Generation of two NAGLU-mutated homozygous cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo B syndrome

Mutations in the NAGLU gene cause Sanfilippo B syndrome (mucopolysaccharidosis IIIB), a rare lysosomal storage disorder whose main symptom is a severe and progressive neurodegeneration for which no treatment is still available. Here, we generated two homozygous NAGLU-mutated cell lines using CRISPR/Cas9 editing in a healthy human induced pluripotent stem cell (hiPSC) line. These novel cell lines express pluripotency specific markers and maintain their capability to differentiate into all three germ layers in vitro while exhibit a normal karyotype. These mutated lines in combination with the isogenic control line will be useful to model in vitro Sanfilippo B syndrome

Bone development and remodeling in metabolic disorders

There are many metabolic disorders that present with bone phenotypes. In some cases, the pathological bone symptoms are the main features of the disease whereas in others they are a secondary characteristic. In general, the generation of the bone problems in these disorders is not well understood and the therapeutic options for them are scarce. Bone development occurs in the early stages of embryonic development where the bone formation, or osteogenesis, takes place. This osteogenesis can be produced through the direct transformation of the pre-existing mesenchymal cells into bone tissue (intramembranous ossification) or by the replacement of the cartilage by bone (endochondral ossification). In contrast, bone remodeling takes place during the bone's growth, after the bone development, and continues throughout the whole life. The remodeling involves the removal of mineralized bone by osteoclasts followed by the formation of bone matrix by the osteoblasts, which subsequently becomes mineralized. In some metabolic diseases, bone pathological features are associated with bone development problems but in others they are associated with bone remodeling. Here, we describe three examples of impaired bone development or remodeling in metabolic diseases, including work by others and the results from our research. In particular, we will focus on hereditary multiple exostosis (or osteochondromatosis), Gaucher disease, and the susceptibility to atypical femoral fracture in patients treated with bisphosphonates for several years

Mapping the genetic and clinical characteristics of Gaucher disease in the Iberian Peninsula

<p>Abstract</p> <p>Background</p> <p>Gaucher disease (GD) is due to deficiency of the glucocerebrosidase enzyme. It is panethnic, but its presentation reveals ethnicity-specific characteristics.</p> <p>Methods</p> <p>We evaluated the distribution, and clinical and genetic characteristics of GD patients in the Iberian Peninsula (IP). We analysed geographical distribution, demographic, genetic and clinical data, age at diagnosis, type, and years of therapy in 436 GD patients from the IP.</p> <p>Results</p> <p>The prevalence of GD was 1/149,000 inhabitants; 88.3% were type 1, 6.7% type 2, and 5.0% type 3. The mean age at diagnosis in type 1 was 28.7 years. A total of 72.7% were classified as having mild forms, 25.5% moderate, and 1.7% severe. Anemia and thrombocytopenia were present in 56% and 55%, respectively. Bone disease and hepatomegaly were reported in 62% and 68%, respectively, and were more likely in asplenic than in non-splenectomized patients. Sixty-nine mutant alleles were identified, and five mutations accounted for 75% of the <it>GBA </it>alleles. Several patients described in our series had interesting phenotypes. A total of 58.7% of patients had received enzyme replacement therapy and 12.6% were treated with miglustat.</p> <p>Conclusions</p> <p>A broad spectrum of <it>GBA </it>mutations is present in the IP, with 98.2% of type 1 GD being mild and 23.0% never treated. These data highlight genetic and phenotypic heterogeneities among geographic populations.</p

The ASXL1 mutation p.Gly646Trpfs*12 found in a Turkish boy with Bohring-Opitz syndrome

Bohring‐Opitz syndrome (BOS, MIM #605039) is a rare and severe disease characterized mainly by intrauterine growth retardation, feeding difficulties, severe to profound developmental delay, nonspecific brain abnormalities, microcephaly, flexion at the elbows with ulnar deviation and flexion of the wrists and metacarpophalangeal joints (known as BOS posture) and distinctive facial features.1 Heterozygous ASXL1 truncating mutations have been identified as the main cause of BOS.1, 2 A recent publication 3 called the attention to the fact that mutations associated with BOS are also present in the ExAC (Exome Aggregation Consortium) database.4 As ASXL1 is one of the genes most commonly mutated during hematopoietic clonal expansion of cells, the authors hypothesized that the presence of this mutation in public databases could be due to somatic mosaicism, and they could confirm the hypothesis by manual examination of the ExAC WES reads

