Value of Vitamin D Metabolite Ratios in 3 Patients as Diagnostic Criteria to Assess Vitamin D Status.

Although clinical guidelines recommend measuring total plasma 25-hydroxyvitamin D (25[OH]D) to assess vitamin D (VitD) status, this index does not account for 3-fold inter-individual variation in VitD binding protein (VDBP) level. We present 3 individuals with total plasma 25(OH)D levels of 10.8 to 12.3 ng/mL (27-30.7 nmol/L). Because Endocrine Society guidelines define VitD deficiency as 25(OH)D ≤ 20 ng/mL (50 nmol/L), all 3 would be judged to be VitD deficient. VitD3 supplementation increased 25(OH)D to the range of 31.7 to 33.8 ng/mL (79.1-84.4 nmol/L). Patient #1 exhibited secondary hyperparathyroidism; VitD3 supplementation decreased parathyroid hormone (PTH) by 34% without a clinically significant change in PTH levels in the other 2 individuals. Thus, 25(OH)D level did not distinguish between the 1 patient who had secondary hyperparathyroidism and the 2 who did not. We therefore inquired whether VitD metabolite ratios (which are VDBP-independent) might distinguish among these 3 individuals. Of all the assessed ratios, the 1,25(OH)2D/24,25(OH)2D ratio was the most informative, which had a value of 102 pg/ng in the individual with secondary hyperparathyroidism but lower values (41 and 20 pg/ng) in the other 2 individuals. These cases illustrate the value of the 1,25(OH)2D/24,25(OH)2D ratio to provide clinically relevant information about VitD status.

Acute pharmacodynamic responses to sitagliptin: Drug-induced increase in early insulin secretion in oral glucose tolerance test.

DPP4 inhibitors are widely prescribed as treatments for type 2 diabetes. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses. Sitagliptin (100 mg) was administered to 47 healthy volunteers. Several endpoints were measured to assess clinically relevant responses - including the effect of sitagliptin on glucose and insulin levels during an oral glucose tolerance test (OGTT). This pilot study confirmed that sitagliptin (100 mg) decreased the area under the curve for glucose during an OGTT (p = 0.0003). Furthermore, sitagliptin promoted insulin secretion during the early portion of the OGTT as reflected by an increase in the ratio of plasma insulin at 30 min divided by plasma insulin at 60 min (T30:T60) from mean ± SEM 0.87 ± 0.05 to 1.62 ± 0.36 mU/L (p = 0.04). The magnitude of sitagliptin's effect on insulin secretion (as judged by the increase in the T30:T60 ratio for insulin) was correlated with the magnitude of sitagliptin-induced increase in the area under the curve for intact plasma GLP1 levels during the first hour of the OGTT. This study confirmed previously reported sex differences in glucose and insulin levels during an OGTT. Specifically, females exhibited higher levels of glucose and insulin at the 90-180 min time points. However, we did not detect significant sex-associated differences in the magnitude of sitagliptin-induced changes in T30:T60 ratios for either glucose or insulin. In conclusion, T30:T60 ratios for insulin and glucose during an OGTT provide useful indices to assess pharmacodynamic responses to DPP4 inhibitors.

Critical Role for 24-Hydroxylation in Homeostatic Regulation of Vitamin D Metabolism.

