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.

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.

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.

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.

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.

A post glycosylphosphatidylinositol (GPI) attachment to proteins, type 2 (PGAP2) variant identified in Mabry syndrome index cases: Molecular genetics of the prototypical inherited GPI disorder.

We report that recessive inheritance of a post-GPI attachment to proteins 2 (PGAP2) gene variant results in the hyperphosphatasia with neurologic deficit (HPMRS) phenotype described by Mabry et al., in 1970. HPMRS, or Mabry syndrome, is now known to be one of 21 inherited glycosylphosphatidylinositol (GPI) deficiencies (IGDs), or GPI biosynthesis defects (GPIBDs). Bi-allelic mutations in at least six genes result in HPMRS phenotypes. Disruption of four phosphatidylinositol glycan (PIG) biosynthesis genes, PIGV, PIGO, PIGW and PIGY, expressed in the endoplasmic reticulum, result in HPMRS 1, 2, 5 and 6; disruption of the PGAP2 and PGAP3 genes, necessary for stabilizing the association of GPI anchored proteins (AP) with the Golgi membrane, result in HPMRS 3 and 4. We used exome sequencing to identify a novel homozygous missense PGAP2 variant NM_014489.3:c.881C > T, p.Thr294Met in two index patients and targeted sequencing to identify this variant in an unrelated patient. Rescue assays were conducted in two PGAP2 deficient cell lines, PGAP2 KO cells generated by CRISPR/Cas9 and PGAP2 deficient CHO cells, in order to examine the pathogenicity of the PGAP2 variant. First, we used the CHO rescue assay to establish that the wild type PGAP2 isoform 1, translated from transcript 1, is less active than the wild type PGAP2 isoform 8, translated from transcript 12 (alternatively spliced to omit exon 3). As a result, in our variant rescue assays, we used the more active NM_001256240.2:c.698C > T, p.Thr233Met isoform 8 instead of NM_014489.3:c.881C > T, p.Thr294Met isoform 1. Flow cytometric analysis showed that restoration of cell surface CD59 and CD55 with variant PGAP2 isoform 8, driven by the weak (pTA FLAG) promoter, was less efficient than wild type isoform 8. Therefore, we conclude that recessive inheritance of c.881C > T PGAP2, expressed as the hypomorphic PGAP2 c.698C > T, p.Thr233Met isoform 8, results in prototypical Mabry phenotype, HPMRS3 (GPIBD 8 [MIM: 614207]). This study highlights the need for long-term follow up of individuals with rare diseases in order to ensure that they benefit from innovations in diagnosis and treatment.

Relationship of Total and Free 25-Hydroxyvitamin D to Biomarkers and Metabolic Indices in Healthy Children.

CONTEXT: Vitamin D status is usually assessed by serum total 25-hydroxyvitamin D (t25-OHD). Whether free 25-hydroxyvitamin D measures better correlate with various clinical outcomes is unclear. OBJECTIVE: To identify correlations between t25-OHD, calculated and direct measures of free 25-OHD, and to identify associations of these measures with other outcomes in children, across the 6 common GC haplotypes. DESIGN: Healthy urban-dwelling children underwent measurement of relevant variables. SETTING: Academic medical center. PARTICIPANTS: The study included 203 healthy, urban-dwelling children, aged 6 months to 10 years, predominantly of Hispanic background and representative of all common GC haplotypes. INTERVENTION: None. MAIN OUTCOME MEASURES: Total and free 25-OHD and 1,25(OH)2D, calcium, phosphate, parathyroid hormone (PTH), glucose, insulin, aldosterone, and renin. RESULTS: Mean t25-OHD [26.3 ±â€…6.7ng/ml; 65.8 ±â€…16.8nmol/L] were lowest in the GC2 genotype. Mean t1,25(OH)2D [57.6 ±â€…16.5pg/ml; 143.9 ±â€…41.3pmol/L], were lowest in GC1f/1f, GC1f/2, and GC2/2 groups. T25-OHD correlated strongly with calculated free 25-OHD (cf25-OHD) (r = 0.89) and moderately with directly measured free 25-OHD (dmf25-OHD) (r = 0.69). Cf25-OHD correlated with dmf25-OHD (r = 0.69) (P < 0.001 for all). t25-OHD inversely correlated with body mass index (BMI) (r=-0.191; P = 0.006), skin reflectometry, and systolic blood pressure. T25-OHD correlated with fasting insulin and the homeostatic model assessment for insulin resistance (HOMA-IR), however significance for these correlations was not evident after adjustment for BMI. PTH inversely correlated with all measures of 25-OHD, but most strongly with t25-OHD. CONCLUSIONS: Measures of circulating total and free 25-OHD are comparable measures of vitamin D status in heathy children. Correlations are similar with other outcome variables, however t25-OHD remains the strongest correlate of circulating PTH and other variables. These data argue against routine refinement of the t25-OHD measure using currently available assessments of free 25-OHD. CLINICAL TRIAL INFORMATION: Clinicaltrials.gov registration no: NCT01050387 (January 15, 2010).

