Ville-Valtteri Välimäki

Molecularly Defined Lactose Malabsorption, Peak Bone Mass and Bone Turnover Rate in Young Finnish Men

Lactose malabsorption (LM; adult-type hypolactasia), an autosomal recessive condition, results from the down-regulation of the activity of lactase enzyme in the intestinal wall. In previous studies the effect of LM on bone mass, bone turnover rate, development of osteoporosis and osteoporotic fractures has remained controversial. We have recently identified a single nucleotide polymorphism (SNP), a C to T change residing 13910 base pairs upstream of the lactase (LCT) gene at chromosome 2q21-22, which shows complete association with lactase persistence, with the C/C–13910 genotype defining LM and the genotypes C/T–13910 and T/T–13910 lactase persistence. The present study was undertaken to examine the relationship of the C/T–13910 polymorphism to peak bone mass, bone turnover rate, and stress fractures among young Finnish men. The study population comprised 234 young men, aged 18.3 to 20.6 years, 184 men were recruits of the Finnish Army, and 50 were men of similar age who had postponed their military service for reasons not related to health. Bone mineral content (BMC), density (BMD), and scan area were measured in the lumbar spine and upper femur by dual-energy X-ray absorptiometry (DXA). Blood was sampled for genotyping of the C/T–13910 polymorphism and determination of serum 25-hydroxyvitamin D (25OHD), intact parathyroid hormone (iPTH), type I procollagen aminoterminal propeptide (PINP), and tartrate-resistant acid phosphatase 5b (TRACP5b). Second-void urine samples were collected for the determination of type I collagen aminoterminal telopeptide (NTX). The prevalence of the C/C–13910-genotype of these young adults did not differ significantly from the corresponding population prevalence of C/C–13910 (17.1% vs 18.1%) among Finnish blood donors. Fifteen recruits of the army experienced a stress fracture; 3 of them (20%) had the C/C–13910-genotype. Calcium intake was similar for the three genotypes as were the unadjusted BMCs, scan areas, and BMDs at different measurement sites. The adjustments for age, height, weight, smoking, alcohol consumption, and physical exercise in the multiple regression analysis did not reveal any significant relationships between the lactase genotypes and BMDs at lumbar (P = 0.16), femoral neck (P = 0.99) or total hip (P = 0.96) sites. Serum 25OHD, iPTH, and bone marker levels were similar for the C/C–13910 C/T–13910 and T/T–13910 genotypes. In summary, in young Finnish men, molecularly defined lactose malabsorption does not alter bone turnover rate and impair the acquisition of peak bone mass. Moreover, the C/C–13910 genotype does not seem to be a risk factor for stress fractures in army recruits.

A Novel Splice Mutation in PLS3 Causes X-linked Early Onset Low-Turnover Osteoporosis

Genetic factors play an important role in the development of osteoporosis. Several monogenic forms of osteoporosis have been recognized, most recently an X‐chromosomal form resulting from mutations in the gene encoding plastin 3 (PLS3). PLS3 is a protein involved in actin bundle formation in the cytoskeleton. We present a large family with early onset osteoporosis and X‐linked inheritance. Phenotyping was performed on 19 family members and whole‐exome sequencing on 7 family members (5 with a diagnosis of early onset osteoporosis and 2 with normal bone parameters). Osteoporosis had its onset in childhood and was characterized by recurrent peripheral fractures, low bone mineral density (BMD), vertebral compression fractures, and significant height loss in adulthood. Males were in general more severely affected than females. Bone histomorphometry findings in 4 males and 1 female showed severe trabecular osteoporosis, low amount of osteoid, and decreased mineral apposition rate, indicating impaired bone formation; resorption parameters were increased in some. All affected subjects shared a single base substitution (c.73‐24T > A) in intron 2 of PLS3 on Xq23. The mutation, confirmed by Sanger sequencing, segregated according to the skeletal phenotype. The mutation introduces a new acceptor splice site with a predicted splice score of 0.99 and, thereby, as confirmed by cDNA sequencing, induces the insertion of 22 bases between exons 2 and 3, causing a frameshift and premature termination of mRNA translation (p.Asp25Alafs*17). The mutation affects the first N‐terminal calcium‐binding EF‐hand domain and abolishes all calcium‐ and actin‐binding domains of the protein. Our results confirm the role of PLS3 mutations in early onset osteoporosis. The mechanism whereby PLS3 affects bone health is unclear, but it may be linked to osteocyte dendrite function and skeletal mechanosensing. Future studies are needed to elucidate the role of PLS3 in osteoporosis and to define optimal treatment.

