Betty Y.L. Wong

Common genetic variants of the vitamin D binding protein (DBP) predict differences in response of serum 25-hydroxyvitamin D [25(OH)D] to vitamin D supplementation.

BACKGROUND To determine the effect of vitamin D binding protein (DBP) genotypes on 25-hydroxyvitamin D [25(OH)D] changes with vitamin D supplements, we studied 98 adults receiving 600 or 4000 IU/d vitamin D(3) for one year. METHODS The DBP functional variant, T436K, was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS Mean 25(OH)D increases were 97% for TT (n=48), 151% for TK (n=31) and 307% (n=6) for KK genotypes (p=.004). CONCLUSIONS As with baseline 25(OH)D, T436K genotype predicts 25(OH)D changes after long-term vitamin D supplementation.

Phase II trial evaluating the palliative benefit of second-line zoledronic acid in breast cancer patients with either a skeletal-related event or progressive bone metastases despite first-line bisphosphonate therapy.

PURPOSE This study evaluated whether additional palliative benefits could be derived from the second-line use of the more potent bisphosphonate zoledronic acid in metastatic breast cancer patients with either progressive bone metastases or skeletal-related events (SRE), despite first-line therapy with either pamidronate or clodronate. PATIENTS AND METHODS This prospective study evaluated the impact of second-line zoledronic acid on pain, quality of life, and markers of bone turnover (for example, urinary N-telopeptide [NTX]). Patients received monthly zoledronic acid (4 mg) for 3 months. Study evaluations were made weekly during the first month and again at week 8. No changes in chemotherapy or endocrine therapy were allowed in the month before or after commencing study treatment. RESULTS Thirty-one women completed this study. By week 8, patients had experienced significant improvements in pain control (P < .001). There was a downward trend in urinary NTX levels over the same time period (P = .008). Overall, there was a trend towards a positive correlation between improvement in pain control and reduction in week one urinary NTX relative to baseline (Spearmans rho r = 0.27; P = .15). CONCLUSION This is the first study to demonstrate that patients with either progressive bone metastases or SREs while on clodronate or pamidronate can have relevant palliative benefits with a switch to the more potent bisphosphonate zoledronic acid. This is reflected by significant improvements in pain control and bone turnover markers. If confirmed in randomized trials, these findings have major implications to the use of bisphosphonates in both the metastatic and adjuvant settings.

Association of vitamin D binding protein (VDBP) polymorphisms and serum 25(OH)D concentrations in a sample of young Canadian adults of different ancestry

Variants of the vitamin D binding protein (VDBP) gene appear to be associated with levels of the main circulating vitamin D metabolite, 25-hydroxyvitamin D [(25(OH)D]. We examined the associations between the common variants of the VDBP (GC) gene and concentrations of 25(OH)D in a sample of young Canadian adults of East Asian, European and South Asian ancestry, taking into account the effect of vitamin D intake, skin pigmentation, sex, BMI, sun exposure and season. Three hundred and fifty-one (351) healthy young adults were genotyped for two non-synonymous single nucleotide polymorphisms (SNPs), T436K (rs4588) and D432E (rs7041), using a method that ascertains the GC diplotypes of each individual. After controlling for relevant predictor variables in multiple regression models, the number of GC-2 (436K) alleles was found to be associated with lower 25(OH)D concentrations in the East Asian sample at fall and winter visits. The number of GC-2 alleles also showed a significant negative association with fall 25(OH)D concentration in the European sample. No associations were noted between the number of GC-2 alleles and 25(OH)D in the South Asian sample at either season. Vitamin D intake was also significantly predictive of serum 25(OHD) concentrations, and similarly to what was observed for the GC polymorphisms, the relative strength of the association was influenced by ancestry and season.

Vitamin D binding protein is a key determinant of 25-hydroxyvitamin D levels in infants and toddlers.