Parathyroid carcinoma.

Parathyroid carcinoma (PC) is a rare disease with an indolent behavior due to the low malignant potential. The etiology is unknown. Somatic mutations of CDC73 gene, the same gene involved in the hyperparathyroidism-jaw tumor syndrome, can be identified in up to 70% of patients with PC and in one-third of cases the mutations are germline. Therefore, in patients who carry germline CDC73 gene mutations, its finding permits to identify the carriers among relatives and sometimes to early detect a parathyroid lesion in such subjects. The diagnosis of PC is commonly made after surgery, however there are some clinical/biochemical features that should raise the suspicion of PC, namely markedly elevated serum calcium and PTH levels, a large parathyroid lesion with suspected ultrasonographic features of malignancy, the damages of kidney and bones. The best chance of cure is the complete surgical resection with the en-bloc excision at the first operation, however several recurrences are often observed during the follow-up. Since PC is an indolent tumor with long-lasting survival and the death is due to complications of untreatable hypercalcemia, multiple surgical interventions with debulking of tumoral tissues along with medical treatment for reducing hypercalcemia are often needed. Patients with PC should be followed up along their lifetime.

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.

MEN1 gene mutation with parathyroid carcinoma: first report of a familial case.

BACKGROUND: The occurrence of parathyroid carcinoma in multiple endocrine neoplasia type I (MENI) is rare and the 15 cases of malignant parathyroid tumor reported so far have been associated with MENI in individuals and not with multiple members within a family. METHODS: We report on a 61-year-old male, operated for a 7.3 cm parathyroid carcinoma infiltrating the esophagus. In his brother, a 4.6 cm parathyroid carcinoma was diagnosed histologically, while in the daughter, neck ultrasonography revealed 2 extrathyroidal nodules, yet to be excised. RESULTS: Screening of the MEN1 gene identified a known germline heterozygous missense mutation (c.1252G>A; p.D418N) in exon 9, in all affected subjects. CONCLUSIONS: The occurrence of parathyroid carcinoma in more than one affected member of a single MEN1 family represents the first reported familial case. This suggests that additional constitutional genetic mutations may contribute to the variation in malignant potential and clinical behavior of parathyroid tumors in MEN1.

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.

Novel association of MEN1 gene mutations with parathyroid carcinoma.

Inactivating mutations of the multiple endocrine neoplasia 1 (MEN1) gene cause MEN1 syndrome, characterized by primary hyperparathyroidism (pHPT), and parathyroid and gastro-entero-pancreatic pituitary tumors. At present, only 14 cases of malignant parathyroid tumor have been associated with the syndrome, with 6 cases carrying an inactivating mutation of the MEN1 gene. The present study presents the case of a 48-year-old female who presented with multigland pHPT and multiple pancreatic lesions. The patient underwent surgery several times for the excision of parathyroid hyperplasia, carcinoma and adenoma. The MEN1 gene was screened, revealing three variants (in cis) at the intron/exon 3 boundary (IVS2-3G>C, c.497A>T and c.499G>T) detected on the DNA of the proband, not shared by her relatives. RNA sequencing revealed that the IVS2-3C>G variant caused the skipping of the exon 3. Therefore, the present study reports on a novel rare association of MEN1 syndrome and parathyroid carcinoma. The reported splicing mutation was previously identified in subjects who always developed malignant lesions; thus, a possible genotype-phenotype association may be considered.

Association of Directly Measured Plasma Free 25(OH)D With Insulin Sensitivity and Secretion: The IRAS Family Study.

Objectives: We aimed to compare the associations of directly measured plasma free 25-hydroxyvitamin D [25(OH)D] and total 25(OH)D concentrations with insulin sensitivity (SI) and ß-cell function in nondiabetic Hispanics and African Americans. We hypothesized that directly measured free 25(OH)D would be more strongly associated with these measures of glucose homeostasis and that associations would differ by race. Design: We studied 1189 nondiabetic participants in the Insulin Resistance Atherosclerosis Study Family Study using data from baseline examinations from 2000 to 2002. SI, acute insulin response, and disposition index (DI) were determined from frequently sampled intravenous glucose tolerance tests. Plasma free and total 25(OH)D concentrations were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Results: The median concentrations of plasma free 25(OH)D were 3.46 pg/mL for Hispanics and 2.17 pg/mL for African Americans (P < 0.0001), whereas the median concentrations of plasma total 25(OH)D were 16 ng/mL for Hispanics and 10 ng/mL for African Americans (P < 0.0001). Plasma free and total 25(OH)D were both positively associated with SI and DI in generalized estimating equations adjusted for demographic and lifestyle factors. After further adjustment with body mass index, the associations were no longer statistically significant, except for a significant association between plasma free 25(OH)D and SI. There was no effect modification by ethnicity on any of the exposure-outcome associations. Conclusions: Our data showed that plasma free 25(OH)D had a slightly stronger association with SI compared with plasma total 25(OH)D, although the difference was modest and there were no marked differences in the associations between Hispanics and African Americans.