CONTEXT: The body has evolved homeostatic mechanisms to maintain free levels of Ca+2 and 1,25-dihydroxyvitamin D [1,25(OH)2D] within narrow physiological ranges. Clinical guidelines emphasize important contributions of PTH in maintaining this homeostasis. OBJECTIVE: To investigate mechanisms of homeostatic regulation of vitamin D (VitD) metabolism and to apply mechanistic insights to improve clinical assessment of VitD status. DESIGN: Crossover clinical trial studying participants before and after VitD3-supplementation. SETTING: Community. PARTICIPANTS: 11 otherwise healthy individuals with VitD-deficiency (25-hydroxyvitamin D [25(OH)D] ≤20 ng/mL). INTERVENTIONS: VitD3-supplements (50,000 IU once or twice a week depending on BMI, for 4-6 weeks) were administered to achieve 25(OH)D≥30 ng/mL. RESULTS: VitD3-supplementation significantly increased mean 25(OH)D by 2.7-fold and 24,25-dihydroxyvitamin D [24,25(OH)2D] by 4.3-fold. In contrast, mean levels of PTH, FGF23, and 1,25(OH)2D did not change. Mathematical modeling suggested that 24-hydroxylase activity was maximal for 25(OH)D≥50 ng/mL and achieved a minimum (∼90% suppression) with 25(OH)D<10-20 ng/mL. The 1,25(OH)2D/24,25(OH)2D ratio better predicted modeled 24-hydroxylase activity (h) (ρ=-0.85; p=0.001) compared to total plasma 25(OH)D (ρ=0.51; p=0.01) and the 24,25(OH)2D/25(OH)D ratio (ρ=0.37; p=0.3). CONCLUSIONS: Suppression of 24-hydroxylase provides a first line of defense against symptomatic VitD-deficiency by decreasing metabolic clearance of 1,25(OH)2D. The 1,25(OH)2D/24,25(OH)2D ratio provides a useful index of VitD status since it incorporates 24,25(OH)2D levels and therefore, provides insight into 24-hydroxylase activity. When VitD availability is limited, this suppresses 24-hydroxylase activity - thereby decreasing the level of 24,25(OH)2D and increasing the 1,25(OH)2D/24,25(OH)2D ratio. Thus, an increased 1,25(OH)2D/24,25(OH)2D ratio signifies triggering of homeostatic regulation, which occurs at early stages of VitD-deficiency.

Critical Role for 24-Hydroxylation in Homeostatic Regulation of Vitamin D Metabolism.

Context: The body has evolved homeostatic mechanisms to maintain free levels of Ca+2 and 1,25-dihydroxyvitamin D [1,25(OH)2D] within narrow physiological ranges. Clinical guidelines emphasize important contributions of PTH in maintaining this homeostasis. Objective: To investigate mechanisms of homeostatic regulation of vitamin D (VitD) metabolism and to apply mechanistic insights to improve clinical assessment of VitD status. Design: Crossover clinical trial studying participants before and after VitD3-supplementation. Setting: Community. Participants: 11 otherwise healthy individuals with VitD-deficiency (25-hydroxyvitamin D [25(OH)D] ≤20 ng/mL). Interventions: VitD3-supplements (50,000 IU once or twice a week depending on BMI, for 4-6 weeks) were administered to achieve 25(OH)D≥30 ng/mL. Results: VitD3-supplementation significantly increased mean 25(OH)D by 2.7-fold and 24,25-dihydroxyvitamin D [24,25(OH)2D] by 4.3-fold. In contrast, mean levels of PTH, FGF23, and 1,25(OH)2D did not change. Mathematical modeling suggested that 24-hydroxylase activity was maximal for 25(OH)D≥50 ng/mL and achieved a minimum (~90% suppression) with 25(OH)D<10-20 ng/mL. The 1,25(OH)2D/24,25(OH)2D ratio better predicted modeled 24-hydroxylase activity (h) (ρ=-0.85; p=0.001) compared to total plasma 25(OH)D (ρ=0.51; p=0.01) and the 24,25(OH)2D/25(OH)D ratio (ρ=0.37; p=0.3). Conclusions: Suppression of 24-hydroxylase provides a first line of defense against symptomatic VitD-deficiency by decreasing metabolic clearance of 1,25(OH)2D. The 1,25(OH)2D/24,25(OH)2D ratio provides a useful index of VitD status since it incorporates 24,25(OH)2D levels and therefore, provides insight into 24-hydroxylase activity. When VitD availability is limited, this suppresses 24-hydroxylase activity - thereby decreasing the level of 24,25(OH)2D and increasing the 1,25(OH)2D/24,25(OH)2D ratio. Thus, an increased 1,25(OH)2D/24,25(OH)2D ratio signifies triggering of homeostatic regulation, which occurs at early stages of VitD-deficiency.