Circulating 25‐hydroxyvitamin D (25‐OHD) levels vary among human populations. Only limited information regarding determinants of these measures is available for infants and children, particularly in minority groups at greatest risk for vitamin D deficiency. We identified demographic determinants of circulating 25‐OHD in a large cohort of minority children, and now extend our studies to examine potential roles of vitamin D binding protein (DBP) as a determinant of 25‐OHD levels. Serum DBP level and common single nucleotide polymorphisms (SNPs) at positions 432 and 436 in the GC gene, encoding DBP, were examined. We confirmed self‐reported ancestry using ancestry informative markers (AIMs), and included quantitative AIMs scores in the analysis. The multivariate model incorporated previously identified demographic and nutritional determinants of 25‐OHD in this cohort, as well as GC SNPs and circulating DBP. Genetic variants in GC differed by self‐reported ancestry. The 1f allele (D432/T436) was enriched in African Americans, occurring in 71%. Homozygosity for the 1f allele (DDTT) occurred in 53% of African Americans but only 6% of Caucasians and 13% of Hispanics. Circulating DBP was significantly correlated with 25‐OHD. GC SNPs were associated with both circulating DBP and 25‐OHD. It appears that progressive substitution of lysine for threonine at the 436 position results in lower circulating 25‐OHD. Multivariate analysis revealed that genetic variance in GC significantly contributes to circulating DBP as well as 25‐OHD. Moreover, the effect of GC SNPs on 25‐OHD are evident after adjusting for their effects on circulating DBP. Thus in young children genetic variance of the common GC T436K SNP affects circulating levels of the DBP protein, which in turn affects circulating 25‐OHD. However, the GC genotype also affects circulating 25‐OHD independently of its effect on circulating DBP. These findings provide data that may be important in the interpretation of vitamin D status in children of varying ancestral backgrounds.

Recessive NPHS2 (Podocin) Mutations Are Rare in Adult-Onset Idiopathic Focal Segmental Glomerulosclerosis

Recessive NPHS2 (podocin) mutations account for up to approximately 30% of steroid-resistant idiopathic FSGS in children and are associated with a reduced risk for disease recurrence after renal transplantation. R229Q, a missense variant that is present in 3.6% of the white population, has been implicated as a common disease-causing mutation. Given these clinical implications, we examined the role of NPHS2 mutations in a cohort of patients with adult-onset FSGS. We used denaturing HPLC to screen for heterozygous and homozygous gene variants in PCR-amplified DNA fragments that contained all exons and splice junctions of NPHS2. Bidirectional sequencing was performed to define all of the gene variants detected. With the use of the denaturing HPLC in a single-blind pilot study, 40 of 43 known NPHS2 mutations were detected from 22 pediatric patients with FSGS to establish a test sensitivity of 93%. This screen then was applied to 87 adult patients with idiopathic FSGS (15 steroid-sensitive, 63 steroid-resistant, and nine familial cases). In this latter cohort, compound heterozygous mutations were detected only in one patient with steroid-sensitive FSGS (R229Q and Q285fsX302) and no homozygous mutations. Overall, R229Q accounted for eight (80%) of ten of the putative mutant alleles that were detected in the study cohort. Contrary to the pediatric experience, recessive NPHS2 mutations are rare in this study population, suggesting that the pathogenesis of FSGS in adults may differ from that in children. These data do not support R229Q as a disease-causing mutation for steroid-resistant FSGS.

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

Abstract 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.

Alleles of the Estrogen Receptor α‐Gene and an Estrogen Receptor Cotranscriptional Activator Gene, Amplified in Breast Cancer‐1 (AIB1), Are Associated with Quantitative Calcaneal Ultrasound

Quantitative bone ultrasound (QUS) has a significant heritable component. Because estrogen is required for attainment of peak bone mass, we studied alleles of two genes, estrogen receptor α (ER1) and amplified in breast cancer‐1 (AIB1), for their association with QUS. In a volunteer sample of 663 white women aged 18–35 years, bone ultrasound attenuation (BUA), speed of sound (SOS), and heel stiffness index (SI), the latter consisting of the component measures of BUA and SOS, were measured at the right calcaneus by QUS. Subjects were genotyped for the ER1 polymorphisms Xba I and Pvu II and for the AIB1 polyglutamine tract polymorphism. In a multiple regression analysis, ER1 genotype was an independent predictor of QUS‐SI (p = 0.03). Because AIB1 and ER1 enhance gene expression in a coordinate manner, we also searched for interactions. A gene‐by‐gene interaction effect was seen for QUS‐SI (p = 0.009), QUS‐BUA (p = 0.03), and QUS‐SOS (p = 0.004). These remained significant after the inclusion of clinically relevant variables into the final regression model. Overall, these clinical and genetic factors accounted for up to 16% of the variance in peak QUS; the genetic markers alone accounted for 4–7%. This is the first demonstration of specific genetic effects on calcaneal QUS encoded by alleles of genes directly involved in mediating estrogen effects on bone.

