Dofetilide for the treatment of premature ventricular complexes and ventricular tachycardia in patients with structural heart disease.

BACKGROUND: Dofetilide is a class III antiarrhythmic agent approved for the treatment of atrial fibrillation and atrial flutter. Given the efficacy of other class III agents, it has been used off-label for the treatment of premature ventricular complexes (PVCs) and ventricular tachycardias (VTs). OBJECTIVE: The purpose of this study was to determine the efficacy and safety of dofetilide for ventricular arrythmias (VAs). METHODS: In this retrospective cohort study, 81 patients (59 men; age = 60 ± 14 years; LVEF = 0.34 ± 0.16) were admitted for dofetilide initiation to treat PVCs (29), VTs (42) or both (10). A ≥ 80% decrease in PVC burden was defined as a satisfactory response. An ICD was present in 72 patients (89%). Another antiarrhythmic was previously used in 50 patients (62%). Prior catheter ablation had been performed in 33 patients (41%). RESULTS: During intitiation, dofetilide was discontinued in 12 patients (15%) due to QT prolongation (8) and inefficacy to suppress VAs (4). Among the 32 patients with PVCs who successfully started dofetilide, the mean PVC burden decreased from 20 ± 10% to 8 ± 8% at a median follow-up of 2.6 months (p < .001). PVC burden was reduced by ≥80% in only 11/32 patients (34%). During 7 ± 1 years of follow-up, 41/69 patients (59%) continued to have VAs and received appropriate ICD therapies for monomorphic VTs (35) and polymorphic VT/VF (6) at a median of 8.0 (IQR 2.6-33.2) months. Dofetilide had to be discontinued in 50/69 patients (72%) due to inefficacy or intolerance. The composite outcome of VT/VF recurrence, heart transplantation, or death occurred in 6/12 patients (50%) without dofetilide and 49/69 patients (71%) with dofetilide. The event free survival was similar between patients treated with and without dofetilide (log-rank p = .55). CONCLUSIONS: Treatment with dofetilide was associated with a decrease in PVCs, however clinically significant suppression occurred in a minority of patients. Dofetilide failed to suppress the occurrence of VTs in a majority of patients.

Efficacy of a Multimodal Surgical Site Injection for Postoperative Pain Control in Pediatric Patients With Cerebral Palsy Undergoing Hip Reconstruction: A Randomized Controlled Trial.

BACKGROUND: One in 4 children with cerebral palsy (CP) will undergo orthopaedic surgery during their childhood. Despite its ubiquity, postoperative pain control has been poorly studied in this patient population. Moreover, poor pain management has been associated with adverse surgical outcomes. Multimodal analgesic injections have been well studied in the adult population, demonstrating safety and efficacy in reducing postoperative pain and narcotic consumption, but this modality has not been studied in pediatric patients undergoing similarly complex procedures. The objective of this study was to evaluate the efficacy of a multimodal surgical site injection for postoperative pain control following operative management of hip dysplasia in patients with CP. METHODS: After obtaining IRB approval, a multicenter, randomized double-blind placebo control trial was completed. Patients below 18 years old with a diagnosis of CP who were scheduled for varus derotation osteotomy (VDRO) of the proximal femur were randomized to receive a surgical-site injection with either a combination of ropivacaine (3 mg/kg), epinephrine (0.5 mg), and ketorolac (0.5 mg/kg) (experimental group) or normal saline (control). All included patients had identical postoperative care, including immobilization, physical therapy, and standardized, multimodal postoperative pain control. Pain scores and narcotic consumption were recorded at regular intervals and compared between groups utilizing two-tailed t test or a nonparametric Mann-Whitney test for quantitative variables and a Fischer exact test for categorical variables. RESULTS: Thirty-four patients were included, evenly divided between study arms. There were no significant differences in demographic variables, gross motor function classification system (GMFCS), comorbidities, preoperative radiographic parameters, or concomitant surgeries between groups. Patients in the experimental group required significantly lower narcotic medications at all postoperative time points from PACU until hospital discharge compared with controls (0.41 ± 0.42 vs. 1.87 ± 2.05 total morphine mEQ/kg, P=0.01). Similarly, patients in the experimental group were found to have significantly lower pain scores throughout their hospital stays compared with controls (1.0 ± 0.6 vs. 2.4 ± 1.1 mean pain score, P<0.001). There were no significant differences in operative time, OR time, blood transfusion requirements or hospital length of stay between groups. There were no adverse medication reactions or injection site complications in either group. CONCLUSIONS: In patients with CP undergoing hip reconstruction, surgical-site injection with a multimodal analgesic combination improves pain control and reduces narcotic consumption in the early postoperative period with no observed adverse effects. SIGNIFICANCE: Local multimodal analgesic injections should be adopted as part of standard multimodal pain control in this patient population for all osseous surgeries. LEVEL OF EVIDENCE: Level I-therapeutic.

