The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 80 patients from 72 families by means of a PCR-based mutation screening strategy, consisting of heteroduplex analysis, restriction enzyme digest, DNA single strand electrophoresis, and direct sequencing. For a subset of patients mutation screening with DHPLC was established which turned out to be as fast and as sensitive as the more conventional methods. Fifteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in smaller previous studies, p.A150P (65%), p.A175D (11%) and p.N335K (8%) were the most common mutated alleles, followed by c.360_363delCAAA, p.R60X, p.Y204X, and c.865delC. Eight novel mutations were identified in eight families with HFI: a small indel mutation (c.1044_1049delTTCTGGinsACACT), two small deletions (c.345_372del28; c.841_842delAC), two splice site mutations (c.113-1G>A, c.799+2T>A), one nonsense mutation (c.612T>G (p.Y204X)), and two missense mutations (c.532T>C (p.C178R), c.851T>C (p.L284P)). By mutation screening for the three most common ALDOB mutations by DHPLC in 2,000 randomly selected newborns we detected 21 heterozygotes. Based on these data and after correction for less common and private ALDOB mutations, HFI prevalence in central Europe is estimated to be 1:26,100 (95% confidence interval 1: 12,600-79,000).     

5′-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma

 DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However, the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5′-cytosine DNA methyltransferase in sporadic colon carcinoma have been published. We used a competitive RT-PCR assay for quantification of mRNA of 5′-cytosine DNA methyltransferase in colon biopsies obtained from patients with hereditary colon carcinoma syndromes and compared the results with those obtained in a control group. No significant difference was found between the flat mucosa of FAP patients and the mucosa of the control group. In FAP and HNPCC patients, the 5′-cytosine DNA methyltransferase mRNA levels of adenomas were significantly higher (P<0.05) than of flat mucosa in the same group, but both showed great variability from patient to patient. Our findings suggest that the mRNA levels of methyltransferase cannot be used as predictive marker for screening in families affected by hereditary colon carcinoma. Received: 20 July 1998 / Accepted: 21 September 1998     

Two-dimensional NMR spectroscopy of urinary glycosaminoglycans from patients with different mucopolysaccharidoses

Patients with different types of mucopolysaccharidoses (MPS) lack specific lysosomal enzymes, which leads to tissue accumulation and urinary excretion of glycosaminoglycans (GAGs). Since little is known about the molecular composition of the excreted GAG fragments, we used two-dimensional [1H,13C]-correlation nuclear magnetic resonance (NMR) spectroscopy for a detailed analysis of the urinary GAGs of patients with MPS types I, II, IIIA, IVA and VI. The method revealed that the molecular structures of the excreted GAGs, i.e. heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and keratan sulfate (KS) are clearly distinct for the different MPS types. The chain terminal residues that are the normal substrates for the defective enzymes constitute characteristic sets of signals for each MPS type. The GAG chains show variations in carbohydrate composition and sulfation patterns that can be related to the different MPS types and clinical features. For example, two patients with MPS IIIA (M. Sanfilippo) with signs of CNS degeneration but only mild somatic features excrete a highly sulfated variant of HS, resembling HS in porcine brain, whereas a patient with MPS I (M. Scheie) and two patients with MPS II (M. Hunter), who present primarily with coarse facial features, joint contractures and skeletal deformities excrete a different type of HS with lower sulfation. In another case study, a patient with MPS IVA (M. Morquio), who presented mainly with skeletal dysplasia, excreted not only excessive amounts of KS but also a highly sulfated CS variant, resembling CS in articular cartilage. The high-resolution NMR analysis of urinary GAGs presented here for the first time provides a solid basis for future studies with a larger number of patients to further explore pathogenesis and course of the MPS diseases.     

