Temporal activation of β-catenin signaling in the chondrogenic process of mesenchymal stem cells affects the phenotype of the cartilage generated

Adult mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering. In vitro predifferentiation of MSCs has been explored as a means to enhance MSC-based articular cartilage repair. However, there remain challenges to control and prevent the premature progression of MSC-derived chondrocytes to the hypertrophy. This study investigated the temporal effect of transforming growth factor (TGF)-β and β-catenin signaling co-activation during MSC chondrogenic differentiation and evaluated the influence of these predifferentiation conditions to subsequent phenotypic development of the cartilage. MSCs were differentiated in chondrogenic medium that contained either TGFβ alone, TGFβ with transient β-catenin coactivation, or TGFβ with continuous β-catenin coactivation. After in vitro differentiation, the pellets were transplanted into SCID mice. Both coactivation protocols resulted in the enhancement of chondrogenic differentiation of MSCs. Compared with TGFβ activation, transient coactivation of TGFβ-induction with β-catenin activation resulted in heightened hypertrophy and formed highly ossified tissues with marrow-like hematopoietic tissue in vivo. The continuous coactivation of the 2 signaling pathways, however, resulted in inhibition of progression to hypertrophy, marked by the suppression of type X collagen, Runx2, and alkaline phosphatase expression, and did not result in ossified tissue in vivo. Chondrocytes of the continuous co-activation samples secreted significantly more parathyroid hormone-related protein (PTHrP) and expressed cyclin D1. Our results suggest that temporal co-activation of the TGFβ signaling pathway with β-catenin can yield cartilage of different phenotype, represents a potential MSC predifferentiation protocol before clinical implantation, and has potential applications for the engineering of cartilage tissue.     

Place of genotyping in addition to the phenotype and the assay of serum α-1 antitrypsin

The diagnosis of deficiency of alpha-1 antitrypsin (A1AT) is based on isoelectric focusing of serum proteins and the extent of serum. However, the focusing is technically difficult and a greatly reduced concentration in abnormal A1AT tapeless does not differentiate an unstable variant of a variant called 'null' (that is to say without any phenotypic expression) to 'heterozygous' state. In this study, we compared the results of the assay, the phenotype and genotype of A1AT in 50 patients. Normal A1AT alleles (Pi*M1 to Pi*M4) or loss of the most common (Pi*S and Pi*Z) were clearly identified in phenotyping. However, genotyping was necessary to characterize: (i) certain alleles rarer A1AT (S-Munich, X-Christchurch); (ii) a null allele and; (iii) two new alleles A1AT not yet described in the literature. In conclusion, although the A1AT genotyping is generally not necessary, it is necessary to resolve complex cases and to obtain witnesses validated for isoelectric focusing.     

Does loss of hormonal receptors influence the pathophysiological characteristics of the HER-2 breast cancer phenotype?

Background

Some breast carcinomas (BC) of the HER-2 type respond poorly to endocrine therapy, indicating that hormonal receptor (HR) status possibly impacts the biological criteria of this tumor class. The aim of this study was to compare the clinicopathological characteristics of HR-positive and HR-negative tumors occurring in HER-2 and non-HER-2 BC.

Amplification of the Xq28 FRAXE repeats: Extreme phenotype variability?

We report on a new case of FRAXE mutation identified through the screening of a population of FRAXA-negative mentally retarded individuals. The index case, a 4-year-old boy with distinct minor anomalies and mental retardation with severe verbal impairment, his older brother, referred to as normal, and the mother have undergone careful clinical and molecular evaluation. The molecular defect, characterized by standard Southern blot analysis, is represented by a hypermethylated “full mutation” in the 2 boys and by a unique, altered, presumably unmethylated, band in the mother, which is interpreted as a “premutation.” The cytogenetic analysis failed to detect a folate-sensitive Xq27-28 fragile site in either “fully mutated” individual. The phenotype and intellectual performance of the 15-year-old brother of the propositus appeared completely normal. Our propositus shares some traits with previously described FRAXE-mutated subjects, suggesting an association with the Xq28 molecular defect; nevertheless, we find it difficult to reconcile the molecular identity and phenotypic difference in these mutated members of the same family. This could be a case of extreme phenotypic variability or a result of a more complicated molecular mechanism. © 1996 Wiley-Liss, Inc.     

