.99); BDNF SNPs did not interact with MC4R status to predict serum BDNF. In the Hispanic cohort, plasma BDNF was not significantly different among 21 LOF subjects, 20 GOF subjects and 28 controls (P=0.79); plasma BDNF was not predicted by BDNF genotype or BDNF-x-MC4R genotype interaction. CONCLUSIONS: Circulating BDNF concentrations were not significantly associated with MC4R functional status, suggesting that peripheral BDNF does not directly reflect hypothalamic BDNF secretion and/or that MC4R signaling is not a significant regulator of the bulk of BDNF expression in humans.

HLA-DRB1 reduces the risk of type 2 diabetes mellitus by increased insulin secretion.

AIMS/HYPOTHESIS: We sought to identify the physiological implications of genetic variation at the HLA-DRB1 region in full-heritage Pima Indians in Arizona. METHODS: Single-nucleotide polymorphisms from the HLA region on chromosome 6p were tested for association with skeletal muscle mRNA expression of HLA-DRB1 and HLA-DRA, and with type 2 diabetes mellitus and prediabetic traits. RESULTS: The A allele at rs9268852, which tags HLA-DRB1 02(1602), was associated both with higher HLA-DRB1 mRNA expression (n = 133, p = 4.27 × 10(-14)) and decreased risk of type 2 diabetes (n = 3,265, OR 0.723, p = 0.002). Among persons with normal glucose tolerance (n = 266) this allele was associated with a higher mean acute insulin response during an intravenous glucose tolerance test (p = 0.005), higher mean 30 min insulin concentration during an oral glucose tolerance test (p = 0.017) and higher body fat percentage (p = 0.010). The polymorphism was not associated with HLA-DRA mRNA expression or insulin sensitivity. CONCLUSIONS/INTERPRETATION: HLA-DRB1*02 is protective for type 2 diabetes, probably by enhancing self tolerance, thereby protecting against the autoimmune-mediated reduction of insulin secretion.

Variants in ACAD10 are associated with type 2 diabetes, insulin resistance and lipid oxidation in Pima Indians.

AIMS/HYPOTHESIS: A prior genome-wide association study in Pima Indians identified a variant within the ACAD10 gene that is associated with early-onset type 2 diabetes. Acylcoenzyme A dehydrogenase 10 (ACAD10) catalyses mitochondrial fatty acid beta-oxidation, which plays a pivotal role in developing insulin resistance and type 2 diabetes. Therefore, ACAD10 was analysed as a positional and biological candidate for type 2 diabetes. METHODS: Twenty-three SNPs were genotyped in 1,500 Pima Indians to determine the linkage disequilibrium pattern across ACAD10. Association with type 2 diabetes was determined by genotyping four tag single nucleotide polymorphisms (SNPs) in a population-based sample of 3,501 full-heritage Pima Indians; two associated SNPs were further genotyped in a second population-based sample of 3,723 American Indians. Associations with quantitative traits were assessed in 415 non-diabetic full heritage Pima individuals who had been metabolically phenotyped. RESULTS: SNPs rs601663 and rs659964 were associated with type 2 diabetes in the full-heritage Pima Indian sample (p=0.04 and 0.0006, respectively), and rs659964 was further associated with type 2 diabetes in the second American Indian sample (p=0.04). Combination of these two samples provided the strongest evidence for association (p=0.009 and 0.00007, for rs601663 and rs659964, respectively). Quantitative trait analyses identified nominal associations with both lower lipid oxidation rate and larger subcutaneous abdominal adipocyte size, which is consistent with the known physiology of ACAD10, and also identified associations with increased insulin resistance. CONCLUSIONS/INTERPRETATION: We propose that ACAD10 variation may increase type 2 diabetes susceptibility by impairing insulin sensitivity via abnormal lipid oxidation.

Design and analysis of genetic association studies to finely map a locus identified by linkage analysis: sample size and power calculations.

Association (e.g. case-control) studies are often used to finely map loci identified by linkage analysis. We investigated the influence of various parameters on power and sample size requirements for such a study. Calculations were performed for various values of a high-risk functional allele (fA), frequency of a marker allele associated with the high risk allele (f1), degree of linkage disquilibrium between functional and marker alleles (D') and trait heritability attributable to the functional locus (h2). The calculations show that if cases and controls are selected from equal but opposite extreme quantiles of a quantitative trait, the primary determinants of power are h2 and the specific quantiles selected. For a dichotomous trait, power also depends on population prevalence. Power is optimal if functional alleles are studied (fA= f1 and D'= 1.0) and can decrease substantially as D' diverges from 1.0 or as f(1) diverges from fA. These analyses suggest that association studies to finely map loci are most powerful if potential functional polymorphisms are identified a priori or if markers are typed to maximize haplotypic diversity. In the absence of such information, expected minimum power at a given location for a given sample size can be calculated by specifying a range of potential frequencies for fA (e.g. 0.1-0.9) and determining power for all markers within the region with specification of the expected D' between the markers and the functional locus. This method is illustrated for a fine-mapping project with 662 single nucleotide polymorphisms in 24 Mb. Regions differed by marker density and allele frequencies. Thus, in some, power was near its theoretical maximum and little additional information is expected from additional markers, while in others, additional markers appear to be necessary. These methods may be useful in the analysis and interpretation of fine-mapping studies.