Cardiac Involvement and Arrhythmias Associated with Myotonic Dystrophy.

Myotonic dystrophy is an autosomal dominant genetic disease of nucleotide expansion resulting in neuromuscular disease with two distinct subtypes. There are significant systemic manifestations of this condition including progressive muscular decline, neurologic abnormalities, and cardiac disease. Given the higher prevalence of cardiac dysfunction compared to the general population, there is significant interest in early diagnosis and prevention of cardiac morbidity and mortality. Cardiac dysfunction has an origin in abnormal and unstable nucleotide repeats in the DMPK and CNBP genes which have downstream effects leading to an increased propensity for arrhythmias and left ventricular systolic dysfunction. Current screening paradigms involve the use of routine screening electrocardiograms, ambulatory electrocardiographic monitors, and cardiac imaging to stratify risk and suggest further invasive evaluation. The most common cardiac abnormality is atrial arrhythmia, however there is significant mortality in this population from high-degree atrioventricular block and ventricular arrhythmia. In this review, we describe the cardiac manifestations of myotonic dystrophy with an emphasis on arrhythmia which is the second most common cause of death in this population after respiratory failure.

Low-cost, high-precision micro-lensed optical fiber providing deep-micrometer to deep-nanometer-level light focusing.

A new type of micro-lensed optical fiber through stacking appropriate high-refractive microspheres at designed locations with respect to the cleaved end of an optical fiber is numerically and experimentally demonstrated. This new type of micro-lensed optical fiber can be precisely constructed with low cost and high speed. Deep micrometer-scale and submicrometer-scale far-field light spots can be achieved when the optical fibers are multimode and single mode, respectively. By placing an appropriate teardrop dielectric nanoscale scatterer at the far-field spot of this new type of micro-lensed optical fiber, a deep-nanometer near-field spot can also be generated with high intensity and minimum joule heating, which is valuable in high-speed, high-resolution, and high-power nanoscale detection compared with traditional near-field optical fibers containing a significant portion of metallic material.

A Rapid MRI Protocol for the Evaluation of Acute Pediatric Musculoskeletal Infections: Eliminating Contrast and Decreasing Anesthesia, Scan Time, and Hospital Length of Stay and Charges.