Cantú syndrome: report of nine new cases and expansion of the clinical phenotype

Cantú syndrome, a rare disorder of congenital hypertrichosis, characteristic facial anomalies, cardiomegaly, and osteochondrodysplasia was first described in 1982 by Cantú. Twenty-three cases of Cantú syndrome have been reported to date. The pathogenesis of this rare autosomal dominant condition is unknown. We describe 10 patients with Cantú syndrome (9 new cases and the long-term follow-up of a 10th case reported by Robertson in 1999) comparing the phenotype with that of the previously reported cases. We describe how the distinctive facial appearance evolves with time and report several new findings including recurrent infections with low immunoglobulin levels and gastric bleeding in some of our patients. The cardiac manifestations include patent ductus arteriosus, septal hypertrophy, pulmonary hypertension, and pericardial effusions. They may follow a benign course, but of the 10 cases we report, 4 patients required surgical closure of the patent ductus arteriosus and 1 patient a pericardectomy. Long-term follow-up of these patients has shown reassuring neuro-developmental outcome and the emergence of a behavior phenotype including obsessive traits and anxiety.     

Is the maintainance of the C-terminus domain of dystrophin enough to ensure a milder Becker muscular dystrophy phenotype?

The severe Duchenne muscular dystrophy (DMD) and the more benignBecker type (BMD) are allelic conditions, controlled by a defectivegene at Xp21, caused by the absence (DMD) or a defect in quantityor quality (BMD) of the protein dystrophin. It has been suggestedthat the C-terminus domain of dystrophin is fundamental to ensurethe proper protein sub-cellular localization and function. Wewish to report our dystrophin findings in 4 among 142 DMD patientsstudied for DNA deletions and dystrophin analysis. Althoughthey have a severe clinical course, a positive dystrophin immunofluorescencepattern was seen using C-terminal antibody, and a dystrophinband of reduced molecular weight (corresponding to their DNAdeletions), but which maintained the C-terminus was seen throughWestern blot (WB). Based on these findings, we suggest thatin order to partially maintain its function, resulting in amilder phenotype, dystrophin may carry large internal deletionsbut in addition to the C-terminus, the region encompassing boththe N-terminus and the proximal region of the rod domain cannotbe absent. Therefore, the prognosis of a Becker phenotype ina young patient should be done with caution if based only onthe presence or not of dystrophin.     

HLA-B phenotype modifies the course of Behçet's disease in Moroccan patients

In Moroccan patients, predisposition to Beh?et's disease is associated with HLA-B*51, mostly in males with young age at disease onset. In addition, the disease is associated with B*15 both in females and in males with late disease onset. We analyzed the clinical presentation, the severity and the course of the disease in 86 Moroccan patients according to their HLA-B phenotype. The presence of the B*51 or B*15 did not predispose to a particular clinical manifestation, nor to a more severe presentation of the disease. By contrast, outcome of the disease significantly differed depending on HLA-B phenotype, with an increase of symptoms in most B*51+ patients and in half of B*15 patients, and a remission or a decrease of symptoms in all B*51-B*15- patients. This variable course was mostly observed for ocular lesions, skin lesions, articular symptoms, and neurological symptoms. These data may suggest that treatment should be given early in the course of the disease in B*51 or B*15-positive patients in order to stabilize the inflammatory process.     