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty‐two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794‐bp deletion (c.532−37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8‐bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty-two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794-bp deletion (c.532-37-736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions. © 2011 Wiley-Liss, Inc.Fil: Cozar, Mónica. Universidad de Barcelona; EspañaFil: Urreizti, Roser. Universidad de Barcelona; EspañaFil: Vilarinho, Laura. Instituto de Genética Médica Jacinto Magalhaes; PortugalFil: Grosso, Carola. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Estudios de las Metabolopatías Congénitas; ArgentinaFil: Dodelson de Kremer, Raquel. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Estudios de las Metabolopatías Congénitas; ArgentinaFil: Asteggiano, Carla Gabriela. Gobierno de la Provincia de Córdoba. Ministerio de Salud. Hospital de Niños de la Santísima Trinidad; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dalmau, Jaime. Hospital Infantil La Fe; EspañaFil: García, Ana María. Hospital Infantil La Fe; EspañaFil: Vilaseca, María Antonia. Hospital Sant Joan de De ́u; EspañaFil: Grinberg Vaisman, Daniel Raúl. Universidad de Barcelona; EspañaFil: Balcells, Susana. Universidad de Barcelona; Españ

Suzaku observations of the HMXB 1A 1118-61

We present broad band analysis of the Be/X-ray transient 1A 1118-61 by Suzaku at the peak of its 3rd observed outburst in January 2009 and 2 weeks later when the source flux had decayed by an order of magnitude. The continuum was modeled with a \texttt{cutoffpl} model as well as a compTT model, with both cases requiring an additional black body component at lower energies. We confirm the detection of a cyclotron line at ~5 keV and discuss the possibility of a first harmonic at ~110 keV. Pulse profile comparisons show a change in the profile structure at lower energies, an indication for possible changes in the accretion geometry. Phase resolved spectroscopy in the outburst data show a change in the continuum throughout the pulse period. The decrease in the CRSF centroid energy also indicates that the viewing angle on the accretion column is changing throughout the pulse period.Comment: accepted by Ap

MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones

Background MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. Methods Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). Results Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. Conclusions We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy

De Novo PORCN and ZIC2 mutations in a highly consanguineous family

We present a Turkish family with two cousins (OC15 and OC15b) affected with syndromic developmental delay, microcephaly, and trigonocephaly but with some phenotypic traits distinct between them. OC15 showed asymmetrical skeletal defects and syndactyly, while OC15b presented with a more severe microcephaly and semilobal holoprosencephaly. All four progenitors were related and OC15 parents were consanguineous. Whole Exome Sequencing (WES) analysis was performed on patient OC15 as a singleton and on the OC15b trio. Selected variants were validated by Sanger sequencing. We did not identify any shared variant that could be associated with the disease. Instead, each patient presented a de novo heterozygous variant in a different gene. OC15 carried a nonsense mutation (p.Arg95*) in PORCN, which is a gene responsible for Goltz-Gorlin syndrome, while OC15b carried an indel mutation in ZIC2 leading to the substitution of three residues by a proline (p.His404_Ser406delinsPro). Autosomal dominant mutations in ZIC2 have been associated with holoprosencephaly 5. Both variants are absent in the general population and are predicted to be pathogenic. These two de novo heterozygous variants identified in the two patients seem to explain the major phenotypic alterations of each particular case, instead of a homozygous variant that would be expected by the underlying consanguinity

2008 official positions of the Brazilian Society for Clinical Densitometry - SBDens

With the evolution of bone densitometry, differences in technologies, acquisition techniques, reference databases, reporting methods, diagnostic criteria and terminology have developed and the International Society for Clinical Densitometry (ISCD) periodically holds Position Development Conferences, the latest in 2007. The Brazilian Society for Clinical Densitometry (SBDens), with support from many Brazilian societies interested in bone health, gathered numerous specialists to discuss the ISCD proposals and to evaluate the validity of the extension of those norms to Brazilian population. The SBDens reunion of consensus made a very utile document to help the understanding and interpretation of bone densitometry and other methods of bone assessment.A evolução dos métodos de avaliação da massa óssea trouxe diferentes tecnologias, modos de aquisição de imagens, bancos de dados de referência, terminologias, critérios diagnósticos fez com que a International Society for Bone Densitometry (ISCD) tomasse a iniciativa de promover reuniões periódicas de consenso, a última em 2007. A Sociedade Brasileira de Densitometria Clínica (SBDens), com apoio de várias sociedades brasileiras ligadas ao estudo da saúde óssea, reuniu diversos especialistas para discutir as propostas da ISCD e validar a aplicação destas normas à população brasileira. A reunião de Posições Oficiais da SBDens produziu um documento extremamente útil para a compreensão e interpretação da densitometria e de outros métodos de avaliação da massa óssea.Sociedade Brasileira de Densitometria ClínicaSociedade Brasileira para o Estudo do Metabolismo Ósseo e MineralUniversidade Federal de São Paulo (UNIFESP)Universidade Federal de Minas Gerais Hospital Mater DeiSociedade Brasileira de ReumatologiaSociedade Brasileira de OsteoporoseUniversidade de São Paulo Faculdade de Medicina Hospital das ClínicasAssociação Brasileira de Medicina Física e ReabilitaçãoUniversidade Federal do Espírito SantoUNIFESPColégio Brasileiro de RadiologiaSociedade Brasileira de Ortopedia e TraumatologiaUNIFESPSciEL