Establishment of iPSC lines and zebrafish with loss-of-function AHDC1 variants: Models for Xia-Gibbs syndrome.

Xia-Gibbs syndrome (XGS) is a syndromic form of intellectual disability caused by heterozygous AHDC1 variants, but the pathophysiological mechanisms underlying this syndrome are still unclear. In this manuscript, we describe the development of two different functional models: three induced pluripotent stem cell (iPSC) lines with different loss-of-function (LoF) AHDC1 variants, derived by reprogramming peripheral blood mononuclear cells from XGS patients, and a zebrafish strain with a LoF variant in the ortholog gene (ahdc1) obtained through CRISPR/Cas9-mediated editing. The three iPSC lines showed expression of pluripotency factors (SOX2, SSEA-4, OCT3/4, and NANOG). To verify the capacity of iPSC to differentiate into the three germ layers, we obtained embryoid bodies (EBs), induced their differentiation, and confirmed the mRNA expression of ectodermal, mesodermal, and endodermal markers using the TaqMan hPSC Scorecard. The iPSC lines were also approved for the following quality tests: chromosomal microarray analysis (CMA), mycoplasma testing, and short tandem repeat (STR) DNA profiling. The zebrafish model has an insertion of four base pairs in the ahdc1 gene, is fertile, and breeding between heterozygous and wild-type (WT) animals generated offspring in a genotypic proportion in agreement with Mendelian law. The established iPSC and zebrafish lines were deposited on the hpscreg.eu and zfin.org platforms, respectively. These biological models are the first for XGS and will be used in future studies that investigate the pathophysiology of this syndrome, unraveling its underlying molecular mechanisms.

Treatment With Methotrexate Associated With Lipid Core Nanoparticles Prevents Aortic Dilation in a Murine Model of Marfan Syndrome.

In Marfan syndrome (MFS), dilation, dissection, and rupture of the aorta occur. Inflammation can be involved in the pathogenicity of aortic defects and can thus be a therapeutic target for MFS. Previously, we showed that the formulation of methotrexate (MTX) associated with lipid nanoparticles (LDE) has potent anti-inflammatory effects without toxicity. To investigate whether LDEMTX treatment can prevent the development of aortic lesions in the MFS murine model. MgΔloxPneo MFS (n = 40) and wild-type (WT, n = 60) mice were allocated to 6 groups weekly injected with IP solutions of: (1) only LDE; (2) commercial MTX; (3) LDEMTX (dose = 1mg/kg) between 3rd and 6th months of life. After 12 weeks of treatments, animals were examined by echocardiography and euthanatized for morphometric and molecular studies. MFS mice treated with LDEMTX showed narrower lumens in the aortic arch, as well as in the ascending and descending aorta. LDEMTX reduced fibrosis and the number of dissections in MFS but not the number of elastic fiber disruptions. In MFS mice, LDEMTX treatment lowered protein expression of pro-inflammatory factors macrophages (CD68), T-lymphocytes (CD3), tumor necrosis factor-α (TNF-α), apoptotic factor cleaved-caspase 3, and type 1 collagen and lowered the protein expression of the transforming growth factor-ß (TGF-ß), extracellular signal-regulated kinases ½ (ERK1/2), and SMAD3. Protein expression of CD68 and CD3 had a positive correlation with an area of aortic lumen (r 2 = 0.36; p < 0.001), suggesting the importance of inflammation in the causative mechanisms of aortic dilation. Enhanced adenosine availability by LDEMTX was suggested by higher aortic expression of an anti-adenosine A2a receptor (A2a) and lower adenosine deaminase expression. Commercial MTX had negligible effects. LDEMTX prevented the development of MFS-associated aortic defects and can thus be a candidate for testing in clinical studies.

Frequency of the LRRK2 G2019S mutation in late-onset sporadic patients with Parkinson's disease.