Increased levels of α6 integrins are associated with the metastatic phenotype of human breast cancer cells

Integrins play an important role in interactions between cells and the extracellular matrix, and thus have a potential role in metastasis. Expression levels of 6, 1 and 4 integrin sub-units were measured in a panel of human breast cancer cell lines by RT/PCR, immunoprecipitation and flow cytometry. All the lines expressed 6, with the highest levels in the MDA-MB-231 and MDA-MB-435 cells. These grew the most aggressively and were metastastic in nude mice. Low levels of 6 protein were measured in breast cancer cells that were poorly tumorigenic and non-metastatic in nude mice, and there was an inverse relationship between ER and 6 expression. RT/PCR revealed that all lines expressed the 2 isoforms of 6, with the 6A isoform generally more abundant than 6B isoform. Clones of MDA-MB-435 were isolated by sterile sorting for cells with high or low 6 expression, and two variants established from metastases in nude mice were found to differ in 6 expression. When injected into nude mice, the 6-high variants produced significantly more lung metastases than the 6-low variants. 1 was abundant in all lines, while 4 was not detected in MDA-MB-134 cells, and in the MDA-MB-435 cells an alternately spliced variant of 4 was identified. Sequencing of the alternate variant revealed a novel sequence from a splicing event in the cytoplasmic tail of 4. None of the cells with this variant mRNA expressed detectable levels of 4 protein. Our results suggest that high 6 expression in human breast cancer cells is associated with tumorigenicity and metastatic potential.     

Evaluating the quality of Marfan genotype–phenotype correlations in existing FBN1 databases

BackgroundGenetic FBN1 testing is pivotal for confirming the clinical diagnosis of Marfan syndrome. In an effort to evaluate variant causality, FBN1 databases are often used. We evaluated the current databases regarding FBN1 variants and validated associated phenotype records with a new Marfan syndrome geno-phenotyping tool called the Marfan score.Methods and resultsWe evaluated four databases (UMD-FBN1, ClinVar, the Human Gene Mutation Database (HGMD), and Uniprot) containing 2,250 FBN1 variants supported by 4,904 records presented in 307 references. The Marfan score calculated for phenotype data from the records quantified variant associations with Marfan syndrome phenotype. We calculated a Marfan score for 1,283 variants, of which we confirmed the database diagnosis of Marfan syndrome in 77.1%. This represented only 35.8% of the total registered variants; 18.5–33.3% (UMD-FBN1 versus HGMD) of variants associated with Marfan syndrome in the databases could not be confirmed by the recorded phenotype.ConclusionFBN1 databases can be imprecise and incomplete. Data should be used with caution when evaluating FBN1 variants. At present, the UMD-FBN1 database seems to be the biggest and best curated; therefore, it is the most comprehensive database. However, the need for better genotype–phenotype curated databases is evident, and we hereby present such a database.Genet Med advance online publication 01 December 2016     

Copper deficiency myelopathy and subacute combined degeneration of the cord – Why is the phenotype so similar?

Copper deficiency myelopathy (CDM) is an increasingly recognised mimic of subacute combined degeneration (SCD) of the cord due to cobalamin (vitamin B(12)) deficiency. It has been suggested that copper deficiency induces myelopathy through dysfunction of cytochrome oxidase, which is known to be copper-dependent. However, cytochrome oxidase is not cobalamin-dependent, so this hypothesis fails to explain the phenotypic similarity between CDM and SCD. We propose that the first step in a final common pathway of CDM and SCD is dysfunction of the methylation cycle. This cycle includes both copper and cobalamin-dependent enzymes and catalyses the net transfer of a methyl group from methyltetrahydrofolate to a variety of macromolecules, including myelin proteins. Dysfunction of the cycle might therefore cause failure of myelin maintenance and ultimately myelopathy. One step of the methylation cycle is catalysed by methionine synthase, which is known to be cobalamin-dependent. Nitrous oxide specifically inhibits this enzyme by inactivating methylcobalamin, causing SCD in animals and humans. Both animal and human data suggest that methionine synthase also requires copper, implying that the enzyme may be involved in the pathogenesis of CDM. Another enzyme involved in the methylation cycle, S-adenosylhomocysteine hydrolase, may be regulated by copper. Although this enzyme is not cobalamin-dependent, its potential impairment in copper deficiency may contribute to the overall dysfunction of the methylation cycle. In cases of congenital deficiencies of methylation cycle enzymes, spinal and cerebral demyelination was observed, providing further support for a critical role of the methylation cycle in myelination. Biochemical dysfunction of the methylation cycle has been reported in HIV myelopathy, which has pathological parallels with SCD. This raises the possibility that other demyelinating myelopathies might involve an impairment of the methylation cycle. Our hypothesis could be tested by measuring CSF concentrations of methylation cycle intermediates in cases of CDM, as these reflect spinal cord tissue levels. If it were confirmed, the hypothesis would not only provide a plausible explanation for the phenotypic similarity between CDM and SCD, but might also open up further therapeutic options such as methionine and betaine supplementation.     

