A homozygous I684T in GLE1 as a novel cause of arthrogryposis and motor neuron loss.

Mutations in GLE1, RNA export mediator (GLE1) gene have previously been shown to cause motor neuron diseases such as lethal congenital contracture syndrome 1 (LCCS1) and lethal arthrogryposis with anterior horn cell disease (LAAHD), including arthrogryposis, fetal akinesis and motor neuron loss as common clinical features. The homozygous FinMajor mutation p.T144_E145insPFQ has been described as one of the causes for LCCS1 whereas LAAHD is caused by a heterocompound FinMajor mutation together with p.R569H, p.V617M or p.I684T missense mutation. None of these heterocompound missense mutations have previously been reported as homozygous states. Here we present the clinical features of 2 siblings with a homozygous p.I684T mutation in GLE1. The patients suffered from similar, but milder symptoms than in LCCS1 and LAAHD, surviving up to 6 months before they died due to a progressive disease course including respiratory failure. Arthrogryposis, lack of spontaneous movements, and epilepsy were notable in both cases and lack of anterior horn cells was identified in autopsy samples. Our studies on patient-derived fibroblasts show that the homozygous p.I684T impairs the nuclear localization of GLE1 further confirming the pathogenic role of this mutation.

Solute Transport of Negatively Charged Contrast Agents Across Articular Surface of Injured Cartilage.

Solute transport through the extracellular matrix (ECM) is crucial to chondrocyte metabolism. Cartilage injury affects solute transport in cartilage due to alterations in ECM structure and solute-matrix interactions. Therefore, cartilage injury may be detected by using contrast agent-based clinical imaging. In the present study, effects of mechanical injury on transport of negatively charged contrast agents in cartilage were characterized. Using cartilage plugs injured by mechanical compression protocol, effective partition coefficients and diffusion fluxes of iodine- and gadolinium-based contrast agents were measured using high resolution microCT imaging. For all contrast agents studied, effective diffusion fluxes increased significantly, particularly at early times during the diffusion process (38 and 33% increase after 4 min, P < 0.05 for iodine and Gd-DTPA; and 76% increase after 10 min for diatrizoate, P < 0.05). Effective partition coefficients were unaffected in mechanically injured cartilage. Mechanical injury reduced PG content and collagen integrity in cartilage superficial zone. This study suggests that alterations in contrast agent diffusion flux, a non-equilibrium transport parameter, provides a more sensitive indicator for assessment of cartilage matrix integrity than partition coefficient and the equilibrium distribution of solute. These findings may help in developing clinical methods of contrast agent-based imaging to detect cartilage injury.

Effect of collagen cross-linking on quantitative MRI parameters of articular cartilage.

OBJECTIVE: To investigate the sensitivity of quantitative magnetic resonance imaging (MRI) parameters to increase of collagen cross-linking in articular cartilage, a factor possibly contributing to the aging-related development of osteoarthritis (OA). The issue has not been widely studied although collagen cross-links may significantly affect the evaluation of cartilage imaging outcome. DESIGN: Osteochondral samples (n = 14) were prepared from seven bovine patellae. To induce cross-linking, seven samples were incubated in threose while the other seven served as non-treated controls. The specimens were scanned at 9.4 T for T1, T1Gd (dGEMRIC), T2, adiabatic and continuous wave (CW) T1ρ, adiabatic T2ρ and T1sat relaxation times. Specimens from adjacent tissue were identically treated and used for reference to determine biomechanical properties, collagen, proteoglycan and cross-link contents, fixed charge density (FCD), collagen fibril anisotropy and water concentration of cartilage. RESULTS: In the threose-treated sample group, cross-links (pentosidine, lysyl pyridinoline (LP)), FCD and equilibrium modulus were significantly (P < 0.05) higher as compared to the non-treated group. Threose treatment resulted in significantly greater T1Gd relaxation time constant (+26%, P < 0.05), although proteoglycan content was not altered. Adiabatic and CW-T1ρ were also significantly increased (+16%, +28%, P < 0.05) while pre-contrast T1 was significantly decreased (-10%, P < 0.05) in the threose group. T2, T2ρ and T1sat did not change significantly. CONCLUSION: Threose treatment induced collagen cross-linking and changes in the properties of articular cartilage, which were detected by T1, T1Gd and T1ρ relaxation time constants. Cross-linking should be considered especially when interpreting the outcome of contrast-enhanced MRI in aging populations.

Diffusion of ionic and non-ionic contrast agents in articular cartilage with increased cross-linking--contribution of steric and electrostatic effects.

