G protein-coupled receptor (GPCR) gene variants and human genetic disease.

Genetic variations in the genes encoding G protein-coupled receptors (GPCRs) can disrupt receptor structure and function, which can result in human genetic diseases. Disease-causing mutations have been reported in at least 55 GPCRs for more than 66 monogenic diseases in humans. The spectrum of pathogenic and likely pathogenic variants includes loss of function variants that decrease receptor signaling on one extreme and gain of function that may result in biased signaling or constitutive activity, originally modeled on prototypical rhodopsin GPCR variants identified in retinitis pigmentosa, on the other. GPCR variants disrupt ligand binding, G protein coupling, accessory protein function, receptor desensitization and receptor recycling. Next generation sequencing has made it possible to identify variants of uncertain significance (VUS). We discuss variants in receptors known to result in disease and in silico strategies for disambiguation of VUS such as sorting intolerant from tolerant and polymorphism phenotyping. Modeling of variants has contributed to drug development and precision medicine, including drugs that target the melanocortin receptor in obesity and interventions that reverse loss of gonadotropin-releasing hormone receptor from the cell surface in idiopathic hypogonadotropic hypogonadism. Activating and inactivating variants of the calcium sensing receptor (CaSR) gene that are pathogenic in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia have enabled the development of calcimimetics and calcilytics. Next generation sequencing has continued to identify variants in GPCR genes, including orphan receptors, that contribute to human phenotypes and may have therapeutic potential. Variants of the CaSR gene, some encoding an arginine-rich region that promotes receptor phosphorylation and intracellular retention, have been linked to an idiopathic epilepsy syndrome. Agnostic strategies have identified variants of the pyroglutamylated RF amide peptide receptor gene in intellectual disability and G protein-coupled receptor 39 identified in psoriatic arthropathy. Coding variants of the G protein-coupled receptor L1 (GPR37L1) orphan receptor gene have been identified in a rare familial progressive myoclonus epilepsy. The study of the role of GPCR variants in monogenic, Mendelian phenotypes has provided the basis of modeling the significance of more common variants of pharmacogenetic significance.

Ancient human genomes suggest three ancestral populations for present-day Europeans.

We sequenced the genomes of a ∼7,000-year-old farmer from Germany and eight ∼8,000-year-old hunter-gatherers from Luxembourg and Sweden. We analysed these and other ancient genomes with 2,345 contemporary humans to show that most present-day Europeans derive from at least three highly differentiated populations: west European hunter-gatherers, who contributed ancestry to all Europeans but not to Near Easterners; ancient north Eurasians related to Upper Palaeolithic Siberians, who contributed to both Europeans and Near Easterners; and early European farmers, who were mainly of Near Eastern origin but also harboured west European hunter-gatherer related ancestry. We model these populations' deep relationships and show that early European farmers had ∼44% ancestry from a 'basal Eurasian' population that split before the diversification of other non-African lineages.

Stage Dependence, Cell-Origin Independence, and Prognostic Capacity of Serum Glycan Fucosylation, ß1-4 Branching, ß1-6 Branching, and α2-6 Sialylation in Cancer.

Glycans represent a promising but only marginally accessed source of cancer markers. We previously reported the development of a molecularly bottom-up approach to plasma and serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, ß1-6 branching, and core fucosylation as single analytical signals. Based on the behavior of P/S glycans established to date, we hypothesized that the alteration of P/S glycans observed in cancer would be independent of the tissue in which the tumor originated yet exhibit stage dependence that varied little between cancers classified on the basis of tumor origin. Herein, the diagnostic utility of this bottom-up approach as applied to lung cancer patients (n = 127 stage I; n = 20 stage II; n = 81 stage III; and n = 90 stage IV) as well as prostate (n = 40 stage II), serous ovarian (n = 59 stage III), and pancreatic cancer patients (n = 15 rapid autopsy) compared to certifiably healthy individuals (n = 30), nominally healthy individuals (n = 166), and risk-matched controls (n = 300) is reported. Diagnostic performance in lung cancer was stage-dependent, with markers for terminal (total) fucosylation, α2-6 sialylation, ß1-4 branching, ß1-6 branching, and outer-arm fucosylation most able to differentiate cases from controls. These markers behaved in a similar stage-dependent manner in other types of cancer as well. Notable differences between certifiably healthy individuals and case-matched controls were observed. These markers were not significantly elevated in liver fibrosis. Using a Cox proportional hazards regression model, the marker for α2-6 sialylation was found to predict both progression and survival in lung cancer patients after adjusting for age, gender, smoking status, and stage. The potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.