Antisense knockdown of calcium-dependent K+ channels in developing cerebellar Purkinje neurons.

Normal developmental upregulation of K(Ca) channel activity in cultured rat cerebellar Purkinje neurons was selectively inhibited by antisense oligonucleotide sequence (3 microM) targeted against the rslo transcript. The knockdown was specific; delayed rectifier and apamin-sensitive K+ channel abundances in Purkinje neurons were not affected by rslo antisense. Sense oligonucleotides (3 microM), used as a control, had no effect on channel abundance. Quantitative morphometric analyses of anti-calbindin-labeled Purkinje neurons showed no differences between neurons in control, sense and antisense treatment groups, and confirmed that the presence of the added oligonucleotide in the sense and antisense treatment conditions had no discernable toxic effects on neuronal health, for which neurite outgrowth is a sensitive indicator. These results confirm the identification of the developmentally regulated K(Ca) channel as the product of the gene rslo in cerebellar Purkinje neurons.

Characterization of cis-elements required for osmotic response of rat Na(+)/H(+) exchanger-2 (NHE-2) gene.

The Na(+)/H(+) exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na(+) across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD-3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5'-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic-responsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp -1201 to -1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA-protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of trans-acting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.

Differential expression of three classes of voltage-gated Ca(2+) channels during maturation of the rat cerebellum in vitro.

Voltage-gated Ca(2+) channels provide a mode of Ca(2+) influx that is essential for intracellular signaling in many cells. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the relative amounts of mRNAs encoding three classes of Ca(2+) channels (alpha1A, alpha1B and alpha1E) during development, in cultures established from prenatal rat cerebellar cortex. Ca(2+) channel transcript levels were standardized to a constitutive marker (cyclophilin). For all three classes of Ca(2+) channels, transcript levels were highest at early stages (4-10 days in vitro) and declined with age. This developmental pattern was differentially regulated by a depolarizing agent, tetraethylammonium chloride (TEA, 1 mM). Chronic depolarization yielded a significant elevation in transcript levels for alpha1B (N-type) and alpha1E (R-type) Ca(2+) channels during neuronal maturation (10-21 days in vitro), but dramatically suppressed transcript levels for the alpha1A (P-type) Ca(2+) channel at all stages of development. The effects of TEA on alpha1A, alpha1B and alpha1E transcript levels were mimicked by increasing external K(+) (from 5 to 10 mM). The regulatory effects of depolarization on transcript levels were dependent on extracellular Ca(2+) for alpha1E but not for alpha1A. For alpha1B, transcript levels depended on extracellular Ca(2+) only for increased K(+) as the depolarizing stimulus, but not for TEA. These results suggest that levels of Ca(2+) channel transcripts in rat cerebellum are developmentally regulated in vitro and can be influenced differentially by transmembrane signaling via chronic depolarization and Ca(2+) entry. Dynamic regulation of Ca(2+) channel expression may be relevant to the different functional roles of Ca(2+) channels and their regional localization within neurons.

Genetic screening for X-linked hypophosphatemic mice and ontogenic characterization of the defect in the renal sodium-phosphate transporter.

X-linked hypophosphatemic (Hyp) rickets is characterized by short stature, rickets, and bone abnormalities. Biochemically, hypophosphatemia and decreased renal reabsorption of phosphate are the hallmark of the disorder. Mutation of the PEX gene has been linked to human and murine Hyp rickets. Our study showed that phenotypical changes of this disease could be detected in 6-wk-old mice, but not in 2-wk-old mice. Therefore, we developed a PCR method to identify Hyp mice by detecting a lack of the 3' region of the PEX gene. Serum inorganic phosphate (Pi) levels were decreased, whereas alkaline phosphatase activity was increased in 2- and 6-wk-old Hyp mice. Northern blot showed that renal Na(+)-Pi transporter mRNA levels were decreased by 2.1-fold (1.47 +/- 0.21 densitometric units for normals; 0.68 +/- 1.43 for Hyp mice; p < 0.040) in 2-wk-old Hyp mice and by 1.7-fold (2.41 +/- 0.42 for normals; 1.44 +/- 0.33 for Hyp mice; p < 0.027) in 6-wk-old mice. Western blot showed that levels of immunoreactive renal Na(+)-Pi transporter protein were decreased by 4.5-fold (0.90 +/- 0.10 for normals; 0.22 +/- 0.08 for Hyp mice; p < 0.001) in 2-wk-old Hyp mice; and by 4.9-fold (1.47 +/- 0.19 for normals; 0.30 +/- 0.09 for Hyp mice; p < 0.0001) in 6-wk-old Hyp mice. In addition, levels of Na(+)-Pi transporter mRNA and protein were increased between 2- and 6-wk-old normal mice, but not in Hyp mice. This study demonstrates an easy assay to detect Hyp mutation and characterizes the defect during ontogeny of the Na(+)-Pi transporter in Hyp mice.

