Genetic Determinants of Creatine Bioavailability and Responsiveness: Emphasis on Common Low-Impact Variants

Sequence Kernel Association Tests for the Combined Effect of Rare and Common Variants

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.

BackgroundThe interleukin (IL)-1 pathway is primarily associated with innate immunological defense and plays a major role in the induction and regulation of inflammation. Both common and rare genetic variation in this pathway underlies various inflammation-mediated diseases, but the role of rare variants relative to common variants in immune response variability in healthy individuals remains unclear.MethodsWe performed molecular inversion probe sequencing on 48 IL-1 pathway-related genes in 463 healthy individuals from the Human Functional Genomics Project. We functionally grouped common and rare variants, over gene, subpathway, and inflammatory levels and performed the Sequence Kernel Association Test to test for association with in vitro stimulation-induced cytokine responses; specifically, IL-1β and IL-6 cytokine measurements upon stimulations that represent an array of microbial infections: lipopolysaccharide (LPS), phytohaemagglutinin (PHA), Candida albicans (C. albicans), and Staphylococcus aureus (S. aureus).ResultsWe identified a burden of NCF4 rare variants with PHA-induced IL-6 cytokine and showed that the respective carriers are in the 1% lowest IL-6 producers. Collapsing rare variants in IL-1 subpathway genes produces a bidirectional association with LPS-induced IL-1β cytokine levels, which is reflected by a significant Spearman correlation. On the inflammatory level, we identified a burden of rare variants in genes encoding for proteins with an anti-inflammatory function with S. aureus-induced IL-6 cytokine. In contrast to these rare variant findings which were based on different types of stimuli, common variant associations were exclusively identified with C. albicans-induced cytokine over various levels of grouping, from the gene, to subpathway, to inflammatory level.ConclusionsIn conclusion, this study shows that functionally grouping common and rare genetic variants enables the elucidation IL-1-mediated biological mechanisms, specifically, for IL-1β and IL-6 cytokine responses induced by various stimuli. The framework used in this study may allow for the analysis of rare and common genetic variants in a wider variety of (non-immune) complex phenotypes and therefore has the potential to contribute to better understanding of unresolved, complex traits and diseases.

Recent breakthroughs in next-generation sequencing technologies allow cost-effective methods for measuring a growing list of cellular properties, including DNA sequence and structural variation. Next-generation sequencing has the potential to revolutionize complex trait genetics by directly measuring common and rare genetic variants within a genome-wide context. Because for a given gene both rare and common causal variants can coexist and have independent effects on a trait, strategies that model the effects of both common and rare variants could enhance the power of identifying disease-associated genes. To date, little work has been done on integrating signals from common and rare variants into powerful statistics for finding disease genes in genome-wide association studies. In this analysis of the Genetic Analysis Workshop 17 data, we evaluate various strategies for association of rare, common, or a combination of both rare and common variants on quantitative phenotypes in unrelated individuals. We show that the analysis of common variants only using classical approaches can achieve higher power to detect causal genes than recently proposed rare variant methods and that strategies that combine association signals derived independently in rare and common variants can slightly increase the power compared to strategies that focus on the effect of either the rare variants or the common variants.

We have previously reported that maternal creatine supplementation protects the neonate from hypoxic injury. Here, we investigated whether maternal creatine supplementation altered expression of the creatine synthesis enzymes (arginine:glycine amidinotransferase [AGAT], guanidinoaceteate methyltransferase [GAMT]) and the creatine transporter (solute carrier family 6 [neurotransmitter transporter, creatine] member 8: SLC6A8) in the term offspring. Pregnant spiny mice were fed a 5% creatine monohydrate diet from midgestation (day 20) to term (39 days). Placentas and neonatal kidney, liver, heart, and brain collected at 24 hours of age underwent quantitative polymerase chain reaction and Western blot analysis. Maternal creatine had no effect on the expression of AGAT and GAMT in neonatal kidney and liver, but mRNA expression of AGAT in brain tissues was significantly decreased in both male and female neonates born to mothers who were fed the creatine diet. SLC6A8 expression was not affected by maternal dietary creatine loading in any tissues. Maternal dietary creatine supplementation from midgestation in the spiny mouse did not alter the capacity for creatine synthesis or transport.