Predictors of 25-Hydroxyvitamin D Concentration Measured at Multiple Time Points in a Multiethnic Population.

The evidence for a relationship between serum vitamin D levels and nonskeletal health outcomes is inconsistent. The validity of single or predicted measurements of 25-hydroxyvitamin D (25(OH)D) concentration is unknown, as levels of this biomarker are highly seasonally variable. We compared models of 25(OH)D measured at baseline, at multiple time points throughout the year, and averaged over the year among 309 persons in Toronto, Ontario, Canada (43°N latitude) during 2009-2013. Information and blood samples were collected every 2 months. Baseline and average 25(OH)D concentrations were correlated (r = 0.88). Major factors associated with 25(OH)D level were similar across models and included race/ethnicity (concentrations in non-European groups were lower than those in Europeans), vitamin D supplement use of ≥1,000 IU/day (18.9 nmol/L (95% confidence interval (CI): 16.1, 21.8) vs. no supplement use in a full data set with all factors), and the presence of the group-specific component/vitamin D binding protein gene (GC/DBP) rs4588 functional polymorphism (AA vs. CC: -16.7 nmol/L (95% CI: -26.2, -7.1); CA vs. CC: -10.7 nmol/L (95% CI: -14.9, -6.5)). Most factors had similar associations in Europeans and non-Europeans. Genetic factors may play a greater role in average 25(OH)D concentrations. Prediction models for 25(OH)D are challenging and population-specific, but use of genetic factors along with a few common population-relevant, quantifiable nongenetic factors with strong associations may be the most feasible approach to vitamin D assessment over time.

Large intragenic deletion of CDC73 (exons 4-10) in a three-generation hyperparathyroidism-jaw tumor (HPT-JT) syndrome family.

BACKGROUND: Inactivating mutations of CDC73 cause Hyperparathyroidism-Jaw Tumour syndrome (HPT-JT), Familial Isolated Hyperparathyroidism (FIHP) and sporadic parathyroid carcinoma. We conducted CDC73 mutation analysis in an HPT-JT family and confirm carrier status of the proband's daughter. METHODS: The proband had primary hyperparathyroidism (parathyroid carcinoma) and uterine leiomyomata. Her father and daughter had hyperparathyroidism (parathyroid adenoma) but no other manifestations of HPT-JT. CDC73 mutation analysis (sequencing of all 17 exons) and whole-genome copy number variation (CNV) analysis was done on leukocyte DNA of the three affecteds as well as the proband's unaffected sister. RESULTS: A novel deletion of exons 4 to 10 of CDC73 was detected by CNV analysis in the three affecteds. A novel insertion in the 5'UTR (c.-4_-11insG) that co-segregated with the deletion was identified. By in vitro assay the 5'UTR insertion was shown to significantly impair the expression of the parafibromin protein. Screening for the mutated CDC73 confirmed carrier status in the proband's daughter and the biochemistry and ultrasonography led to pre-emptive surgery and resolution of the hyperparathyroidism. CONCLUSIONS: A novel gross deletion mutation in CDC73 was identified in a three-generation HPT-JT family emphasizing the importance of including screening for large deletions in the molecular diagnostic protocol.

Variance Formulae for Correlation Measures of Linkage Disequilibrium.