Vitamin D Deficiency Increases Vulnerability to Canagliflozin-induced Adverse Effects on 1,25-Dihydroxyvitamin D and PTH.

CONTEXT: Canagliflozin has been reported to increase the risk of bone fracture-possibly mediated by decreasing 1,25-dihydroxyvitamin D (1,25(OH)2D) and increasing parathyroid hormone (PTH). OBJECTIVE: This work investigated whether baseline vitamin D (VitD) deficiency renders individuals vulnerable to this adverse effect and whether VitD3 supplementation is protective. METHODS: This community-based, outpatient study had a paired design comparing individual participants before and after VitD3 supplementation. Eleven VitD-deficient (25-hydroxyvitamin D [25(OH)D] ≤ 20 ng/mL) individuals were recruited from the Amish population in Lancaster, Pennsylvania. Participants underwent 2 canagliflozin challenge protocols (300 mg daily for 5 days): the first before and the second after VitD3 supplementation. In the VitD3 supplementation protocol, participants received VitD3 supplementation (50 000 IU once or twice a week depending on body mass index for 4-6 weeks) to achieve 25(OH)D of 30 ng/mL or greater. Two coprimary end points were identified: effects of VitD3 supplementation on canagliflozin-induced changes in 1,25(OH)2D and PTH. Secondary end points included effects of VitD3 supplementation on baseline levels of VitD metabolites and PTH. RESULTS: VitD3 supplementation increased mean 25(OH)D from 16.5 ± 1.6 to 44.3 ± 5.5 ng/mL (P = .0006) and 24,25-dihydroxyvitamin D (24,25(OH)2D) from 1.0 ± 0.1 to 4.3 ± 0.6 ng/mL (P = .0002). Mean 1,25(OH)2D and PTH were unchanged. VitD3 supplementation decreased the magnitude of canagliflozin-induced changes in 1,25(OH)2D (from -31.3%±4.7% to -9.3%±8.3%; P = .04) and PTH (from +36.2%±6.2% to +9.7%±3.7%; P = .005). CONCLUSION: VitD deficiency rendered individuals more vulnerable to adverse effects of canagliflozin on biomarkers associated with bone health. VitD3 supplementation was protective against canagliflozin's short-term adverse effects on 1,25(OH)2D and PTH.

Acute pharmacodynamic responses to sitagliptin: Drug-induced increase in early insulin secretion in oral glucose tolerance test.

Aim: DPP4 inhibitors are widely prescribed as treatments for type 2 diabetes. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses. Methods: Sitagliptin (100 mg) was administered to 47 healthy volunteers. Several endpoints were measured to assess clinically relevant responses - including the effect of sitagliptin on glucose and insulin levels during an oral glucose tolerance test (OGTT). Results: This pilot study confirmed that sitagliptin (100 mg) decreased the area under the curve for glucose during an OGTT (p=0.0003). Furthermore, sitagliptin promoted insulin secretion during the early portion of the OGTT as reflected by an increase in the ratio of plasma insulin at 30 min divided by plasma insulin at 60 min (T30:T60) from 0.87+/-0.05 to 1.62+/-0.36 mU/L (p=0.04). The magnitude of sitagliptin's effect on insulin secretion (as judged by the increase in the T30:T60 ratio for insulin) was correlated with the magnitude of sitagliptin-induced increase in the area under the curve for intact plasma GLP1 levels during the first hour of the OGTT. This study confirmed previously reported sex differences in glucose and insulin levels during an OGTT. Specifically, females exhibited higher levels of glucose and insulin at the 90-180 min time points. However, we did not detect significant sex-associated differences in the magnitude of sitagliptin-induced changes in T30:T60 ratios for either glucose or insulin. Conclusions: T30:T60 ratios for insulin and glucose during an OGTT provide useful indices to assess pharmacodynamic responses to DPP4 inhibitors.