Pharmacologic blockade of 5-lipoxygenase improves the amyloidotic phenotype of an Alzheimer's disease transgenic mouse model involvement of γ-secretase

The 5-lipoxygenase (5-LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD) and that its genetic absence results in a reduction of amyloid β (Aβ) levels in Tg2576 mice. In the present study, we examined the effect of 5-LO pharmacological inhibition on the amyloidotic phenotype of these mice. Aβ deposition in the brains of mice receiving zileuton, a selective and specific 5-LO inhibitor, was significantly reduced when compared with control Tg2576 mice receiving vehicle. This reduction was associated with a similar decrease in brain Aβ peptides levels. Zileuton treatment did not induce any change in the steady state levels of amyloid-β precursor protein (APP), BACE1 or ADAM10. By contrast, it resulted in a significant reduction of presenilin 1 (PSEN1, alias PS1), nicastrin (NCSTN) , presenilin enhancer 2 homolog (PSNEN, alias, Pen-2), and anterior pharynx defective 1 (APH-1), the four components of the γ-secretase complex-at the protein and message level. Furthermore, in vitro studies confirmed that zileuton prevents Aβ formation by modulating γ-secretase complex levels without affecting Notch signaling. These data establish a functional role for 5-LO in the pathogenesis of AD-like amyloidosis, whereby it modulates the γ-secretase pathway. They suggest that pharmacological inhibition of 5-LO could provide a novel therapeutic opportunity for AD.     

Expanding the phenotype of 12q21 deletions: A role of BTG1 in speech development?

We report on a female individual with feeding difficulties, constipation, poor overall growth, periventricular lesions resembling gliosis in brain MRI, recurrent otitis media with palsy of facial nerve, distinct facial features, and pronounced delay in speech development. The latter was the most prominent feature. Molecular karyotyping revealed a heterozygous de novo deletion of 4.353 Mb at chromosome 12q21.33q22. This report expands the number of described individuals with heterozygous deletions at 12q21.33, their clinical spectrum and highlights the clinical variability, even in individuals with deletion of the same genes. Furthermore, our findings indicate a role of BTG1 (OMIM *109580) in speech development.     

Growth charts for Marfan syndrome in the Netherlands and analysis of genotype–phenotype relationships

To optimize care for children with Marfan syndrome (MFS) in the Netherlands, Dutch MFS growth charts were constructed. Additionally, we aimed to investigate the effect of FBN1 variant type (haploinsufficiency [HI]/dominant negative [DN]) on growth, and compare MFS-related height increase across populations. Height and weight data of individuals with MFS aged 0–21 years were retrospectively collected. Generalized Additive Models for Location, Scale and Shape (GAMLSS) was used for growth chart modeling. To investigate genotype–phenotype relationships, FBN1 variant type was included as an independent variable in height-for-age and BMI-for-age models. MFS-related height increase was compared with that of previous MFS growth studies from the United States, Korea, and France. Height and weight data of 389 individuals with MFS were included (210 males). Height-for-age, BMI-for-age, and weight-for-height charts reflected the tall and slender MFS habitus throughout childhood. Mean increase in height of individuals with MFS compared with the general Dutch population was significantly lower than in the other three MFS populations compared to their reference populations. FBN1-HI variants were associated with taller height in both sexes, and decreased BMI in females (p-values <0.05). This Dutch MFS growth study broadens the notion that genetic background and MFS variant type (HI/DN) influence tall and slender stature in MFS.     