BACKGROUND: Linkage disequilibrium (LD) is the non-random association between alleles at different loci and remains important for disease mapping studies in humans. A common measure of LD is the sample correlation between indicator variables for alleles at the 2 loci. Knowledge of LD estimate precision may help inform biomedical decisions based on those estimates. OBJECTIVES AND METHODS: Variance formulae are obtained for correlation measures of LD in 4 scenarios. These scenarios include data in the form of gametic and genotypic counts, with different assumptions used to simplify the analysis. RESULTS: The formulae are expressed as polynomials (or ratios of polynomials) in higher-order disequilibrium coefficients with constants which are functions of the allele frequencies and Hardy-Weinberg disequilibrium coefficients. With genotypic data, the variance is the same as with gametic data when the phase is known and there is random mating. When the phase is unknown, the correlation LD has variance which is twice as large. CONCLUSIONS: Symbolic computation proved to be effective in facilitating algebraic derivations which would otherwise have been intractable.

Neurogenetic Aspects of Hyperphosphatasia in Mabry Syndrome.

An autosomal recessive syndrome of hyperphosphatasia (elevated circulating alkaline phosphatase (AP), seizures and neurologic deficits) was first described by Mabry and colleagues in 1970. Over the ensuing four decades, few cases were reported. In 2010, however, new families were identified and the syndromic nature of the disorder confirmed. Shortly thereafter, next generation sequencing was used to characterize causative defects in the glycosyl phosphatidylinositol (GPI) biosynthetic pathway, based partly on our understanding of how AP is anchored by GPI to the plasma membrane. Whether the seizures and cognitive defects seen in Mabry syndrome patients are attributable in part to the constant hyperphosphatasia is not known, as there are more than 250 other proteins dependent on GPI for their anchoring to the plasma membrane. However, Mabry syndrome may provide a new window on AP function in growth and development.

The causal effect of vitamin D binding protein (DBP) levels on calcemic and cardiometabolic diseases: a Mendelian randomization study.

BACKGROUND: Observational studies have shown that vitamin D binding protein (DBP) levels, a key determinant of 25-hydroxy-vitamin D (25OHD) levels, and 25OHD levels themselves both associate with risk of disease. If 25OHD levels have a causal influence on disease, and DBP lies in this causal pathway, then DBP levels should likewise be causally associated with disease. We undertook a Mendelian randomization study to determine whether DBP levels have causal effects on common calcemic and cardiometabolic disease. METHODS AND FINDINGS: We measured DBP and 25OHD levels in 2,254 individuals, followed for up to 10 y, in the Canadian Multicentre Osteoporosis Study (CaMos). Using the single nucleotide polymorphism rs2282679 as an instrumental variable, we applied Mendelian randomization methods to determine the causal effect of DBP on calcemic (osteoporosis and hyperparathyroidism) and cardiometabolic diseases (hypertension, type 2 diabetes, coronary artery disease, and stroke) and related traits, first in CaMos and then in large-scale genome-wide association study consortia. The effect allele was associated with an age- and sex-adjusted decrease in DBP level of 27.4 mg/l (95% CI 24.7, 30.0; n = 2,254). DBP had a strong observational and causal association with 25OHD levels (p = 3.2 × 10(-19)). While DBP levels were observationally associated with calcium and body mass index (BMI), these associations were not supported by causal analyses. Despite well-powered sample sizes from consortia, there were no associations of rs2282679 with any other traits and diseases: fasting glucose (0.00 mmol/l [95% CI -0.01, 0.01]; p = 1.00; n = 46,186); fasting insulin (0.01 pmol/l [95% CI -0.00, 0.01,]; p = 0.22; n = 46,186); BMI (0.00 kg/m(2) [95% CI -0.01, 0.01]; p = 0.80; n = 127,587); bone mineral density (0.01 g/cm(2) [95% CI -0.01, 0.03]; p = 0.36; n = 32,961); mean arterial pressure (-0.06 mm Hg [95% CI -0.19, 0.07]); p = 0.36; n = 28,775); ischemic stroke (odds ratio [OR]  = 1.00 [95% CI 0.97, 1.04]; p = 0.92; n = 12,389/62,004 cases/controls); coronary artery disease (OR = 1.02 [95% CI 0.99, 1.05]; p = 0.31; n = 22,233/64,762); or type 2 diabetes (OR = 1.01 [95% CI 0.97, 1.05]; p = 0.76; n = 9,580/53,810). CONCLUSIONS: DBP has no demonstrable causal effect on any of the diseases or traits investigated here, except 25OHD levels. It remains to be determined whether 25OHD has a causal effect on these outcomes independent of DBP. Please see later in the article for the Editors' Summary.