BACKGROUND: Acute musculoskeletal infection affects >1 in 6,000 children in the United States annually. Magnetic resonance imaging (MRI) is the gold standard for the diagnosis of musculoskeletal infection, but it traditionally requires contrast and anesthesia for children, delaying management. A rapid MRI protocol involves MRI without anesthesia and with limited non-contrast sequences optimized for fluid detection and diffusion-weighted images to identify abscesses. We hypothesized that a rapid MRI protocol would improve imaging and treatment efficiency for pediatric patients undergoing musculoskeletal infection evaluation without substantially affecting accuracy. METHODS: This was a single-center, retrospective study of patients undergoing evaluation for musculoskeletal infection before (60 patients in the traditional cohort [TC]) and after (68 patients in the rapid cohort [RC]) implementation of the rapid MRI protocol. Sociodemographic and clinical variables were extracted from electronic health records, and statistical comparisons were performed. RESULTS: The anesthesia rates were 53% for the TC and 4% for the RC, and the contrast administration rates were 88% for the TC and 0% for the RC. The median time to MRI after ordering was 6.5 hours (95% confidence interval [CI], 5.0 to 8.6 hours) for the TC and 2.2 hours (95% CI, 1.4 to 3.6 hours) for the RC (p < 0.01). The median duration of MRI was 63.2 minutes (95% CI, 56.8 to 69.6 minutes) for the TC and 24.0 minutes (95% CI, 21.1 to 29.5 minutes) for the RC (p < 0.01). The median hospital length of stay was 5.3 days (95% CI, 3.7 to 6.9 days) for the TC and 3.7 days (95% CI, 1.9 to 4.1 days) for the RC (p < 0.01). The median hospital charges were $47,309 (95% CI, $39,137 to $58,769) for the TC and $32,824 (95% CI, $22,865 to $45,339) for the RC (p < 0.01). Only 2 positive cases of musculoskeletal infection in the RC were missed on the initial imaging, but these instances were not attributable to the rapid protocol itself. Although 10 of 68 rapid MRI scans resulted in nondiagnostic outcomes due to patient motion, only 6 of 68 required repeat MRI with anesthesia. CONCLUSIONS: In patients evaluated for musculoskeletal infection, the rapid MRI protocol eliminated contrast and minimized anesthesia while improving MRI access and decreased scan and interpretation times, hospital length of stay, and hospital charges. The rapid MRI protocol had high sensitivity for diagnosing musculoskeletal infection and a low rate of imaging failure. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Complications of catheter ablation for ventricular tachycardia.

With the increasing literature demonstrating benefits of catheter ablation for ventricular tachycardia (VT), the number of patients undergoing VT ablation has increased dramatically. As VT ablation is being performed more routinely, operators must be aware of potential complications of VT ablation. This review delves deeper into the practice of VT ablation with a focus on periprocedural complications.

Effect of Transcutaneous Magnetic Stimulation in Patients With Ventricular Tachycardia Storm: A Randomized Clinical Trial.

IMPORTANCE: Autonomic neuromodulation provides therapeutic benefit in ventricular tachycardia (VT) storm. Transcutaneous magnetic stimulation (TcMS) can noninvasively and nondestructively modulate a patient's nervous system activity and may reduce VT burden in patients with VT storm. OBJECTIVE: To evaluate the safety and efficacy of TcMS of the left stellate ganglion for patients with VT storm. DESIGN, SETTING, AND PARTICIPANTS: This double-blind, sham-controlled randomized clinical trial took place at a single tertiary referral center between August 2019 and July 2021. The study included 26 adult patients with 3 or more episodes of VT in 24 hours. INTERVENTIONS: Patients were randomly assigned to receive a single session of either TcMS that targeted the left stellate ganglion (n = 14) or sham stimulation (n = 12). MAIN OUTCOMES AND MEASURES: The primary outcome was freedom from VT in the 24-hour period following randomization. Key secondary outcomes included safety of TcMS on cardiac implantable electronic devices, as well as burden of VT in the 72-hour period following randomization. RESULTS: Among 26 patients (mean [SD] age, 64 [13] years; 20 [77%] male), a mean (SD) of 12.7 (10.3) episodes of VT occurred within the 24 hours preceding randomization. Patients had recurrent VT despite taking a mean (SD) of 2.0 (0.6) antiarrhythmic drugs (AADs), and 11 patients (42%) required mechanical hemodynamic support at the time of randomization. In the 24-hour period after randomization, VT recurred in 4 of 14 patients (29% [SD 47%]) in the TcMS group vs 7 of 12 patients (58% [SD 51%]) in the sham group (P = .20). In the 72-hour period after randomization, patients in the TcMS group had a mean (SD) of 4.5 (7.2) episodes of VT vs 10.7 (13.8) in the sham group (incidence rate ratio, 0.42; P < .001). Patients in the TcMS group were taking fewer AADs 24 hours after randomization compared with baseline (mean [SD], 0.9 [0.8] vs 1.8 [0.4]; P = .001), whereas there was no difference in the number of AADs taken for the sham group (mean [SD], 2.3 [0.8] vs 1.9 [0.5]; P = .20). None of the 7 patients in the TcMS group with a cardiac implantable electronic device had clinically significant effects on device function. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, findings support the potential for TcMS to safely reduce the burden of VT in the setting of VT storm in patients with and without cardiac implantable electronic devices and inform the design of future trials to further investigate this novel treatment approach. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04043312.