Genotype–phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene

The aims of our research were to define the genotype–phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype–phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-l-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH₄) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.     

Does the bleomycin sensitivity assay express cancer phenotype?

The bleomycin (BLM) sensitivity assay has been associated with the measuring of increased risk of individual susceptibility to cancer, when chromatid breaks per cell (b/c) induced by an in vitro treatment of lymphocytes with BLM are elevated. The high heritability of BLM sensitivity indicates a genetic background. We wished to clarify whether the test characterizes the head and neck cancer phenotype as compared not only with healthy individuals, but also with alcoholic patients (ALCs) whose exposure to tobacco and alcohol consumption were similar to that of head and neck cancer patients (HNCPs), but whose liver diseases were not cancerous. If the BLM test quantifies merely cancer susceptibility on an inherited basis, the mutagen sensitivity of HNCPs should differ from that of ALCs. Conventional chromosome analysis and the BLM assay were carried out on 156 HNCPs, 51 ALCs, 146 healthy non-smokers and non-drinkers and 149 non-drinking smokers. The spontaneous rates of chromosomal aberrations (CAs) in HNCPs, ALCs and healthy smokers were identical (2.8%), but differed significantly from the non-smoking controls (2.25%). Sporadic CAs were clearly associated with tobacco smoking, but not with health status. Mutagen sensitivity measured by the BLM test showed significantly (P < 0.04) elevated values not only in HNCPs (1.13 b/c), but also in ALCs (1.29 b/c) as compared with the controls (1.01 b/c). The main finding of the study was that a considerable proportion (46%) of Hungarian controls were mutagen sensitive, twice as many as in those populations reported by others so far. Our data suggest that the BLM test does not characterize susceptibility to cancer due to insignificant differences between HNCPs and ALCs (P = 0.12) under our conditions. However, the assay might be used as a biomarker to predict cancer susceptibility under circumstances when aberrant cell frequency is >or=2% and b/c is >or=1.     

Satellite DNA β overrides the pathogenicity phenotype of the C4 gene of tomato leaf curl virus but does not compensate for loss of function of the coat protein and V2 genes

We have investigated the ability of satellite DNA beta to complement mutations in the CP, V2 and C4 genes of the monopartite begomovirus, tomato leaf curl virus, which are potentially involved in movement. A mutation in the coat protein was not complemented by DNA beta. Mutations of the C4 and V2 genes attenuated and abolished symptoms, respectively. In the presence of the C4 mutant, but not the V2 mutant, DNA beta induced typical symptoms, confirming that the satellite encodes a dominant symptom determinant. In contrast to the C4 mutant, DNA beta did not enhance the viral DNA levels of the V2 mutant, suggesting that V2 is required for this phenomenon. The significance of these findings is discussed based on our present understanding of the functions of the viral genes and DNA beta.     

The induction of the lupus phenotype by estrogen is via an estrogen receptor-α-dependent pathway

In order to investigate the roles of ER subtypes in the estrogen-induced lupus phenotype, ERα-deficient (ERα^−/−) and wild-type mice (WT) were injected monthly with estradiol (E-2) starting at 8 weeks. In WT mice, E-2 treatment induced a lupus phenotype, with accelerated death and increased kidney damage, as well as Th2-type serum cytokine and autoantibody production. In contrast, only minimal changes were observed in ERα^−/− mice after E-2 treatment. In a separate study, we found that in immune cells of autoimmune-prone SNF₁ and non-autoimmune DBF₁ mice, both ERα and ERβ were differentially expressed and modulated by E-2. In SNF₁ mice, there were more CD4⁺ and CD8⁺ T cells constitutively expressing ERα, and the percentages of ERα+ dendritic cells and macrophages were increased after E-2 exposure compared to DBF₁ mice. Taken together, these observations strongly suggest a role for ERα in E-2-induced development of the lupus phenotype.