The same annual dose of 292000 IU of vitamin D3 (cholecalciferol) on either daily or four monthly basis for elderly women: 1‐year comparative study of the effects on serum 25(OH)D3 concentrations and renal function

Objective  Daily dosing of vitamin D supplements may be difficult among older people. Infrequent administration of ‘megadoses’ controlled by health care personnel may overcome adherence problem. We compared the efficacy and safety of two oral dosages (800 IU daily or 97333 IU four monthly) of vitamin D3 resulting in the equal annual dose of 292000 IU.

Variable number of tandem repeats polymorphism in parathyroid hormone-related protein as predictor of peak bone mass in young healthy Finnish males

OBJECTIVE Mice with osteoblast-specific deletion of parathyroid hormone-related protein (PTHrP) exhibit impaired recruitment and increased apoptosis of osteogenic cells resulting in decreased bone formation and premature osteoporosis. The PTHrP levels within the bone microenvironment are therefore critical in influencing bone mass acquisition. Whether this is applicable in humans has not been established. Here, we studied the association of a variable number of tandem repeats (VNTR) polymorphism in PTHrP with peak bone mass. METHODS Enrolled in the study were 234 young Finnish males, with median age of 19.6 years (range 18.3-20.6 years). Lifestyle factors, serum bone markers, osteodensitometric measurements (lumbar spine and hip) and calcaneal quantitative ultrasound readings were obtained. The PTHrP VNTR length was determined by the PCR amplification of genomic DNA extracted from peripheral blood and correlated to bone parameters by the multiple regression models. RESULTS The presence of at least one 252 bp allele was associated with increased lumbar spine bone mineral density (BMD; P<0.0034), broadband ultrasound attenuation (BUA; P<0.0012) and speed-of-sound (SOS; P<0.0023) measurements. The correlation with increased lumbar spine BMD (P=0.0008), BUA (P=0.005) and SOS (P=0.001) was further strengthened by the pairing of the 252 bp allele with a 460 bp allele in comparison with those without any 252 bp allele. Electrophoretic mobility shift assays were used to illustrate the potential transcriptional functionality of the VNTR sequence. CONCLUSION The results indicate that the PTHrP VNTR sequence likely modulates local PTHrP expression within the skeletal microenvironment and could serve as a diagnostic predictor of peak bone mass acquisition.

Teriparatide Treatment in Patients with WNT1 or PLS3 Mutation-Related Early-Onset Osteoporosis - A Pilot Study

Context: We previously identified 2 Finnish families with dominantly inherited, low-turnover osteoporosis caused by mutations in WNT1 or PLS3. Objective, Design, and Setting: This prospective, longitudinal, uncontrolled study was undertaken to evaluate whether these patients respond to teriparatide. Patients and Intervention: We recruited 6 adults (median age, 54 years); 3 with a WNT1 missense mutation, c.652T>G, and 3 with a PLS3 splice mutation, c.73-24T>A, to receive teriparatide 20 &mgr;g daily for 24 months. Five patients had previously used bisphosphonates. Main Outcome Measures: Outcome measures included lumbar spine and hip bone mineral density (BMD) by dual-energy X-ray absorptiometry, distal radius peripheral quantitative computed tomography, spinal radiography, serum bone turnover markers, paired iliac crest biopsies. Results: All patients showed increases in formation markers procollagen type 1 amino-terminal propeptide (90% to 398%) and osteocalcin (50% to 280%) and in resorption markers cross-linked C-terminal telopeptide of type I collagen (58% to 457%) and tartrate-resistant acid phosphatase 5b (20% to 68%) in first 6 months. Lumbar spine BMD increased 5.2% to 7.9% in 5 patients and femoral neck BMD 2.6% to 7.8% in 4 patients in 24 months. Distal radius cortical volumetric BMD decreased 5.4% to 26.1%. In histomorphometric analyses, osteoid indices increased more consistently in patients with WNT1 vs PLS3 mutation. Eroded surface decreased 44% to 100% in all patients. Adipocyte number increased in 5 patients studied. Conclusions: Patients with WNT1 or PLS3 mutation–related osteoporosis responded to teriparatide treatment. Future studies are needed to evaluate whether observed changes translate to fracture resistance.