Molecular cloning and characterization of the rat NHE-2 gene promoter.

To understand the molecular mechanisms underlying NHE-2 regulation in the mammalian kidney and intestine, we cloned and sequenced 5.6 kb of the 5'-flanking region of the rat NHE-2 gene. DNA sequence analysis revealed multiple putative cis-acting regulatory elements including SP1, CK, NFY-CBF, Tant, GCN4, and one progesterone and several retinoic acid response elements. The upstream sequence lacked TATA and CAAT boxes, but contained a high G/C rich region within the first 300 bp. A single transcriptional initiation site was identified by primer extension in rat kidney and small intestine, approximately 103 bp upstream of the previously identified 5'-end of the rat NHE-2 cDNA. Various regions of the promoter (from [-]5567 to [+]105 bp) were tested for their ability to drive expression of the luciferase reporter gene in transiently transfected murine Inner Medullary Collecting Duct (mIMCD-3) cells. Results demonstrated that [-]289, [-]1271 and [-]2630 bp constructs showed promoter activity that was significantly higher than the negative control construct (20-fold). These results also demonstrated that basal cis-acting elements are contained within [-]289 bp of the transcriptional start site. However, the functional activity of the [-]5567 bp construct was not significantly different from the negative control, suggesting that a negative regulatory element may be present between [-]2630 and [-]5567 bp of the promoter region.

Increased calcium-dependent K+ channel activity contributes to the maturation of cellular firing patterns in developing cerebellar Purkinje neurons.

Developmental changes in neuronal excitability reflect the regulated expression of ion channels and receptors. Purkinje neurons of the rat cerebellum progress from slow irregular firing to a fast pacemaker-like pattern during postnatal development in vivo. In this study, a comparable period of development in culture was investigated at the protein level using cell-attached single channel recordings to quantify the abundance of active calcium-dependent (KCa) and delayed rectifier (KD) potassium channels. In control cultures, KCa channel activity increased whereas KD channel activity was not significantly different with developmental age. The increase in active KCa channels was antagonized by chronic treatment with the blocker, tetraethylammonium (TEA, 1 mM), which also retarded the normal development of cellular firing patterns. The consequences of chronic TEA treatment were assessed in cultures after thorough washout of the TEA-containing culture medium. Current clamp analyses (nystatin-perforated patches) showed that control Purkinje neurons progressed from a single spike mode to a repetitive firing mode, with a concomitant decrease in action potential duration and an increase in maximal firing rate. Chronic TEA treatment prevented these changes; Purkinje neurons retained the slow firing rate and long duration action potentials that are typical of the immature state. These data suggest that the developmental increase in KCa channel activity may be required for the maturation of cellular firing patterns in cerebellar Purkinje neurons.

Regulation of Ca2+-dependent K+ channel expression in rat cerebellum during postnatal development.

Potassium channels govern duration and frequency of excitable membrane events and may regulate signals that are important in neuronal development. This study assesses the developmental expression of the large conductance Ca2+-dependent K+ channel in vivo and in vitro in rat cerebellum. In vivo, transcript levels for the Ca2+-dependent K+ channel (KCa) were shown by Northern analysis to increase during development, whereas transcript levels for the voltage-gated K+ channel Kv3.1, a delayed rectifier (KD), remained relatively constant. A comparable pattern was demonstrated by expression in Xenopus oocytes of poly(A)-enriched RNA isolated from postnatal rat cerebella. In cerebellar cultures, increased external K+ provided a simple manipulation of cell excitability that influenced KCa transcript levels during development. With low external K+ (5.3 mM), the levels of KCa channel transcript (assessed by semiquantitative PCR) remained constant throughout development. However, in culture medium that supported significant dendritic outgrowth (10 mM extracellular K+), an upregulation of KCa transcript level was observed similar to that seen in vivo. Tetraethylammonium (TEA; 1 mM) similarly enhanced KCa expression, suggesting that depolarizing stimuli increased KCa expression. The stimulatory effects of increased K+ or TEA on KCa expression required extracellular Ca2+ and were abolished in low external calcium (0.1 mM, buffered with EGTA), although morphological development and survival were not impaired. The regulation of KCa channel expression by depolarization and Ca2+ entry provides evidence of a logical feedback mechanism governing Ca2+ signals that may be significant in cerebellar development.