Calcium-sensing receptor mutations and denaturing high performance liquid chromatography

The calcium-sensing receptor (CASR), a plasma membrane G-protein-coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism, and activating mutations cause autosomal dominant hypocalcemia (ADH). CASR mutation identification plays an important role in the clinical management of mineral metabolism disorders. We describe here a high-throughput method using screening with denaturing high performance liquid chromatography (DHPLC) to initially interrogate 12 amplicons covering translated exons and exon/intron boundaries, followed by sequencing of any amplicon with a modified melting curve relative to wild type, and direct sequencing of a 13th amplicon encoding the COOH-terminal tail to distinguish causative mutations from three common missense single nucleotide polymorphisms. A blinded analysis of 32 positive controls representing mutations throughout the CASR sequence, as well as 22 negative controls, yielded a concordance rate of 100%. We report eight novel and five recurrent FHH mutations, along with six novel and two recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.

Transforming growth factor beta-1 (TGFB1) and peak bone mass: association between intragenic polymorphisms and quantitative ultrasound of the heel

BackgroundVariance of peak bone mass has a substantial genetic component, as has been shown with twin studies examining quantitative measures such as bone mineral density (BMD) and quantitative ultrasound (QUS). Evidence implicating single nucleotide polymorphisms (SNPs) of the transforming growth factor beta-1 (TGFB1) gene is steadily accumulating. However, a comprehensive look at multiple SNPs at this locus for their association with indices of peak bone mass has not been reported.MethodsA cohort of 653 healthy Caucasian females 18 to 35 years old was genotyped for seven TGFB1 SNPs. Polymorphisms were detected by restriction endonuclease digestion of amplified DNA segments.ResultsThe frequencies of the least common allele at G-800A, C-509T, codon 10 (L10P), codon 25 (R25P), codon 263 (T263I), C861-20T, and 713-8 delC loci were 0.07, 0.33, 0.41, 0.08, 0.04, 0.25 and 0.01, respectively. A significant association was seen between QUS Stiffness Index (QUS-SI) and the SNP at codon 10 and the linked promoter SNP, C-509T. This association remained significant after multiple regression was used to incorporate important clinical covariates – age, BMI, level of activity, family history, and caffeine intake – into the model.ConclusionThe association of QUS-SI with -509T is consistent with a gene-dose effect, while only individuals homozygous for the codon 10P allele showed a significant increase. In this cohort of young healthy Caucasian females, the T allele at position -509 is associated with greater bone mass as measured by calcaneal ultrasound.

Whole genome amplification of buccal cell DNA : genotyping concordance before and after multiple displacement amplification

Abstract While buccal cells provide an easily accessible source of genomic DNA, the amount extracted may be insufficient for many studies. Whole genome amplification (WGA) using multiple displacement amplification (MDA) may optimize buccal cell genomic DNA yield. We compared the usefulness, in epidemiological surveys, of DNA derived from buccal cells collected by alcohol mouthwash and amplified by WGA protocol and standard protocols. Buccal cell collection kits were mailed to 300 randomly selected members of a large cohort study, and 189 subjects returned buccal cell samples. We determined: (i) which QIAamp ® DNA Blood Mini Kit extraction protocol (tissue or blood) produced more DNA; and (ii) whether it is feasible to use MDA to prepare DNA for single nucleotide polymorphism (SNP) genotyping of markers such as the methylenetetrahydrofolate reductase ( MTHFR) and vitamin D receptor ( VDR) genes. The two DNA extraction protocols were tested on 20 different patient samples each. The tissue protocol yielded more DNA than the blood protocol (15.4±8.6 vs. 7.6±7.1μg, p<0.0001). The 20 DNA samples extracted using the tissue protocol were then subjected to pre- and post-MDA genotyping using amplicons for the MTHFR SNP at C677T and the intron 8 VDR SNP. No genotyping discrepancies were detected in pair-wise comparisons of pre- and post-MDA. Genotyping DNA from MDA-based WGA is indistinguishable from routine polymerase chain reaction and offers a stable DNA source for genomic research and clinical diagnosis.