Pharmacogenetics of sodium-glucose co-transporter-2 inhibitors: Validation of a sex-agnostic pharmacodynamic biomarker.

AIM: To validate pharmacodynamic responses to sodium-glucose co-transporter-2 (SGLT2) inhibitors and test for association with genetic variants in SLC5A4, SLC5A9, and SLC2A9. METHODS: Canagliflozin (300 mg), a SGLT2 inhibitor, was administered to 30 healthy volunteers. Several endpoints were measured to assess clinically relevant responses, including drug-induced increases in urinary excretion of glucose, sodium and uric acid. RESULTS: This pilot study confirmed that canagliflozin (300 mg) triggered acute changes in mean levels of several biomarkers: fasting plasma glucose (-4.1 mg/dL; P = 6 × 10-5 ), serum creatinine (+0.05 mg/dL; P = 8 × 10-4 ) and serum uric acid (-0.90 mg/dL; P = 5 × 10-10 ). The effects of sex on glucosuria depended upon how data were normalized. Whereas males' responses were ~60% greater when data were normalized to body surface area, males and females exhibited similar responses when glucosuria was expressed as grams of urinary glucose per gram-creatinine. The magnitude of glucosuria was not significantly correlated with fasting plasma glucose, estimated glomerular filtration rate or age in those healthy individuals without diabetes with an estimated glomerular filtration rate of more than 60 mL/min/1.73m2 . CONCLUSIONS: Normalizing data relative to creatinine excretion will facilitate including data from males and females in a single analysis. Furthermore, because our ongoing pharmacogenomic study (NCT02891954) is conducted in healthy individuals, this will facilitate detection of genetic associations with limited confounding by other factors such as HbA1c and renal function.

Acute pharmacodynamic responses to exenatide: Drug-induced increases in insulin secretion and glucose effectiveness.

AIM: Glucagon-like peptide-1 receptor agonists provide multiple benefits to patients with type 2 diabetes, including improved glycaemic control, weight loss and decreased risk of major adverse cardiovascular events. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses. METHODS: Exenatide (5 µg, subcutaneously) or saline (0.2 ml, subcutaneously) was administered to 62 healthy volunteers. Frequently sampled intravenous glucose tolerance tests were conducted to assess the impact of exenatide on insulin secretion and insulin action. This pilot study was a crossover design in which participants received exenatide and saline in random order. RESULTS: Exenatide increased first phase insulin secretion 1.9-fold (p = 1.9 × 10-9 ) and accelerated the rate of glucose disappearance 2.4-fold (p = 2 × 10-10 ). Minimal model analysis showed that exenatide increased glucose effectiveness (Sg ) by 32% (p = .0008) but did not significantly affect insulin sensitivity (Si ). The exenatide-induced increase in insulin secretion made the largest contribution to interindividual variation in exenatide-induced acceleration of glucose disappearance while interindividual variation in the drug effect on Sg contributed to a lesser extent (ß = 0.58 or 0.27, respectively). CONCLUSIONS: This pilot study provides validation for the value of a frequently sampled intravenous glucose tolerance test (including minimal model analysis) to provide primary data for our ongoing pharmacogenomic study of pharmacodynamic effects of semaglutide (NCT05071898). Three endpoints provide quantitative assessments of the effects of glucagon-like peptide-1 receptor agonists on glucose metabolism: first phase insulin secretion, glucose disappearance rates and glucose effectiveness.

Vitamin D deficiency increases vulnerability to canagliflozin-induced adverse effects on 1,25-dihydroxyvitamin D and PTH.