OBJECTIVE: To investigate the effect of threose-induced collagen cross-linking on diffusion of ionic and non-ionic contrast agents in articular cartilage. DESIGN: Osteochondral plugs (Ø=6mm) were prepared from bovine patellae and divided into two groups according to the contrast agent to be used in contrast enhanced computed tomography (CECT) imaging: (I) anionic ioxaglate and (II) non-ionic iodixanol. The groups I and II contained 7 and 6 sample pairs, respectively. One of the paired samples served as a reference while the other was treated with threose to induce collagen cross-linking. The equilibrium partitioning of the contrast agents was imaged after 24h of immersion. Fixed charge density (FCD), water content, contents of proteoglycans, total collagen, hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent) cross-links were determined as a reference. RESULTS: The equilibrium partitioning of ioxaglate (group I) was significantly (p=0.018) lower (-23.4%) in threose-treated than control samples while the equilibrium partitioning of iodixanol (group II) was unaffected by the threose-treatment. FCD in the middle and deep zones of the cartilage (p<0.05) and contents of Pent and LP (p=0.001) increased significantly due to the treatment. However, the proteoglycan concentration was not systematically altered after the treatment. Water content was significantly (-3.5%, p=0.007) lower after the treatment. CONCLUSIONS: Since non-ionic iodixanol showed no changes in partition after cross-linking, in contrast to anionic ioxaglate, we conclude that the cross-linking induced changes in charge distribution have greater effect on diffusion compared to the cross-linking induced changes in steric hindrance.

Computed tomography detects changes in contrast agent diffusion after collagen cross-linking typical to natural aging of articular cartilage.

OBJECTIVE: The effect of threose-induced collagen cross-linking on the mechanical and diffusive properties of cartilage was investigated in vitro. In particular, we investigated the potential of Contrast Enhanced Computed Tomography (CECT) to detect changes in articular cartilage after increased collagen cross-linking, which is an age-related phenomenon. METHODS: Osteochondral plugs (Ø=6.0 mm, n=28) were prepared from intact bovine patellae (n=7). Two of the four adjacent samples, prepared from each patella, were treated with threose to increase the collagen cross-linking, while the other two specimen served as paired controls. One sample pair was mechanically tested and then mechanically injured using a material testing device. Contrast agent [ioxaglate (Hexabrix™)] diffusion was imaged in the other specimen pair for 25 h using CECT. Water fraction, collagen and proteoglycan content, collagen network architecture and the amount of cross-links [hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent)] of the samples were also determined. RESULTS: Cartilage collagen cross-linking, both Pent and LP, were significantly (P<0.001) increased due to threose treatment. CECT could detect the increased cross-links as the contrast agent penetration and the diffusion flux were significantly (P<0.05) lower in the threose treated than in untreated samples. The equilibrium modulus (+164%, P<0.05) and strain dependent dynamic modulus (+47%, P<0.05) were both significantly greater in the threose treated samples than in reference samples, but there was no association between the initial dynamic modulus and the threose treatment. The water fraction, proteoglycan and collagen contents, as well as collagen architecture, were not significantly altered by the threose treatment. CONCLUSIONS: To conclude, the CECT technique was found to be sensitive at detecting changes in cartilage tissue due to increased collagen cross-linking. This is important since increased cross-linking has been proposed to be related to the increased injury susceptibility of tissue.

Detection of mechanical injury of articular cartilage using contrast enhanced computed tomography.

OBJECTIVE: Osteoarthritic degeneration may be initiated by mechanical overloading of articular cartilage. Mechanical injury increases the permeability of tissue, thereby probably affecting the diffusion of contrast agents in articular cartilage. We investigated whether it is possible to detect acute cartilage injury by measuring contrast agent diffusion into articular cartilage using contrast enhanced computed tomography (CECT). METHODS: Osteochondral plugs (Ø=6.0 mm, n=36) were prepared from intact bovine patellae (n=9). Two of the adjacent samples were injured by impact loading, using a drop tower, while the others served as paired controls. The samples were imaged before immersion in contrast agent solution [ioxaglate (Hexabrix™) or sodium iodide (NaI)] and 1, 3, 5, 7, 10, 15, 20 and 25 h after immersion using a MicroCT-instrument. Contrast agent content, diffusion coefficient and diffusion flux were determined for each sample. RESULTS: Already after 1 h the penetration of contrast agents into cartilage was significantly (P<0.05) greater in the injured samples. The diffusion coefficient was not altered by the injury, which suggests that reaching the diffusion equilibrium takes the same time in injured and intact cartilage. However, the diffusion flux of ioxaglate through the articular surface was significantly higher in injured samples at 30-60 min after immersion. CONCLUSIONS: To conclude, CECT could diagnose articular cartilage injuries, and determination of the diffusion flux of ioxaglate helped to detect tissue injury without waiting for the diffusion equilibrium. These results are encouraging, however, in vivo application of CECT is challenging and systematic further studies are needed to reveal its clinical potential.

Influence of female hormonal factors, in relation to autoantibodies and genetic markers, on the development of rheumatoid arthritis in northern Sweden: a case-control study.