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.

Vitamin D status in primary hyperparathyroidism: effect of genetic background.

Primary hyperparathyroidism (PHPT) is associated with hypovitaminosis D as assessed by serum total 25-hydroxyvitamin D (TotalD) levels. The aim of this study is to evaluate whether this is also the case for the calculated bioavailable 25-hydroxyvitamin D (BioD) or free 25-hydroxyvitamin D (FreeD), and whether the vitamin D status is influenced by genetic background. We compared vitamin D status of 88 PHPT patients each with a matched healthy family member sharing genetic background, i.e., first-degree relative (FDR), or not, namely an in-law relative (ILR). We compared TotalD and vitamin D-binding protein (DBP), using the latter to calculate BioD and FreeD. We also genotyped two common DBP polymorphisms (rs7041 and rs4588) likely to affect the affinity for and levels of vitamin D metabolites. TotalD was lower (p < 0.001) in PHPT (12.3 ± 6.6 ng/mL) than either family member group (FDR: 19.4 ± 12.1 and ILR: 23.2 ± 14.1), whether adjusted for DBP or not. DBP levels were also significantly lower (p < 0.001) in PHPT (323 ± 73 mg/L) versus FDR (377 ± 98) or ILR (382 ± 101). The differences between PHPT and control groups for TotalD, BioD, and FreeD were maintained after adjustment for season, gender, and serum creatinine. 25-hydroxyvitamin D, evaluated as total, free, or bioavailable fractions, is decreased in PHPT. No difference was seen between first-degree relative and in-law controls, suggesting that neither genetic nor non-genetic background greatly influences the genesis of the hypovitaminosis D seen in PHPT.

Rare Noncoding Mutations Extend the Mutational Spectrum in the PGAP3 Subtype of Hyperphosphatasia with Mental Retardation Syndrome.

HPMRS or Mabry syndrome is a heterogeneous glycosylphosphatidylinositol (GPI) anchor deficiency that is caused by an impairment of synthesis or maturation of the GPI-anchor. The expressivity of the clinical features in HPMRS varies from severe syndromic forms with multiple organ malformations to mild nonsyndromic intellectual disability. In about half of the patients with the clinical diagnosis of HPMRS, pathogenic mutations can be identified in the coding region in one of the six genes, one among them is PGAP3. In this work, we describe a screening approach with sequence specific baits for transcripts of genes of the GPI pathway that allows the detection of functionally relevant mutations also including introns and the 5' and 3' UTR. By this means, we also identified pathogenic noncoding mutations, which increases the diagnostic yield for HPMRS on the basis of intellectual disability and elevated serum alkaline phosphatase. In eight affected individuals from different ethnicities, we found seven novel pathogenic mutations in PGAP3. Besides five missense mutations, we identified an intronic mutation, c.558-10G>A, that causes an aberrant splice product and a mutation in the 3'UTR, c.*559C>T, that is associated with substantially lower mRNA levels. We show that our novel screening approach is a useful rapid detection tool for alterations in genes coding for key components of the GPI pathway.

Characterization of additional vitamin D binding protein variants.

The gene (GC) for the vitamin D binding protein (DBP) shows significant genetic variation. Two missense variants, p.D432E and p.T436K, are common polymorphisms and both may influence vitamin D metabolism. However, less common variants, identified biochemically, have been reported previously. This study aimed to identify the underlying mutations by molecular screening and to characterize the mutant proteins by mass spectrometry. Denaturing high performance liquid chromatography (DHPLC) was used for screening genetic variants in GC exons and exon/intron boundaries of genomic DNA samples. Sanger sequencing identified the specific mutations. An immuno-capture coupled mass spectrometry method was used to characterize protein variants in serum samples. Initial molecular screening identified 10 samples (out of 761) containing an alanine deletion at codon 246 in exon 7 (p.A246del, c.737_739delCTG), and 1 sample (out of 97) containing a cysteine to phenylalanine substitution at codon 311 in exon 8 (p.C311F, c.932G>T). The mutant allele proteins and posttranslational modified products were distinguishable from the wild-type proteins by mass spectrum profiling. Loss of a disulfide bond due to loss of cysteine-311 was accompanied by the appearance of a novel mixed disulfide species, consistent with S-cysteinylation of the remaining unpaired cysteine-299 in the mutant protein. We confirm earlier biochemical studies indicating that there are additional deleterious GC mutations, some of which may be low-frequency variants. The major findings of this study indicate that additional mutant proteins are secreted and can be identified in the circulation. By combining molecular screening and mass spectrometric methods, mutant DBP species can be identified and characterized.