Context. Canagliflozin has been reported to increase the risk of bone fracture - possibly mediated by decreasing 1,25-dihydroxyvitamin D [1,25(OH) 2 D] and increasing PTH. Objective. To investigate whether baseline vitamin D (VitD) deficiency renders individuals vulnerable to this adverse effect and whether VitD3 supplementation is protective. Design. This study had a paired design comparing individual participants before and after VitD3 supplementation. Setting. Community-based outpatient. Patients. 11 VitD deficient (25-hydroxyvitamin D [25(OH)D] ≤ 20 ng/mL) individuals recruited from the Amish population in Lancaster PA. Interventions. Participants underwent two canagliflozin challenge protocols (300 mg daily for five days): the first before and the second after VitD3 supplementation. In the VitD3 supplementation protocol, participants received VitD3 supplementation (50,000 IU once or twice a week depending on BMI for 4-6 weeks) to achieve 25(OH)D ≥ 30 ng/mL. Main Outcome Measures. Two co-primary endpoints were identified: effects of VitD3 supplementation on canagliflozin-induced changes in 1,25(OH) 2 D and PTH. Secondary endpoints included effects of VitD3 supplementation on baseline levels of VitD metabolites and PTH. Results. VitD3 supplementation increased mean 25(OH)D from 16.5±1.6 to 44.3±5.5 ng/mL (p=0.0006) and 24,25-dihydroxyvitamin D [24,25(OH) 2 D] from 1.0±0.1 to 4.3±0.6 ng/mL (p=0.0002). Mean 1,25(OH) 2 D and PTH were unchanged. VitD3 supplementation decreased the magnitude of canagliflozin-induced changes in 1,25(OH) 2 D (from -31.3%±4.7% to -9.3%±8.3%; p=0.04) and PTH (from +36.2%±6.2% to +9.7%±3.7%; p=0.005). Conclusions. VitD deficiency rendered individuals more vulnerable to adverse effects of canagliflozin on biomarkers associated with bone health. VitD3 supplementation was protective against canagliflozin's short-term adverse effects on 1,25(OH) 2 D and PTH.

Pharmacogenetics of SGLT2 Inhibitors: Validation of a sex-agnostic pharmacodynamic biomarker.

Aim: SGLT2 inhibitors provide multiple benefits to patients with type 2 diabetes - including improved glycemic control and decreased risks of cardiorenal disease. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses. Methods: Canagliflozin (300 mg) was administered to 30 healthy volunteers. Several endpoints were measured to assess clinically relevant responses - including drug-induced increases in urinary excretion of glucose, sodium, and uric acid. Results: This pilot study confirmed that canagliflozin (300 mg) triggered acute changes in mean levels of several biomarkers: fasting plasma glucose (-4.1 mg/dL; p=6x10), serum creatinine (+0.05 mg/dL; p=8×10 -4 ), and serum uric acid (-0.90 mg/dL; p=5×10 -10 ). The effects of sex on glucosuria depended upon how data were normalized. Whereas males' responses were ∼60% greater when data were normalized to body surface area, males and females exhibited similar responses when glucosuria was expressed as grams of urinary glucose per gram-creatinine. The magnitude of glucosuria was not significantly correlated with fasting plasma glucose, estimated GFR, or age in these healthy non-diabetic individuals with estimated GFR>60 mL/min/1.73m 2 . Conclusions: Normalizing data relative to creatinine excretion will facilitate including data from males and females in a single analysis. Furthermore, because our ongoing pharmacogenomic study ( NCT02891954 ) is conducted in healthy individuals, this will facilitate detection of genetic associations with limited confounding by other factors such as age and renal function. Registration: NCT02462421 ( clinicaltrials.gov ). Funding: Research grants from the National Institute of Diabetes and Digestive and Kidney Diseases: R21DK105401, R01DK108942, T32DK098107, and P30DK072488.

Acute pharmacodynamic responses to exenatide: Drug-induced increases in insulin secretion and glucose effectiveness.