SummaryBackground:Idiopathic pulmonary fibrosis (IPF) and telomere length (TL) are both strongly linked to rare and common genetic variation. Shortened TL itself may be causal for IPF. Whether rare and common variants compete or cooperate to confer genetic risk of IPF uniformly is unknown.Methods:We used whole genome sequencing (WGS) data from a discovery case-control cohort sequenced at Columbia (777 IPF, 2905 controls) and validated findings using WGS data from Trans-Omics for Precision Medicine (TOPMed, 1148 IPF, 5202 controls) and the UK Biobank (UKBB, 2739 IPF, 395331 controls). In all cohorts, we identified rare damaging variants in disease-associated genes and computed control-normalized polygenic risk scores for IPF (IPF-PRS) and telomere length (TL-PRS). Telomere length of blood leukocytes was measured using a qPCR assay for two cohorts. We determined the association of the MUC5B rs35705950 polymorphism, an IPF-PRS excluding MUC5B (IPF-PRS-noMUC5B), and a TL-PRS with IPF risk in the overall cohort and in subgroups stratified by genetic endotypes (rare variant carriers, non-carriers stratified by TL cutoffs). We calculated cross-validated area under the receiver operator curve (AUC) and compared the liability of IPF explained by genetic variables.Findings:We identified independent associations between IPF risk and rare variants, the MUC5B SNP, and both polygenic scores in the discovery cohort and replicated these findings in the TOPMed and UKBB cohorts. The adjusted effect size of the TL-PRS, which includes >180 SNPs not previously associated with IPF, was comparable to the IPF-PRS-noMUC5B in the discovery (ORTL-PRS 1.63 [95% CI 1.47, 1.81] vs. ORIPF-PRS 1.60 [1.44, 1.77]) and replication cohorts (TOPMed ORTL-PRS 1.47 [1.36, 1.59] vs. ORIPF-PRS 1.37 [1.25, 1.50]; UKBB ORTL-PRS 1.24 [1.19, 1.29] vs. ORIPF-PRS 1.25 [1.21, 1.30]). The TL-PRS incrementally improved disease prediction beyond known IPF common and rare genetic predictors and clinical variables in discovery (combined AUC: 0.89, pDelong = 0.006), TOPMed (combined AUC: 0.89, pDelong = 0.01), and UKBB cohorts (combined AUC: 0.77, pDelong = 0.03). Rare and common variants jointly contributed to genetic liability of IPF. The TL-PRS increased liability of IPF explained by 13% in the discovery cohort and 8% and 13% in the TOPMed and UKBB cohorts, respectively. In IPF subjects with damaging rare variants, the TL-PRS was consistently associated with disease risk whereas the IPF-PRS-noMUC5B was not. The TL-PRS also conferred nominally greater odds of disease risk than the IPF-PRS-noMUC5B in patients with shorter TL, in the discovery and UKBB cohorts. Together, 23–43% of IPF cases have damaging rare variants or telomeres <10th percentile, where the TL-PRS represents a major unrecognized genetic risk factor.Interpretation:Common and rare genetic variation confer context-specific genetic risk in IPF competitively and cooperatively. In contrast to known IPF common risk variants, the TL-PRS, which includes >180 genetic loci not previously associated with IPF, increases the risk of disease specifically in certain IPF endotypes. Polygenic risk from telomere-associated common variants is a key feature of IPF genetic heterogeneity.Funding:National Institutes of Health (NIH), Medical Research Council (MRC), National Institute for Health and Care Research (NIHR)