OBJECTIVES: To study the influence of female hormonal factors on the development of rheumatoid arthritis (RA) in relation to the human leucocyte antigen (HLA)-DRB1 shared epitope (SE), the protein tyrosine phosphatase (PTPN22) 1858T variant, anti-citrullinated protein antibodies (ACPAs), and immunoglobulin (Ig)M-rheumatoid factor (IgM-RF). METHODS: A case-control study (1:4) was nested within the Medical Biobank of northern Sweden. Females who had subsequently developed RA (n = 70), median of 2.7 years before the onset of symptoms, and matched controls (n = 280) were identified from among the blood donors. A questionnaire concerning previous exposures until disease onset, including hormonal and reproductive factors, and smoking habits was distributed. RESULTS: Breastfeeding was significantly associated with the development of RA [odds ratio (OR) 4.8, 95% confidence interval (CI) 1.43-15.8]. Increasing time of breastfeeding increased the risk of RA (OR 5.7, 95% CI 1.83-17.95) for breastfeeding ≥ 17 months. In a multiple logistic regression analysis, increasing time of breastfeeding (OR 9.5, 95% CI 2.14-42.43 for ≥ 17 months), seropositivity for ACPAs (OR 19.5, 95% CI 4.47-84.81), and carriage of the PTPN22 1858T variant (OR 3.2, 95% CI 1.36-7.54) remained significant predictors of RA. Users of oral contraceptives (OC) for ≥ 7 years had a decreased risk for development of RA (OR 0.37, 95% CI 0.15-0.93). CONCLUSIONS: A longer duration of breastfeeding increased the risk of developing RA, especially among individuals seropositive for ACPA or IgM-RF or carrying the PTPN22 1858T variant. Use of OC for ≥ 7 years was associated with a decreased risk.

Differentiation of osteoblasts on pectin-coated titanium.

The gold standard for implant metals is titanium, and coatings such as collagen-I, RGD-peptide, chondroitin sulfate, and calcium phosphate have been used to modify its biocompatibility. We investigated how titanium coated with pectins, adaptable bioactive plant polysaccharides with anti-inflammatory effects, supports osteoblast differentiation. MC3T3-E1 cells, primary murine osteoblasts, and human mesenchymal cells (hMC) were cultured on titanium coated with rhamnogalacturonan-rich modified hairy regions (MHR-A and MHR-B) of apple pectin. Alkaline phosphatase (ALP) expression and activity, calcium deposition, and cell spreading were investigated. MHR-B, but not MHR-A, supports osteoblast differentiation. The MHR-A surface was not mineralized, but on MHR-B, the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The ALP activity of hMCs on MHR-A was 58.3% at day 7 and 9.3% from that of MHR-B at day 10. These data indicate that modified pectin nanocoatings may enhance the biocompatibility of bone and dental implants.

Decreased binding of [11C]flumazenil in Angelman syndrome patients with GABA(A) receptor beta3 subunit deletions.

We used positron emission tomography (PET) to study brain [11C]flumazenil (FMZ) binding in four Angelman syndrome (AS) patients. Patients 1 to 3 had a maternal deletion of 15q11-q13 leading to the loss of beta3 subunit of gamma-aminobutyric acidA/benzodiazepine (GABA(A)/BZ) receptor, whereas Patient 4 had a mutation in the ubiquitin protein ligase (UBE3A) saving the beta3 subunit gene. [11C]FMZ binding potential in the frontal, parietal, hippocampal, and cerebellar regions was significantly lower in Patients 1 to 3 than in Patient 4. We propose that the 15q11-q13 deletion leads to a reduced number of GABA(A)/BZ receptors, which could partly explain the neurological deficits of the AS patients.

An unexpected recurrence of Angelman syndrome suggestive of maternal germ-line mosaicism of del(15)(q11q13) in a Finnish family.

Angelman syndrome is a neuro-developmental disorder caused by genetic abnormalities affecting the maternal gene expression in the chromosome region 15q11-q13. In a study group of 45 Finnish Angelman patients, a recurrence of a del(15)(q11q13) was detected in one family. The mother's chromosomes 15 were structurally normal, whereas the patients and their unaffected brother shared an identical maternally derived haplotype outside the deletion region. These findings are suggestive of maternal germ-line mosaicism of del(15)(q11q13).

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation.

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.

Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.

A molecular and cytogenetic study in Finnish Prader-Willi patients.

The Prader-Willi syndrome (PWS) is a developmental disorder caused by a deficiency of paternal contributions, arising from differently sized deletions, uniparental disomy or rare imprinting mutations, in the chromosome region 15q11-q13. We studied 41 patients with suspected PWS and their parents using cytogenetic and molecular techniques. Of the 27 clinically typical PWS patients, 23 (85%) had a molecular deletion that could be classified into four size categories. Only 15 of them (71%) could be detected cytogenetically. Maternal uniparental heterodisomy was observed in four cases. The rest of the patients showed no molecular defects including rare imprinting mutations. In our experience, the use of the methylation test with the probe PW71 (D15S63), together with the probe hN4HS (SNRPN), which distinguishes between a deletion and uniparental disomy, is the method of choice for the diagnosis of PWS.