How well are the optimal serum 25OHD concentrations reached in high‐dose intermittent vitamin D therapy? a placebo‐controlled study on comparison between 100 000 IU and 200 000 IU of oral D3 every 3 months in elderly women

Intermittent dosing may improve adherence to vitamin D therapy. Dosing regimen should maintain optimal serum 25‐hydroxyvitamin D (25OHD) levels over all the year. We compared two dosing regimens, the primary outcome being the percentage of 25OHD measurements reaching the targets of 75 nmol/l or 50 nmol/l after baseline.

Zoledronic acid in vivo increases in vitro proliferation of rat mesenchymal stromal cells

Background and purpose — Bisphosphonates are widely used in the treatment of bone loss, but they might also have positive effects on osteoblastic cells and bone formation. We evaluated the effect of in vivo zoledronic acid (ZA) treatment and possible concomitant effects of ZA and fracture on the ex vivo osteogenic capacity of rat mesenchymal stromal cells (MSCs). Methods — A closed femoral fracture model was used in adult female rats and ZA was administered as a single bolus or as weekly doses up to 8 weeks. Bone marrow MSCs were isolated and cultured for in vitro analyses. Fracture healing was evaluated by radiography, micro-computed tomography (μCT), and histology. Results — Both bolus and weekly ZA increased fracture-site bone mineral content and volume. MSCs from weekly ZA-treated animals showed increased ex vivo proliferative capacity, while no substantial effect on osteoblastic differentiation was observed. Fracture itself did not have any substantial effect on cell proliferation or differentiation at 8 weeks. Serum biochemical markers showed higher levels of bone formation in animals with fracture than in intact animals, while no difference in bone resorption was observed. Interestingly, ex vivo osteoblastic differentiation of MSCs was found to correlate with in vivo serum bone markers. Interpretation — Our data show that in vivo zoledronic acid treatment can influence ex vivo proliferation of MSCs, indicating that bisphosphonates can have sustainable effects on cells of the osteoblastic lineage. Further research is needed to investigate the mechanisms.

Osteocyte Protein Expression Is Altered in Low-Turnover Osteoporosis Caused by Mutations in WNT1 and PLS3

Context Osteocytes express proteins that regulate bone remodeling and mineralization. Objective To evaluate the relationship between osteocyte-specific protein expression and bone histology in patients with monogenic osteoporosis due to wingless integration site 1 (WNT1) or plastin 3 (PLS3) mutations. Design and Setting Cross-sectional cohort study at a university hospital. Participants Six patients (four males; ages: 14 to 72 years) with a heterozygous WNT1 mutation and five patients (four males; ages: 9 to 70 years) with a heterozygous/hemizygous PLS3 mutation. Methods and Main Outcome Measures Immunohistochemistry was performed for fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP1), sclerostin, and phosphorylated (phospho-)β-catenin in iliac crest samples and compared with bone histomorphometry. Results FGF23 expression in WNT1 patients was 243% that observed in PLS3 patients (P < 0.01). DMP1, sclerostin, and phospho-β-catenin expression did not differ between groups. Serum phosphate correlated inversely with FGF23 expression (r = -0.79, P = 0.01) and serum ionized calcium correlated inversely with sclerostin expression (r = -0.60, P = 0.05). Phospho-β-catenin expression correlated inversely with DMP1 expression (r = -0.88, P < 0.001), osteoid volume/bone volume (r = -0.68, P = 0.02), and bone formation rate (r = -0.78, P < 0.01). FGF23 expression did not correlate with DMP1 expression, sclerostin expression, or bone histomorphometry. Marrow adiposity was higher in WNT1 than in PLS3 patients (P = 0.04). Conclusions Mutations that disrupt WNT signaling and osteocytic mechanosensing affect osteocyte protein expression. Abnormal osteocyte function may play a role in the pathogenesis of monogenetic forms of osteoporosis.