DSM-5 and Psychiatric Genetics — Round Hole, Meet Square Peg

Despite the discovery of at least five pathogenic genes in Parkinson disease (PD), the genetic etiology in the vast majority of PD remains to be clarified. Common genetic variants could act as susceptibility risk factors. Our previous meta-analysis of PD genetic association studies, over a 30-year period yielded four genes (N-acetylcysteine 2, monoamine oxidase B, glutathione transferase, and mitochondrial tRNA), as their common variants were found to be associated with PD. More recently, international collaborative studies and meta-analysis have identified the S18Y variant of ubiquitin carboxy-terminal hydrolase L1, Rep 1 variant of alpha-synuclein and tau H1 haplotype to be genetic susceptibility risk/protective factors. However, the most significant, common genetic risk factor in PD has been its association with the leucine-rich repeat kinase-2 (LRRK2) G2385R variant. We conducted an analysis of independent studies involving 2205 PD and 1817 controls and found the average carrier rate of G2385R variant to be about 9% in PD and 4% in controls (p < 0.001; odds ratio: 2.27; 95% confidence interval: 1.78-2.9). A higher frequency of G2385R carriers has been observed in familial PD when compared with sporadic patients. Based on current evidence, certain common genetic variants are likely to modulate the risk of PD.

Marked Interindividual Variability in the Response to Selective Inhibitors of Cyclooxygenase-2

Background/Objectives: Prostate cancer is the most common cancer among men globally and a leading cause of cancer-related death. Germline genetic evaluation is increasingly recognized as essential for men with high-risk features such as a strong family history or advanced disease. Methods: Comprehensive genetic risk assessment should integrate three components: family history (FH), rare pathogenic mutations (RPMs), and polygenic risk scores (PRS). RPMs in DNA repair genes (e.g., BRCA2, CHEK2, ATM) can inform screening, prognosis, and treatment strategies, particularly for metastatic or aggressive disease. PRS, derived from common genetic variants, provides a personalized and independent measure of prostate cancer risk and may guide decisions on screening intensity and timing. Results: Although PRS cannot yet differentiate between indolent and aggressive cancer, it has the potential to stratify men into low and high-risk categories more effectively than FH or RPMs alone. Knowledge of specific RPMs can influence treatment decisions in clinically advanced prostate cancer. Challenges in clinical implementation include limited provider awareness, underutilization of genetic counseling, and lack of diversity in genomic datasets, which can lead to misdiagnoses. Emerging technologies and digital tools are being developed to streamline genetic testing and counseling. Population-level strategies and tailored screening protocols based on genetic risk are under active investigation. Conclusions: While early evidence suggests high satisfaction with genetic testing among patients, further studies in diverse populations are needed. Integration of germline genetic information into prostate cancer management offers promising avenues for personalized screening, surveillance, and treatment, ultimately aiming to reduce morbidity and mortality.

Creatine is a nitrogen containing compound that serves as an energy shuttle between the mitochondrial sites of ATP production and the cytosol where ATP is utilized. There are two known disorders of creatine synthesis (both transmitted as autosomal recessive traits: arginine: glycine amidinotransferase (AGAT) deficiency; OMIM 602360; and guanidinoacetate methyltransferase (GAMT) deficiency (OMIM 601240)) and one disorder of creatine transport (X-linked recessive SLC6A8 creatine transporter deficiency (OMIM 300036)). All these disorders are characterized by brain creatine deficiency, detectable by magnetic resonance spectroscopy. Affected patients can have mental retardation, hypotonia, autism or behavioral problems and seizures. The diagnosis of these conditions relies on the measurement of plasma and urine creatine and guanidinoacetate. Creatine levels in plasma are reduced in both creatine synthesis defects and guanidinoacetate is increased in GAMT deficiency. The urine creatine/creatinine ratio is elevated in creatine transporter deficiency with normal plasma levels of creatine and guanidinoacetate. The diagnosis is confirmed in all cases by DNA testing or functional studies. Defects of creatine biosynthesis are treated with creatine supplements and, in GAMT deficiency, with ornithine and dietary restriction of arginine through limitation of protein intake. No causal therapy is yet available for creatine transporter deficiency and supplementation with the guanidinoacetate precursors arginine and glycine is being explored. The excellent response to therapy of early identified patients with GAMT or AGAT deficiency candidates these condition for inclusion in newborn screening programs.