Background: GLP1R agonists provide multiple benefits to patients with type 2 diabetes - including improved glycemic control, weight loss, and decreased risk of major adverse cardiovascular events. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses. Methods: Exenatide (5 µg, sc) or saline (0.2 mL, sc) was administered to 62 healthy volunteers. Frequently sampled intravenous glucose tolerance tests were conducted to assess the impact of exenatide on insulin secretion and insulin action. This pilot study was designed as a crossover study in which participants received exenatide and saline in random order. Results: Exenatide increased first phase insulin secretion 1.9-fold (p=1.9×10 -9 ) and accelerated the rate of glucose disappearance 2.4-fold (p=2×10 -10 ). Minimal model analysis demonstrated that exenatide increased glucose effectiveness (S g ) by 32% (p=0.0008) but did not significantly affect insulin sensitivity (S i ). The exenatide-induced increase in insulin secretion made the largest contribution to inter-individual variation in exenatide-induced acceleration of glucose disappearance while inter-individual variation in the drug effect on S g contributed to a lesser extent (ß=0.58 or 0.27, respectively). Conclusions: This pilot study provides validation for the value of an FSIGT (including minimal model analysis) to provide primary data for our ongoing pharmacogenomic study of pharmacodynamic effects of semaglutide ( NCT05071898 ). Three endpoints provide quantitative assessments of GLP1R agonists' effects on glucose metabolism: first phase insulin secretion, glucose disappearance rates, and glucose effectiveness. Registration: NCT02462421 (clinicaltrials.gov). Funding: American Diabetes Association (1-16-ICTS-112); National Institute of Diabetes and Digestive and Kidney Disease (R01DK130238, T32DK098107, P30DK072488).

Model for Integration of Monogenic Diabetes Diagnosis Into Routine Care: The Personalized Diabetes Medicine Program.

OBJECTIVE: To implement, disseminate, and evaluate a sustainable method for identifying, diagnosing, and promoting individualized therapy for monogenic diabetes. RESEARCH DESIGN AND METHODS: Patients were recruited into the implementation study through a screening questionnaire completed in the waiting room or through the patient portal, physician recognition, or self-referral. Patients suspected of having monogenic diabetes based on the processing of their questionnaire and other data through an algorithm underwent next-generation sequencing for 40 genes implicated in monogenic diabetes and related conditions. RESULTS: Three hundred thirteen probands with suspected monogenic diabetes (but most diagnosed with type 2 diabetes) were enrolled from October 2014 to January 2019. Sequencing identified 38 individuals with monogenic diabetes, with most variants found in GCK or HNF1A. Positivity rates for ascertainment methods were 3.1% for clinic screening, 5.3% for electronic health record portal screening, 16.5% for physician recognition, and 32.4% for self-referral. The algorithmic criterion of non-type 1 diabetes before age 30 years had an overall positivity rate of 15.0%. CONCLUSIONS: We successfully modeled the efficient incorporation of monogenic diabetes diagnosis into the diabetes care setting, using multiple strategies to screen and identify a subpopulation with a 12.1% prevalence of monogenic diabetes by molecular testing. Self-referral was particularly efficient (32% prevalence), suggesting that educating the lay public in addition to clinicians may be the most effective way to increase the diagnosis rate in monogenic diabetes. Scaling up this model will assure access to diagnosis and customized treatment among those with monogenic diabetes and, more broadly, access to personalized medicine across disease areas.

Impact of parental relatedness on reproductive outcomes among the Old Order Amish of Lancaster County.