Investigations of the Y chromosome, male founder structure and YSTR mutation rates in the Old Order Amish.

OBJECTIVES: Using Y chromosome short tandem repeat (YSTR) genotypes, (1) evaluate the accuracy and completeness of the Lancaster County Old Order Amish (OOA) genealogical records and (2) estimate YSTR mutation rates. METHODS: Nine YSTR markers were genotyped in 739 Old Order Amish males who participated in several ongoing genetic studies of complex traits and could be connected into one of 28 all-male lineage pedigrees constructed using the Anabaptist Genealogy Database and the query software Ped-Hunter. A putative founder YSTR haplotype was constructed for each pedigree, and observed and inferred father-son transmissions were used to estimate YSTR mutation rates. RESULTS: We inferred 27 distinct founder Y chromosome haplotypes in the 28 male lineages, which encompassed 27 surnames accounting for 98% of Lancaster OOA households. Nearly all deviations from founder haplotypes were consistent with mutation events rather than errors. The estimated marker-specific mutation rates ranged from 0 to 1.09% (average 0.33% using up to 283 observed meioses only and 0.28% using up to 1,232 observed and inferred meioses combined). CONCLUSIONS: These data confirm the accuracy and completeness of the male lineage portion of the Anabaptist Genealogy Database and contribute mutation rate estimates for several commonly used Y chromosome STR markers.

Osteoporosis-pseudoglioma syndrome: description of 9 new cases and beneficial response to bisphosphonates.

Osteoporosis-pseudoglioma syndrome (OPPG) is a rare autosomal recessive disorder of severe juvenile osteoporosis and congenital blindness, due to mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Approximately fifty cases of OPPG have been reported. We report 9 new cases of OPPG, in three related nuclear families of Conservative Mennonites in Pennsylvania. All 9 children with OPPG were blind and had osteoporosis. Four of six parents had low bone mineral density (BMD) or osteoporosis; 2 were normal. Sequence analysis from genomic DNA revealed homozygosity for a nonsense mutation of exon 6 of LRP5 (W425X) in four OPPG cases tested in families A and C. In family B, OPPG cases were compound heterozygotes for the exon 6 W425X LRP5 mutation and a second exon 6 mutation (T409A); bone phenotype was milder than in family A. Neither of these mutations was present in an unrelated normal. The four treated OPPG patients all responded to bisphosphonates (duration 1.5-6.5 years) with improvement in Z-scores. One patient had a negligible response to teriparatide. In summary, we report 9 new cases of OPPG due to two novel LRP5 mutations, note a milder bone phenotype but similar ocular phenotype in LRP5 W425X/T409A compound heterozygotes than in W425X homozygotes and describe positive response to bisphosphonate treatment in four cases.

Role of genetic background in determining phenotypic severity throughout postnatal development and at peak bone mass in Col1a2 deficient mice (oim).

Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disease characterized by extreme bone fragility. Although fracture numbers tend to decrease post-puberty, OI patients can exhibit significant variation in clinical outcome, even among related individuals harboring the same mutation. OI most frequently results from mutations in type I collagen genes, yet how genetic background impacts phenotypic outcome remains unclear. Therefore, we analyzed the phenotypic severity of a known proalpha2(I) collagen gene defect (oim) on two genetic backgrounds (congenic C57BL/6J and outbred B6C3Fe) throughout postnatal development to discern the phenotypic contributions of the Col1a2 locus relative to the contribution of the genetic background. To this end, femora and tibiae were isolated from wildtype (Wt) and homozygous (oim/oim) mice of each strain at 1, 2 and 4 months of age. Femoral geometry was determined via muCT prior to torsional loading to failure to assess bone structural and material biomechanical properties. Changes in mineral composition, collagen content and bone turnover were determined using neutron activation analyses, hydroxyproline content and serum pyridinoline crosslinks. muCT analysis demonstrated genotype-, strain- and age-associated changes in femoral geometry as well as a marked decrease in the amount of bone in oim/oim mice of both strains. Oim/oim mice of both strains, as well as C57BL/6J (B6) mice of all genotypes, had reduced femoral biomechanical strength properties compared to Wt at all ages, although they improved with age. Mineral levels of fluoride, magnesium and sodium were associated with biomechanical strength properties in both strains and all genotypes at all ages. Oim/oim animals also had reduced collagen content as compared to Wt at all ages. Serum pyridinoline crosslinks were highest at two months of age, regardless of strain or genotype. Strain differences in bone parameters exist throughout development, implicating a role for genetic background in determining biomechanical strength. Age-associated improvements indicate that oim/oim animals partially compensate for their weaker bone material, but never attain Wt levels. These studies indicate the importance of genetic background in determining phenotypic severity, but the presence of the proalpha2(I) collagen gene defect and age of the animal are the primary determinants of phenotypic severity.

Quantitative trait loci for BMD identified by autosome-wide linkage scan to chromosomes 7q and 21q in men from the Amish Family Osteoporosis Study.

UNLABELLED: Using autosome-wide linkage analysis in 964 Amish, strong evidence was found for the presence of genes affecting hip and spine BMD in men on chromosomes 7q31 and 21q22 (LOD = 4.15 and 3.36, respectively). INTRODUCTION: BMD is highly heritable, with genetic factors accounting for 60-88% of variation. The goal of this study was to localize genes contributing to BMD variation. MATERIALS AND METHODS: The Amish Family Osteoporosis Study was designed to identify genes affecting bone health. The Amish are a genetically closed population with a homogeneous lifestyle. BMD was measured at the spine, hip, and radius using DXA in 964 participants (mean age, 50.2 +/- 16.3 [SD] years; range, 18-99 years) from large multigenerational families. Genotyping of 731 highly polymorphic microsatellite markers (average spacing of 5.4 cM) and autosome-wide multipoint linkage analysis were performed. RESULTS: In the overall study population, no strong evidence for linkage was detected to any chromosomal region (peak LOD: 2.11 for radius BMD on chromosome 3q26). In a subgroup analysis of men (n = 371), strong evidence was detected for a quantitative trait locus (QTL) influencing BMD variation on chromosome 7q31 at the total hip (LOD = 4.15) and femoral neck (LOD = 3.09) and for a second QTL influencing spine BMD at 21q22 (LOD = 3.36). Suggestive evidence of linkage was found in men for a QTL at 12q24 affecting total hip BMD (LOD = 2.60) and at 18p11 for femoral neck (LOD = 2.07), and in women (n = 593) at 1p36 for femoral neck BMD (LOD = 2.02) and at 1q21 for spine BMD (LOD = 2.11). In age subgroup analyses, suggestive evidence for linkage was found for those <50 years of age (n = 521) on chromosomes 11q22 and 14q23 (LODs = 2.11 and 2.16, respectively) and for those >50 years of age (n = 443) on 3p25.2 (LOD = 2.32). CONCLUSIONS: These results strongly suggest the presence of genes affecting hip and spine BMD in men on chromosomes 7q31 and 21q22. Modest evidence was found for genes affecting BMD in women on chromosomes 1p36 and 1q21 and in men at 12q24, replicating results from other populations.

The relationship between parity and bone mineral density in women characterized by a homogeneous lifestyle and high parity.