Idiopathic pulmonary fibrosis (IPF) and telomere length are both strongly linked to rare and common genetic variants. Shortened telomere length might itself be causal for IPF. We aimed to evaluate whether rare and common variants compete or cooperate to confer genetic risk of IPF uniformly. In this genetic analysis, we used whole-genome sequencing (WGS) data from a discovery case-control cohort sequenced at Columbia University and validated findings using WGS data from Trans-Omics for Precision Medicine (TOPMed) and UK Biobank. In all cohorts, we identified rare damaging variants in disease-associated genes and computed control-normalised non-overlapping polygenic risk scores (PRS) for IPF and telomere length. We assessed the MUC5B rs35705950 single-nucleotide polymorphism (SNP), an IPF common risk variant with a large effect, independently from the polygenic scores. Telomere length in blood leukocytes was measured using a quantitative PCR assay for the discovery cohort and UK Biobank validation cohort. We conducted logistic regression (adjusting for age, sex, and principal components of ancestry) to evaluate the association between IPF risk and the MUC5B SNP, the IPF PRS excluding MUC5B (IPF-PRS-noMUC5B), and the PRS for telomere length in the overall cohort and analysed their effects in patient subgroups for IPF endotypes (carriers and non-carriers of rare variants stratified by telomere length cutoffs). To assess disease prediction, we calculated cross-validated area under the receiver operating receiver operating curve (AUC). We also compared the liability of IPF explained by genetic variables. The discovery cohort was recruited between April 23, 2003 and June 19, 2019 and included 777 patients with IPF and 2905 controls. We replicated the analyses in the TOPMed (1148 patients with IPF and 5202 controls) and UK Biobank (2739 patients with IPF and 395 331 controls) cohorts. 23-43% of patients with IPF had damaging rare variants or telomeres shorter than the tenth percentile. Analysis of the association of genetic variables with IPF diagnosis yielded odds ratios of 1·63 (95% CI 1·47-1·81) for telomere length PRS and 1·60 (1·44-1·77) for IPF-PRS-noMUC5B in the discovery cohort, with similar effect sizes for the two variables in the replication cohorts (1·47, 1·36-1·59 vs 1·37, 1·25-1·50 in TOPMed; 1·24, 1·19-1·29 vs 1·25, 1·21-1·30 in UK Biobank). The telomere length PRS had the greatest effect on disease risk in patients with IPF not harbouring rare variants and with telomere length shorter than the tenth percentile in the discovery cohort (2·02, 1·76-2·33) and UK Biobank replication cohort (1·70, 1·56-1·85). Accounting for clinical variables and all genetic variables (rare variants, MUC5B SNP, IPF PRS, and telomere length PRS) led to the best disease prediction in the discovery cohort (combined AUC 0·89), TOPMed cohort (0·89), and UK Biobank cohort (0·77). Rare and common variants contributed jointly to the genetic liability of IPF. The telomere length PRS accounted for 13% of the explained genetic liability of IPF in the discovery cohort and 8% and 13% in the TOPMed and UK Biobank cohorts, respectively. Common and rare genetic variation confer context-specific genetic risk in patients with IPF both competitively and cooperatively. In contrast to known IPF common risk variants, the telomere length PRS, which includes more than 180 genetic loci not previously associated with IPF, is associated with increased risk of disease in patients with specific IPF endotypes. Polygenic risk from telomere-associated common variants is a key feature of genetic heterogeneity in IPF. US National Institutes of Health, UK Medical Research Council, and UK National Institute for Health and Care Research.