Effect of vitamin D-binding protein genotype on the development of asthma in children.

BACKGROUND: Potential vitamin D-related influences on inflammatory diseases such as asthma are controversial, including the suggestion that vitamin D insufficiency is associated with increased asthma morbidity. Vitamin D-binding protein transports vitamin D metabolites in the circulation. Single nucleotide polymorphisms in the GC gene encoding vitamin D-binding protein are associated with circulating vitamin D metabolite levels in healthy infants and toddlers. OBJECTIVE: To test the hypothesis that GC single nucleotide polymorphisms encoding the D432E and T436K variants predict subsequent development of asthma in healthy children. METHODS: A retrospective medical record review was performed to determine the development of asthma in 776 children in whom GC genotype, vitamin D-binding protein concentration, and circulating 25-hydroxyvitamin D had been determined at 6 to 36 months of age. Demographic and detailed current clinical data were collected and criteria for asthma were recorded. RESULTS: GC genotype was available for 463 subjects. After an initial analysis of all subject data, the analysis was limited to the predominant Hispanic population (72.1%) to minimize potential confounding effects of ethnicity. Asthma was diagnosed in 87 children (26%). Subjects with the GC genotype encoding the ET/ET (Gc1s/Gc1s) variant had lower odds of developing asthma, representing a protective effect compared with subjects with the DT/DT (Gc1f/Gc1f) variant. CONCLUSION: In the Hispanic population of inner-city New Haven, Connecticut, the ET/ET (Gc1s/Gc1s) genotype of vitamin D-binding protein might confer protection against the development of asthma compared with the wild-type genotype DT/DT (Gc1f/Gc1f).

Urinary Vitamin D Binding Protein: A Marker of Kidney Tubular Dysfunction in Patients at Risk for Type 2 Diabetes.

Context: Recent studies have reported elevated urinary vitamin D binding protein (uVDBP) concentrations in patients with diabetic kidney disease, although the utility of uVDBP to predict deterioration of kidney function over time has not been examined. Objective: Our objective was to assess the association of uVDBP with longitudinal changes in kidney function. Methods: Adults at-risk for type 2 diabetes from the Prospective Metabolism and Islet Cell Evaluation (PROMISE) study had 3 assessments over 6 years (n = 727). Urinary albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) were used as measures of kidney function. Measurements of uVDBP were performed with enzyme-linked immunosorbent assay and normalized to urine creatinine (uVDBP:cr). Generalized estimating equations (GEEs) evaluated longitudinal associations of uVDBP and uVDBP:cr with measures of kidney function, adjusting for covariates. Results: Renal uVDBP loss increased with ACR severity at baseline. Individuals with normoalbuminuria, microalbuminuria, and macroalbuminuria had median log uVDBP:cr concentrations of 1.62 µg/mmol, 2.63 µg/mmol, and 2.48 µg/mmol, respectively, and ACR positively correlated with uVDBP concentrations (r = 0.37; P < .001). There was no significant association between uVDBP and eGFR at baseline. Adjusted longitudinal GEE models indicated that each SD increase both in baseline and longitudinal uVDBP:cr was significantly associated with higher ACR over 6 years (ß = 30.67 and ß = 32.91, respectively). Conversely, neither baseline nor longitudinal uVDBP:cr measures showed a significant association with changes in eGFR over time. These results suggest that loss of uVDBP:cr over time may be a useful marker for predicting renal tubular damage in individuals at risk for diabetes.

Common variants of the vitamin D binding protein gene and adverse health outcomes.