CONTEXT: We reported previously that Old Order Amish (OOA) women have fewer hip fractures and higher bone mineral density (BMD) than non-Amish Caucasian women. OBJECTIVE: The objective of this study was to determine whether the high parity characteristic of OOA women contributes to their relative bone health. Previous data on the long-term effects of parity on BMD have yielded conflicting results with few data from very high parity populations. This observational study included participants in the Amish Family Osteoporosis Study, begun in 1997 to identify genetic and clinical determinants of osteoporosis in the OOA. We measured BMD by dual-energy x-ray absorptiometry at the spine, hip, and distal radius in 424 parous OOA women aged 40 and older (mean age, 57.7 +/- 12 yr; mean parity, 7.6 +/- 2.9). RESULTS: Increasing parity was associated with later menopause (P = 0.001) and modestly, but not significantly, higher body mass index (BMI) (P = 0.09). Increasing parity was associated with higher BMD at the total hip and trochanter (age-adjusted P = 0.02 and 0.03), no longer statistically significant after accounting for BMI. Among women aged 50-59 yr, parity was strongly associated with BMD even after accounting for age and BMI (age-adjusted P = 0.02), although this was not true for women younger than 50 or at least 60 yr old. CONCLUSIONS: We conclude that high parity is associated with increased hip BMD in OOA women, largely mediated by higher BMI. The parity-hip BMD association remained statistically significant after accounting for age and BMI only in women aged 50-59 yr, partially explained by a later menopausal age with high parity. The benefit of high parity on BMD appeared to be lost soon after the menopausal transition, and, therefore, these data provide evidence of neither a detrimental nor beneficial effect of high parity on long-term bone health.

Changes in thermal stability and microunfolding pattern of collagen helix resulting from the loss of alpha2(I) chain in osteogenesis imperfecta murine.

Homozygous mutations resulting in formation of alpha1(I)(3) homotrimers instead of normal type I collagen cause mild to severe osteogenesis imperfecta (OI) in humans and mice. Limited studies of changes in thermal stability of type I homotrimers were reported previously, but the results were not fully consistent. We revisited this question in more detail using purified tendon collagen from wild-type (alpha1(I)(2)alpha2(I) heterotrimers) and oim (alpha1(I)(3)) mice as well as artificial alpha1(I)(3) homotrimers obtained by refolding of rat-tail-tendon collagen. We found that at the same heating rate oim homotrimers completely denature at approximately 2.5deg.C higher temperature than wild-type heterotrimers, as determined by differential scanning calorimetry. At the same, constant temperature, homotrimers denature approximately 100 times slower than heterotrimers, as determined by circular dichroism. Detailed analysis of proteolytic cleavage at different temperatures revealed that microunfolding of oim homotrimers and wild-type heterotrimers occurs at similar rate but within a number of different sites. In particular, the weakest spot on the oim triple helix is located approximately 100 amino acid residues from the C-terminal end within the cyanogen bromide peptide CB6. The same microunfolding site is also present in wild-type collagen, but the weakest spot of the latter is located close to the N-terminal end of CB8. Amino acid analysis and differential gel electrophoresis showed virtually no posttranslational overmodification of oim mouse tendon collagen. Moreover, thermal stability and microunfolding of artificial rat-tail-tendon homotrimers were similar to oim homotrimers. Thus, the observed changes are associated with difference in the amino acid composition of alpha1(I) and alpha2(I) chains rather than posttranslational overmodification.

Reduced incidence of hip fracture in the Old Order Amish.

UNLABELLED: The incidence of hip fracture was estimated in a community of Old Order Amish and compared with available data from non-Amish whites. Hip fracture rates were 40% lower in the Amish, and the Amish also experienced higher BMD. INTRODUCTION: Understanding the patterns of fracture risk across populations could reveal insights about bone health and lead to the earlier detection and prevention of osteoporosis. Toward this aim, we compared hip fracture incidence and bone mineral density (BMD) between an Old Order Amish (OOA) community, characterized by a rural and relatively active lifestyle, and non-Amish U.S. whites. MATERIALS AND METHODS: All hospital admissions for hip fracture among OOA individuals in Lancaster County, PA, were identified between 1995 and 1998 from four area hospitals. Hip fracture incidence was calculated by cross-referencing an available Anabaptist genealogy database with communities located within these hospital service areas and compared with non-Amish whites obtained from National Hospital Discharge data. Additionally, BMD at the hip was compared between 287 Amish subjects and non-Amish whites from the National Health and Nutrition Examination Survey III survey. RESULTS AND CONCLUSIONS: OOA experienced 42% fewer hip fractures than would be expected had they experienced the same rate of hip fracture as observed in non-Amish whites (p < 0.01) and a higher mean BMD that was significant in women (p < 0.05) but not men. Further evaluation of lifestyle and/or genetic differences between Amish and non-Amish populations may shed insights into etiologic factors influencing hip fracture risk.

A possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to "off-target" effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A common variant in fibroblast growth factor binding protein 1 (FGFBP1) is associated with bone mineral density and influences gene expression in vitro.

We previously detected strong evidence for linkage of forearm bone mineral density (BMD) to chromosome 4p (lod=4.3) in a set of 29 large Mexican American families. Fibroblast growth factor binding protein 1 (FGFBP1) is a strong candidate gene for bone homeostasis in this region. We sequenced the coding region of FGFBP1 in a subset of our Mexican American study population and performed association studies with BMD on SNPs genotyped in the entire cohort. We then attempted to replicate these findings in an independent study cohort and performed in vitro functional studies on replicated, potentially functional polymorphisms using a luciferase reporter construct to evaluate influence on gene expression. Several SNPs spanning the gene, all in one large block of linkage disequilibrium, were significantly associated with BMD at various skeletal sites (n=872, p=0.001-0.04). The associations were then replicated in an independent population of European ancestry (n=972; p=0.02-0.04). Sex-stratified association analyses in both study populations suggest this association is much stronger in men. Subsequent luciferase reporter gene assays revealed marked differences in FGFBP1 expression among the three common haplotypes. Further experiments revealed that a promoter polymorphism, rs12503796, results in decreased expression of FGFBP1 and inhibits upregulation of the gene by testosterone in vitro. Collectively, these findings suggest that sequence variation in FGFBP1 may contribute to variation in BMD, possibly influencing osteoporosis risk.

Variable bone fragility associated with an Amish COL1A2 variant and a knock-in mouse model.

Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1-4) areal bone mineral density (aBMD) Z-scores demonstrated that 73% had moderate to severe disease (less than -2), 23% had mild disease (-1 to -2), and 4% were in the unaffected range (greater than -1). A line of knock-in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F(1) lines of knock-in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI.

Segregation of type I collagen homo- and heterotrimers in fibrils.

Normal type I collagen is a heterotrimer of two alpha1(I) and one alpha2(I) chains, but various genetic and environmental factors result in synthesis of homotrimers that consist of three alpha1(I) chains. The homotrimers completely replace the heterotrimers only in rare recessive disorders. In the general population, they may compose just a small fraction of type I collagen. Nevertheless, they may play a significant role in pathology; for example, synthesis of 10-15% homotrimers due to a polymorphism in the alpha1(I) gene may contribute to osteoporosis. Homotrimer triple helices have different stability and less efficient fibrillogenesis than heterotrimers. Their fibrils have different mechanical properties. However, very little is known about their molecular interactions and fibrillogenesis in mixtures with normal heterotrimers. Here we studied the kinetics and thermodynamics of fibril formation in such mixtures by combining traditional approaches with 3D confocal imaging of fibrils, in which homo- and heterotrimers were labeled with different fluorescent colors. In a mixture, following a temperature jump from 4 to 32 degrees C, we observed a rapid increase in turbidity most likely caused by formation of homotrimer aggregates. The aggregates promoted nucleation of homotrimer fibrils that served as seeds for mixed and heterotrimer fibrils. The separation of colors in confocal images indicated segregation of homo- and heterotrimers at a subfibrillar level throughout the process. The fibril color patterns continued to change slowly after the fibrillogenesis appeared to be complete, due to dissociation and reassociation of the pepsin-treated homo- and heterotrimers, but this remixing did not significantly reduce the segregation even after several days. Independent homo- and heterotrimer solubility measurements in mixtures confirmed that the subfibrillar segregation was an equilibrium property of intermolecular interactions and not just a kinetic phenomenon. We argue that the subfibrillar segregation may exacerbate effects of a small fraction of alpha1(I) homotrimers on formation, properties, and remodeling of collagen fibers.