Genetically isolated populations that arise due to recent bottleneck events have reduced genetic variation reflecting the common set of founders. Increased genetic relatedness among members of isolated populations puts them at increased risk for some recessive disorders that are rare in outbred populations. To assess the burden on reproductive health, we compared frequencies of adverse reproductive outcomes between Amish couples who were both heterozygous carriers of a highly penetrant pathogenic or likely pathogenic variant and noncarrier couples from the same Amish community. In addition, we evaluated whether overall genetic relatedness of parents was associated with reproductive outcomes. Of the 1824 couples included in our study, 11.1% were at risk of producing a child with an autosomal recessive disorder. Carrier couples reported a lower number of miscarriages compared to noncarrier couples (p = 0.02), although the number of stillbirths (p = 0.3), live births (p = 0.9), and number of pregnancies (p = 0.9) did not differ significantly between groups. In contrast, higher overall relatedness between spouses was positively correlated with number of live births (p < 0.0001), pregnancies (p < 0.0001), and stillbirths (p = 0.03), although not with the number of miscarriages (p = 0.4). These results highlight a complex association between relatedness of parents and reproductive health outcomes in this community.

Revisiting the Case of Sarah Newbury's Death from Mollities Ossium.

Multiple myeloma and its precursor and variant types represent some of the most common hematologic malignancies in adults. These plasma cell dyscrasias are well-known in modern medicine. There are well-established clinical, laboratory, and pathologic criteria for diagnosis and staging. There is debate about the diagnosis of some of the earliest cases of myeloma described in the literature. We present a critical review of one such case.

Evaluating the heterogeneous effect of a modifiable risk factor on suicide: The case of vitamin D deficiency.

OBJECTIVES: To illustrate the use of machine learning methods to search for heterogeneous effects of a target modifiable risk factor on suicide in observational studies. The illustration focuses on secondary analysis of a matched case-control study of vitamin D deficiency predicting subsequent suicide. METHODS: We describe a variety of machine learning methods to search for prescriptive predictors; that is, predictors of significant variation in the association between a target risk factor and subsequent suicide. In each case, the purpose is to evaluate the potential value of selective intervention on the target risk factor to prevent the outcome based on the provisional assumption that the target risk factor is causal. The approaches illustrated include risk modeling based on the super learner ensemble machine learning method, Least Absolute Shrinkage and Selection Operator (Lasso) penalized regression, and the causal forest algorithm. RESULTS: The logic of estimating heterogeneous intervention effects is exposited along with the illustration of some widely used methods for implementing this logic. CONCLUSIONS: In addition to describing best practices in using the machine learning methods considered here, we close with a discussion of broader design and analysis issues in planning an observational study to investigate heterogeneous effects of a modifiable risk factor.

Genetic and functional evidence links a missense variant in B4GALT1 to lower LDL and fibrinogen.

Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 10­19) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 10­5). B4GALT1 gene­based analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.

The burden of pathogenic variants in clinically actionable genes in a founder population.

Founder populations may be enriched with certain genetic variants of high clinical impact compared to nonfounder populations due to bottleneck events and genetic drift. Using exome sequencing (ES), we quantified the load of pathogenic variants that may be clinically actionable in 6136 apparently healthy adults living in the Lancaster, PA Old Order Amish settlement. We focused on variants in 78 genes deemed clinically actionable by the American College of Medical Genetics and Genomics (ACMG) or Geisinger's MyCode Health Initiative. ES revealed 3191 total variants among these genes including 480 nonsynonymous variants. After quality control and filtering, we applied the ACMG/AMP guidelines for variant interpretation and classified seven variants, across seven genes, as either pathogenic or likely pathogenic. Through genetic drift, all seven variants, are highly enriched in the Amish compared to nonfounder populations. In total, 14.7% of Lancaster Amish individuals carry at least one of these variants, largely explained by the 13% who harbor a copy of a single variant in APOB. Other studies report combined frequencies of pathogenic/likely pathogenic (P/LP) variants in actionable genes between 2.0% and 6.2% in outbred populations. The Amish population harbors fewer actionable variants compared to similarly characterized nonfounder populations but have a higher frequency of each variant identified, offering opportunities for efficient and cost-effective targeted precision medicine.

Surveillance of Depleted Uranium-exposed Gulf War Veterans: More Evidence for Bone Effects.