Mendelian randomization (MR) has become an important technique for establishing causal relationships between risk factors and health outcomes. By using genetic variants as instrumental variables, it can mitigate bias due to confounding and reverse causation in observational studies. Current MR analyses have predominantly used common genetic variants as instruments, which represent only part of the genetic architecture of complex traits. Rare variants, which can have larger effect sizes and provide unique biological insights, have been understudied due to statistical and methodological challenges. We introduce MR-common and annotation-informed rare variants (MR-CARV), a novel framework integrating common and rare genetic variants in two-sample MR. This method leverages comprehensive genetic data made available by high-throughput sequencing technologies and large-scale consortia. Rare variants are aggregated into functional categories, such as gene-coding, gene-noncoding, and nongene regions, by leveraging variant annotations and biological impact as weights. The effects of rare variant sets are then estimated with STAARpipeline and combined with the estimated effects of common variants by the existing MR methods. Simulation studies demonstrate that MR-CARV maintains robust type I error and achieves higher statistical power, with up to a 66.3% relative increase compared with existing methods only based on common variants. Consistent with these findings, application to real data on high-density lipoprotein cholesterol (HDL-C) and preeclampsia showed that MR-CARV [inverse variance weighted (IVW)] yielded a more precise and statistically significant effect estimate (–0.020, SE = 0.0102, P =.0470) than IVW using only common variants (–0.023, SE = 0.0123, P =.0659).

Mendelian randomization (MR) has become an important technique for establishing causal relationships between risk factors and health outcomes. By using genetic variants as instrumental variables, it can mitigate bias due to confounding and reverse causation in observational studies. Current MR analyses have predominantly used common genetic variants as instruments, which represent only part of the genetic architecture of complex traits. Rare variants, which can have larger effect sizes and provide unique biological insights, have been understudied due to statistical and methodological challenges. We introduce MR-CARV, a novel framework integrating common and rare genetic variants in two-sample Mendelian randomization. This method leverages comprehensive genetic data made available by high-throughput sequencing technologies and large-scale consortia. Rare variants are aggregated into functional categories, such as gene-coding, gene-noncoding, and non-gene regions, by leveraging variant annotations and biological impact as weights. The effects of rare variant sets are then estimated with STAARpipeline and combined with the estimated effects of common variants by the existing MR methods. Simulation studies demonstrate that MR-CARV maintains robust type I error and achieves higher statistical power, with up to a 66.3% relative increase compared to existing methods only based on common variants. Consistent with these findings, application to real data on HDL-C and preeclampsia showed that MR-CARV(IVW) yielded a more precise and statistically significant effect estimate (−0.021, SE = 0.0101, P = 0.0365) than IVW using only common variants (−0.024, SE = 0.0123, P = 0.0538).

BackgroundBRCA1 and BRCA2 mutations are found in a proportion of families with multiple early-onset breast cancers. There are a large number of different deleterious mutations in both genes, none of which would be detectable using standard genetic association studies. Single common variants and haplotypes of common variants may capture groups of deleterious mutations since some low prevalence haplotypes of common variants occur more frequently among chromosomes that carry rare, deleterious mutations than chromosomes that do not.MethodsDNA sequence data for BRCA1 and BRCA2 was obtained from 571 participants from the Australian Breast Cancer Family Study. Genetic variants were classified as either deleterious mutations or common genetic variants. Variants tagging common polymorphisms were selected and haplotypes resolved using Haploview. Their frequency was compared to those with and without deleterious mutations using a permutation test.ResultsA common genetic variant in BRCA1 (3232A > G) was found to be over-represented in deleterious mutation carriers (p = 0.05), whereas a common genetic variant in BRCA2 (1342A > C) occurred less frequently in deleterious mutation carriers (p = 0.04). All four of the common BRCA1 variants used to form haplotypes occurred more frequently in the deleterious mutation carriers when compared to the non-carriers, but there was no evidence of a difference in the distributions between the two groups (p = 0.34). In BRCA2, all four common variants were found to occur less frequently in the deleterious mutation carriers when compared to non-carriers, but the evidence for difference in the distribution between the two groups was weak (p = 0.16). Several less common haplotypes of common BRCA1 variants were found to be over-represented among deleterious mutation carriers but there was no evidence for this at the population level. In BRCA2, only the most common haplotype was found to occur more frequently in deleterious mutation carriers, with again no evidence at the population level.ConclusionsWe observed differences in the frequency of common genetic variants of the BRCA1 and BRCA2 and their haplotypes between early-onset breast cancer cases who did and did not carry deleterious mutations in these genes. Although our data provide only weak evidence for a difference in frequencies at the population level, the number of deleterious mutation carriers was low and the results may yet be substantiated in a larger study using pooled data.