Morphomechanics of the humero-ulnar joint: II. Concave incongruity determines the distribution of load and subchondral mineralization

Background: A deeper joint socket (concave incongruity) is found at most angles of flexion of the humero-ulnar joint and maintained over a wide range of physiological loading. It is, however, unclear how far this incongruity affects the distribution of load and subchondral mineralization of this joint as compared with a congruous configuration. Methods: Two nonlinear, axisymmetrical finite element models with two cartilage layers were constructed, one congruous and one incongruous, with a joint space of realistic magnitude. The distribution of subchondral mineralization was determined by computed tomography osteoabsorptiometry in the same six specimens that were investigated in the first part of the study, and compared with the biomechanical data obtained there and the predictions of the models. Results: In the congruous case, the center of the socket is highly loaded, whereas the periphery does not experience mechanical stimulation. A central bone density maximum is predicted. With concave incongruity the position of the contact areas shifts from the joint margin towards the center as the load increases, and the peak stresses are considerably lower. A bicentric ventro-dorsal distribution pattern of subchondral mineralization is predicted, and this is actually found in the six specimens. Conclusions: Concave incongruity is shown to determine load transmission and subchondral mineralization of the humero-ulnar joint. It is suggested that this shape leads to a more even distribution of stress, provides intermittent stimulation of the cartilaginous tissue, and has beneficial effects on the metabolism, nutrition, and lubrication of the articular cartilage during cyclic loading. © 1995 Wiley-Liss, Inc.     

α6 integrins participate in pro-T cell homing to the thymus

During embryogenesis, colonization of the thymic rudiment by hemopoietic progenitor cells depends on the adhesion of these cells to the jugular endothelium. Previously, we showed that progenitor T cells (pro-T cells) interact with α₆ integrins present on vascular endothelium. Here, we demonstrate that anti-α₆ integrin antibodies reduced the number of thymocytes up to 80 % in a congenic mouse model for thymus colonization by pro-T cells. In organotypic thymus cultures, the anti-α₆ integrin antibodies did not influence T cell development and proliferation. From this, we conclude that α₆ integrin participates in thymus homing. During mouse thymus ontogeny, α₆ integrin mRNA and protein expression was found as early as day 10 of development; at day 11, perithymic endothelial cells were α₆ integrin positive. Two α₆ integrin mRNA exist which are produced by alternative exon usage. The longer form, α₆, integrin, predominates during early embryonic stages, while the shorter α_6A form was present later during development. Although α₆, integrins can be displayed by immature thymocytes, strongest expression was found on intra- and perithymic vascular endothelium. These data suggest that α₆ integrins are involved in the homing of pro-T cells to the developing thymus by mediating adhesion of pro-T cells to the vascular endothelium.     

In vivo efficacy of bone-marrow-coated polycaprolactone scaffolds for the reconstruction of orbital defects in the pig

Alloplastic materials offer a number of advantages over bone autografts in the reconstruction of craniofacial defects. These include: lack of donor site morbidity, unlimited quantities of available material, and the possibility to conform exactly to the defect. An ideal bioresorbable material would degrade slowly, and have osteoconductive properties to allow replacement and remodeling by osseous tissue. This is seldom observed, the materials instead being replaced by fibrous tissue. Polycaprolactone (PCL), an FDA-approved bioresorbable polymer, has several properties that might make it suitable for reconstruction of craniofacial defects. The technique of fused deposition modeling (FDM) allows for the fabrication of highly reproducible bioresorbable 3D scaffolds. The nature of the fully interconnected pore network might enhance vascular ingrowth and osteoconductive properties. It was hypothesized that coating the scaffolds in bone marrow might enhance bone formation due to the osteoinductive nature of the bone-marrow mesenchymal cells. This study aimed to test these hypotheses in the pig model. Defects measuring 2 x 2 cm were surgically created in each orbit of eight Yorkshire pigs. The orbits were divided into three groups: Group 1 (n=4), no reconstruction (control); Group 2 (n=6), reconstruction with no coated PCL scaffolds; and Group 3 (n=6) reconstruction with bone-marrow-coated PCL scaffolds. The results were evaluated at 3 months by histological and histomorphometric analyses. The defects in Group 1 were covered with fibrous scar tissue. The shape of the reconstructed area was insufficient. The defects in Groups 2 and 3 were reconstructed correctly. In Group 2 the noncoated scaffolds showed 4.5% of new bone formation compared with 14.1% in Group 3, which is statistically significant (p<0.05). The entirely interconnected 3D polycaprolactone scaffold seems to be a promising material. It induces the bone ingrowth required for reconstructing craniofacial and orbital defects. Further long-term evaluations of these PCL scaffolds must be made in order to confirm these conclusions.     