ABSTRACT: Gulf War I veterans who were victims of depleted uranium (DU) "friendly-fire" incidents have undergone longitudinal health surveillance since 1994. During the spring of 2019, 36 members of the cohort were evaluated with a monitoring protocol including exposure assessment for total and isotopic uranium concentrations in urine and a comprehensive review of health outcomes, including measures of bone metabolism and bone mineral density (BMD) determination. Elevated urine U concentrations were observed in cohort members with retained depleted uranium (DU) shrapnel fragments. In addition, a measure of bone resorption, N-telopeptide, showed a statistically significant increase in those in the high DU subgroup, a finding consistent with a statistically significant decrease in bone mass also observed in this high DU subgroup compared to the low DU subgroup. After more than 25 y since first exposure to DU, an aging cohort of military veterans continues to show few U-related health effects in known target organs of U toxicity. The new finding of impaired BMD in the high DU subgroup has now been detected in two consecutive surveillance visits. While this is a biologically plausible uranium effect, it is not reflected in other measures of bone metabolism in the full cohort, which have largely been within normal limits. However, ongoing accrual of the U burden from fragment absorption over time and the effect of aging further impairing BMD suggest the need for future surveillance assessments of this cohort.

Heterozygosity for a Pathogenic Variant in SLC12A3 That Causes Autosomal Recessive Gitelman Syndrome Is Associated with Lower Serum Potassium.

BACKGROUND: Potassium levels regulate multiple physiologic processes. The heritability of serum potassium level is moderate, with published estimates varying from 17% to 60%, suggesting genetic influences. However, the genetic determinants of potassium levels are not generally known. METHODS: A whole-exome sequencing association study of serum potassium levels in 5812 subjects of the Old Order Amish was performed. A dietary salt intervention in 533 Amish subjects estimated interaction between p.R642G and sodium intake. RESULTS: A cluster of variants, spanning approximately 537 kb on chromosome 16q13, was significantly associated with serum potassium levels. Among the associated variants, a known pathogenic variant of autosomal recessive Gitelman syndrome (p.R642G SLC12A3) was most likely causal; there were no homozygotes in our sample. Heterozygosity for p.R642G was also associated with lower chloride levels, but not with sodium levels. Notably, p.R642G showed a novel association with lower serum BUN levels. Heterozygotes for p.R642G had a two-fold higher rate of self-reported bone fractures and had higher resting heart rates on a low-salt diet compared with noncarriers. CONCLUSIONS: This study provides evidence that heterozygosity for a pathogenic variant in SLC12A3 causing Gitelman syndrome, a canonically recessive disorder, contributes to serum potassium concentration. The findings provide insights into SLC12A3 biology and the effects of heterozygosity on electrolyte homeostasis and related subclinical phenotypes that may have implications for personalized medicine and nutrition.

KCNQ1 and Long QT Syndrome in 1/45 Amish: The Road From Identification to Implementation of Culturally Appropriate Precision Medicine.

BACKGROUND: In population-based research exome sequencing, the path from variant discovery to return of results is not well established. Variants discovered by research exome sequencing have the potential to improve population health. METHODS: Population-based exome sequencing and agnostic ExWAS were performed 5521 Amish individuals. Additional phenotyping and in vitro studies enabled reclassification of a KCNQ1 variant from variant of unknown significance to pathogenic. Results were returned to participants in a community setting. RESULTS: A missense variant was identified in KCNQ1 (c.671C>T, p.T224M), a gene associated with long QT syndrome type 1, which can cause syncope and sudden cardiac death. The p.T224M variant, present in 1/45 Amish individuals is rare in the general population (1/248 566 in gnomAD) and was highly associated with QTc on electro-cardiogram (P=5.53E-24, ß=20.2 ms/allele). Because of the potential importance of this variant to the health of the population, additional phenotyping was performed in 88 p.T224M carriers and 54 noncarriers. There was stronger clinical evidence of long QT syndrome in carriers (38.6% versus 5.5%, P=0.0006), greater history of syncope (32% versus 17%, P=0.020), and higher rate of sudden cardiac death in first degree relatives