The vitamin D binding protein (DBP) is the major plasma carrier for vitamin D and its metabolites, but it is also an actin scavenger, and is the precursor to the immunomodulatory protein, Gc-MAF. Two missense variants of the DBP gene - rs7041 encoding Asp432Glu and rs4588 encoding Thr436Lys - change the amino acid sequence and alter the protein function. They are common enough to generate population-wide constitutive differences in vitamin D status, based on assay of the serum metabolite, 25-hydroxyvitamin D (25OHD). Whether these variants also influence the role of vitamin D in an immunologic milieu is not known. However, the issue is relevant, given the immunomodulatory effects of DBP and the role of protracted innate immune-related inflammation in response to tissue injury or repeated infection. Indeed, DBP and vitamin D may jointly or independently contribute to a variety of adverse health outcomes unrelated to classical notions of their function in bone and mineral metabolism. This review summarizes the reports to date of associations between DBP variants, and various chronic and infectious diseases. The available information leads us to conclude that DBP variants are a significant and common genetic factor in some common disorders, and therefore, are worthy of closer attention. In view of the heightened interest in vitamin D as a public health target, well-designed studies that look simultaneously at vitamin D and its carrier in relation to genotypes and adverse health outcome should be encouraged.

Genotype-based association analysis using discordant pairs: a penetrance odds ratio approach.

Genotypic counts of paired relatives discordant for a complex late-onset disease are often used to test for genetic association. The power of the various statistical test options, when data on covariates are unavailable, has been the focus of recent research. Comparison of the Cochran-Armitage, Bhapkar, and McNemar tests indicates that none is superior to the others in all cases. Using an alternative approach, we found that the theoretical genotypic frequencies of the discordant pairs depend only on the penetrance odds ratios, after conditioning. These odds ratios can be estimated by maximizing a product binomial likelihood and provide insight into the mode of inheritance. We identified cases where exact maximum likelihood (ML) estimates can be explicitly obtained. This approach led us to two tests for association which depend on likelihood ratio (LR) or score statistics. We quantified the power of these tests analytically and examined their performance through simulation. We explored the utility of these tests with an example from the literature-the association between complement factor H (CFH) polymorphisms and age-related macular degeneration. The LR and Score tests serve as simple and effective ways of interpreting paired case-control data sets.

Genetic variants in the vitamin D pathway and their association with vitamin D metabolite levels: Detailed studies of an inner-city pediatric population suggest a modest but significant effect in early childhood.

OBJECTIVES: In a large cohort of healthy infants and toddlers 6-36 months of age (n = 776), we have been exploring the potential role of genetic variation in predisposition to vitamin D insufficiency. The genes encoding the key cytochrome P450 hydroxylases (CYP2R1, CYP24A1, and CYP27B1) harbour recurrent mutations of uncertain effect. This study was undertaken to look for biochemically relevant associations of these variants with inter-individual differences in vitamin D metabolism in an at-risk pediatric population. METHODS: Genotyping for CYP2R1-CT (c.-1127 C>T, rs10741657), CYP24A1-AG (c.-686A>G, rs111622401), and CYP27B1-CA (c.-1261 C>A, rs10877012) mutations were performed using SNaPshot assay, followed by Sanger sequencing confirmation. Vitamin D metabolites and vitamin D binding protein (DBP) were measured by established methods. RESULTS: In a multivariate regression model, with corrections for co-variates, subjects with the homozygous CYP2R1-TT variant had significantly higher concentrations of 25(OH)D, free 25(OH)D, and 24,25(OH)2D levels. In subjects with the CYP24A1-AG mutation, concentrations of 25(OH)D were significantly higher. CONCLUSIONS: The CYP2R1-TT and CYP24A1-AG variants have measurable effects on the vitamin D pathway. It seems unlikely that they will be clinically relevant in isolation, but they may be members of the large pool of infrequent mutations contributing to different risks for the vitamin D deficiency phenotype.

Phenotypic variability in hyperphosphatasia with seizures and neurologic deficit (Mabry syndrome).