Short- and long-term impact of neutropenia within the first year after kidney transplantation

The aim of this retrospective single-center study was to investigate the short- and long-term impact of neutropenia occurring within the first year after kidney transplantation, with a special emphasis on different neutropenia grades. In this unselected cohort, 225/721 patients (31%) developed 357 neutropenic episodes within the first year post-transplant. Based on the nadir neutrophil count, patients were grouped as neutropenia grade 2 (<1.5–1.0*10⁹/l; n = 105), grade 3 (<1.0–0.5*10⁹/l; n = 65), and grade 4 (<0.5*10⁹/l; n = 55). Most neutropenia episodes were presumably drug-related (71%) and managed by reduction/discontinuation of potentially responsible drugs (mycophenolic acid [MPA] 51%, valganciclovir 25%, trimethoprim/sulfamethoxazole 19%). Steroids were added/increased as replacement for reduced/discontinued MPA. Granulocyte colony-stimulating factor was only used in 2/357 neutropenia episodes (0.6%). One-year incidence of (sub)clinical rejection, one-year mortality, and long-term patient and graft survival were not different among patients without neutropenia and neutropenia grade 2/3/4. However, the incidence of infections was about 3-times higher during neutropenia grade 3 and 4, but not increased during grade 2. In conclusion, neutropenia within the first year after kidney transplantation represents no increased risk for rejection and has no negative impact on long-term patient and graft survival. Adding/increasing steroids as replacement for reduced/discontinued MPA might supplement management of neutropenia.     

The utility of in-bore multiparametric magnetic resonance-guided biopsy in men with negative multiparametric magnetic resonance-ultrasound software-based fusion targeted biopsy

ObjectivesTo evaluate the utility of in-bore multiparametric magnetic resonance-guided biopsy of the prostate (IB) in patients with visible lesion/s and previous negative software-based multiparametric magnetic resonance imaging/ultrasonography fusion-targeted biopsy of the prostate (FTB).Patients and methodsWe retrospectively analysed prospectively maintained database including consecutive men undergoing IB from March 2013 to October 2017 in 2 European centres expert in this procedure. We selected men with the following criteria: No previous treatment for prostate cancer (CaP), multiparametric magnetic resonance imaging (mpMRI) lesion(s) PIRADS score ≥ 3, FTB showing no clinically significant cancer (csCaP), and subsequent IB. Patient's characteristics, mpMRI findings, biopsy technique, and histopathological results were extracted. The primary outcome was to determine the detection rate of csCaP, defined as any Gleason pattern ≥ 4. A multivariable analysis was performed to identify predictors of positive findings at IB.ResultsFifty-three men were included. Median age was 68 years (interquartile range [IQR] 64–68), median Prostate-Specific Antigen (PSA) was 7.6 ng/ml (IQR 5.2–10.9), and median prostate volume was 59 ml (IQR 44–84). Fifty-six lesions with PIRADS score 3 in 9 cases (16%), 4 in 30 cases (54%), and 5 in 17 cases (30%) were detected. FTB was performed in all cases using a transrectal approach with 3 different platforms (Toshiba, Koelis, and Artemis). Median time between FTB and IB was 3 months (IQR 1–7). A median of 2 cores per lesion were collected with IB (IQR 2–3). No cancer, clinically insignificant and clinically significant cancer were found in 33 (59%), 9 (16%), and 14 (25%) targeted lesions, respectively. Median maximum cancer core length and maximum positive percentage were 9 mm (3–13) and 55% (21%–80%). The only predictor of csCaP on IB was prostate volume (P = 0.026) with an ideal cut-off at 70 ml.ConclusionOne in 4 patients with previous negative FTB, IB was able to detect csCaP. According to this study, IB would be of particularly useful in patients with large glands.