Hyperphosphatasia with neurologic deficit (Mabry syndrome) was first described in a single family (OMIM#239300) by Mabry et al. [1970]. Although considered rare at the time, more than 20 individuals with the triad of developmental disability, seizures, and hyperphosphatasia have been identified world-wide. The 1-6 mannosyltransferase 2, phosphatidylinositol glycan V (PIGV) gene has been found to be disrupted in some patients with the additional feature of brachytelephalangy. In the present report we identify three patients compound homozygous for PIGV mutations. Two siblings were found to be compound heterozygotes for c.467G > A and c.494C > A in exon 3 of PIGV (the c.494C > A PIGV variant is novel). A third patient with similar phenotype, was a compound heterozygote for the known c.1022C > A/c.1022C > T (p.Ala341Glu/p.Ala341Val) mutation. This patient was also noted to have lysosomal storage in cultured fibroblasts. In contrast, the fourth patient who had no apparent hand abnormality, was found to be heterozygous for a previously unclassified c.1369C > T mutation in exon 4 of the PIGV gene, resulting in a p.Leu457Phe substitution in the catalytic domain of the enzyme. Unless this variant has a dominant negative effect, however, it seems likely that another GPI biosynthesis gene variant may contribute to the disorder, possibly through digenic inheritance. Since slightly fewer than half of the nine cases presented in this report and our previous report [Thompson et al., 2010] have PIGV mutations, we suggest that other genes critical to GPI anchor biosynthesis are likely to be disrupted in some patients.

Novel Glial Cells Missing-2 (GCM2) variants in parathyroid disorders.

OBJECTIVE: The aim of this study was to analyze variants of the gene glial cells missing-2 (GCM2), encoding a parathyroid cell-specific transcription factor, in familial hypoparathyroidism and in familial isolated hyperparathyroidism (FIHP) without and with parathyroid carcinoma. DESIGN: We characterized 2 families with hypoparathyroidism and 19 with FIHP in which we examined the mechanism of action of GCM2 variants. METHODS: Leukocyte DNA of hypoparathyroid individuals was Sanger sequenced for CASR, PTH, GNA11 and GCM2 mutations. DNA of hyperparathyroid individuals underwent MEN1, CDKN1B, CDC73, CASR, RET and GCM2 sequencing. The actions of identified GCM2 variants were evaluated by in vitro functional analyses. RESULTS: A novel homozygous p.R67C GCM2 mutation which failed to stimulate transcriptional activity in a luciferase assay was identified in affected members of two hypoparathyroid families. Oligonucleotide pull-down assay and in silico structural modeling indicated that this mutant had lost the ability to bind the consensus GCM recognition sequence of DNA. Two novel (p.I383M and p.T386S) and one previously reported (p.Y394S) heterozygous GCM2 variants that lie within a C-terminal conserved inhibitory domain were identified in three affected individuals of the hyperparathyroid families. One family member, heterozygous for p.I138M, had parathyroid carcinoma (PC), and a heterozygous p.V382M variant was found in another patient affected by sporadic PC. These variants exerted significantly enhanced in vitrotranscriptional activity, including increased stimulation of the PTH promoter. CONCLUSIONS: We provide evidence that two novel GCM2 R67C inactivating mutations with an inability to bind DNA are causative of hypoparathyroidism. Additionally, we provide evidence that two novel GCM2 variants increased transactivation of the PTH promoter in vitro and are associated with FIHP. Furthermore, our studies suggest that activating GCM2 variants may contribute to facilitating more aggressive parathyroid disease.

Mild infantile hypercalcemia: diagnostic tests and outcomes.

OBJECTIVE: To assess outcome in a cohort of patients with infantile hypercalcemia followed over 3 years. STUDY DESIGN: Patients (n = 32) presenting to the calcium clinic between July 2002 and September 2008 were studied. In addition to tests of calcium phosphate metabolism, serum insulin-like growth factor-1, calcitonin, urine citrate, and calcium-sensing receptor gene analysis were obtained. RESULTS: Mean age at presentation was 6.0 ± 6.3 months. Mean calcium level was 11.4 ± 0.7 mg/dL (2.84 ± 0.17 mmol/L). A recognized cause was found in 14% and a probable cause in 14% of the cohort. Those with nephrocalcinosis (n = 11) had significantly lower mean weight SDS and higher mean calcium levels. The biochemical profile of those in whom no cause could be determined included nonsuppressed parathyroid hormone with either normal or increased 1,25(OH)(2)D. Hypercalcemia resolved in 20 patients. However, in approximately a third, there was persistence in hypercalcemia, hypercalciuria, or nephrocalcinosis. CONCLUSIONS: The addition of 1,25(OH)(2)D and calcium-sensing receptor mutation analysis to a panel of investigations may improve diagnostic yield. Clinical outcome is overall good, however, one-third need ongoing follow-up.

Neonatal severe hyperparathyroidism: further clinical and molecular delineation.

UNLABELLED: We report a newborn female from a consanguineous Sri Lankan family with clinical and biochemical features of neonatal severe hyperparathyroidism (NSHPT). Mutation screening of the calcium-sensing receptor (CASR) gene in genomic DNA revealed a homozygous truncating mutation (c.679C>T, predicting p.R227X), confirming the clinical diagnosis. Other mutations at the R227 position are reported to cause varying degrees of hypercalcemia and hyperparathyroidism, but this nonsense variant is novel and expected to induce unremitting hyperparathyroidism from birth onward. In our patient with NSHPT, early bisphosphonate therapy was crucial in counteracting the marked hypercalcemia and allowed for safe surgical intervention ("total" parathyroidectomy, "thymectomy and hemithyroidectomy") at 3 months of age. CONCLUSION: This report highlights the continuing challenges in diagnosis and management of this life-threatening condition.

25-OHD response to vitamin D supplementation in children: effect of dose but not GC haplotype.

OBJECTIVE: GC/DBP effects on response to vitamin D supplementation have not been well-studied. Thus we assessed free and total 25-OHD after vitamin D treatment across the six common GC haplotypes. DESIGN: This double-blind, randomized study compared two vitamin D3 doses in healthy, urban-dwelling 6-month to 10-year-old children at-risk for vitamin D deficiency. Randomization was stratified by GC haplotype. METHODS: Children were randomized to receive 2800 or 7000 International Units of vitamin D3 weekly. 25-OHD and 1,25(OH)2D were sampled at baseline and after 1-6 months of supplementation. RESULTS AND CONCLUSIONS: One hundred ninety-two of 225 enrolled subjects completed the study. After one month, total 25-OHD increased with both doses and were higher with 7000 IU/week (85.5 ± 22.8 nmol/L) compared to 2800 IU/week (76.8 ± 18.0 nmol/L), despite equivalent baseline levels. No further significant increase occurred at 6 months (89.8 ± 35.5 and 74.3 ± 18.3 nmol/L, respectively). Free 25-OHD similarly changed. 25-OHD differed among GC groups at baseline. Although no significant effects of individual GC haplotypes on incremental changes were evident, a trend toward an effect of combined 'at risk' GC alleles on response was evident (P = 0.06). Total 1,25(OH)2D showed modest increases, moreso with the larger dose. In urban-dwelling children at-risk for vitamin D deficiency, 1 month of vitamin D3 2800 IU/week increased 25-OHD across all GC haplotype groups, and somewhat enhanced with 7000 IU/week with no further significant increases after 6 months of supplementation. Free 25-OHD measures offer no monitoring advantage over total 25-OHD.

Attributing Hardy-Weinberg disequilibrium to population stratification and genetic association in case-control studies.

Loci exhibiting Hardy-Weinberg disequilibrium (HWD) are often excluded from association studies, because HWD may indicate genotyping error, population stratification or selection bias. For case-control studies, HWD can result from a genetic effect at the locus. We extend the modelling to accommodate both stratification and genetic effects. Theoretical genotype frequencies and HWD coefficients are derived under a general genetic model for a population with two strata. Maximum likelihood is used to estimate model parameters and a test for lack of fit identifies the models most consistent with the data. Simulations were used to assess the method. The technique was applied to a group of ethnically and clinically heterogeneous kidney stone formers and controls, both exhibiting HWD for the R990G SNP of the CASR gene. Results indicate the best fitting model incorporates both stratification and genetic association. The ability of our method to apportion HWD to stratification and genetic effects may well be a significant advance in dealing with heterogeneity in case-control genetic association studies.