UNLABELLED: Mutations in the LRRK2 gene, predominantly G2019S, have been reported in individuals with autosomal dominant inheritance and sporadic Parkinson's disease (PD). The G2019S mutation has an age-dependent penetrance and evidence shows common ancestry. The clinical manifestations are indistinguishable from idiopathic PD. Its prevalence varies according to the population studied ranging from less than 0.1% in Asians to 41% in North African Arabs. This study aimed to identify G2019S mutation in Brazilian idiopathic PD patients. METHOD: We sampled 100 PD patients and 100 age- and gender-matched controls. Genetical analysis was accomplished by polymerase chain reaction (PCR). RESULTS: No G2019S mutations were found in both patients with sporadic PD and controls. CONCLUSIONS: Our results may be explained by the relatively small sample size.

Frequency of the LRRK2 G2019S mutation in late-onset sporadic patients with Parkinson’s disease / Frequência da mutação G2019S do LRRK2 em pacientes com doença de Parkinson esporádica e início tardio

Mutations in the LRRK2 gene, predominantly G2019S, have been reported in individuals with autosomal dominant inheritance and sporadic Parkinson’s disease (PD). The G2019S mutation has an age-dependent penetrance and evidence shows common ancestry. The clinical manifestations are indistinguishable from idiopathic PD. Its prevalence varies according to the population studied ranging from less than 0.1% in Asians to 41% in North African Arabs. This study aimed to identify G2019S mutation in Brazilian idiopathic PD patients. Method: We sampled 100 PD patients and 100 age- and gender-matched controls. Genetical analysis was accomplished by polymerase chain reaction (PCR). Results: No G2019S mutations were found in both patients with sporadic PD and controls. Conclusions: Our results may be explained by the relatively small sample size. .

Mutação no gene LRRK2, predominantemente G2019S, foi descrita em indivíduos com doença de Parkinson (DP) esporádica ou herança autossômica dominante. A penetrância da mutação varia com a idade e há evidências de ancestral comum. As manifestações clínicas são indistinguíveis da DP idiopática. Sua prevalência depende da população estudada e varia de 0,1% em asiáticos a 41% em árabes do norte africano. O objetivo desse estudo foi identificar a mutação G2019S em brasileiros com DP esporádica. Método: Foram testados 100 pacientes com DP e 100 controles pareados por idade e sexo. A análise genética foi realizada pela reação em cadeia por polimerização (PCR). Resultados: Não foi encontrada a mutação G2019S nem nos pacientes com DP nem nos controles. Conclusão: É possível que nossos resultados sejam devidos ao pequeno número de pacientes incluídos. .

Mesenchymal and embryonic characteristics of stem cells obtained from mouse dental pulp.

OBJECTIVE: Several studies have demonstrated that human dental pulp is a source of mesenchymal stem cells. To better understand the biological properties of these cells we isolated and characterized stem cells from the dental pulp of EGFP transgenic mice. METHODS: The pulp tissue was gently separated from the roots of teeth extracted from C57BL/6 mice, and cultured under appropriate conditions. Flow cytometry, RT-PCR, light microscopy (staining for alkaline phosphatase) and immunofluorescence were used to investigate the expression of stem cell markers. The presence of chromosomal abnormalities was evaluated by G banding. RESULTS: The mouse dental pulp stem cells (mDPSC) were highly proliferative, plastic-adherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were positive for alkaline phosphatase. The karyotype was normal until the 5th passage. The Pou5f1/Oct-4 and ZFP42/Rex-1, but not Nanog transcripts were detected in mDPSC. Flow cytometry and fluorescence analyses revealed the presence of a heterogeneous population positive for embryonic and mesenchymal cell markers. Adipogenic, chondrogenic and osteogenic differentiation was achieved after two weeks of cell culture under chemically defined in vitro conditions. In addition, some elongated cells spontaneously acquired a contraction capacity. CONCLUSIONS: Our results reinforce that the dental pulp is an important source of adult stem cells and encourage studies on therapeutic potential of mDPSC in experimental disease models.

Genotype-phenotype correlation in Brazillian Rett syndrome patients.

BACKGROUND: Rett syndrome (RS) is a severe neurodevelopmental X-linked dominant disorder caused by mutations in the MECP2 gene. PURPOSE: To search for point mutations on the MECP2 gene and to establish a correlation between the main point mutations found and the phenotype. METHOD: Clinical evaluation of 105 patients, following a standard protocol. Detection of point mutations on the MECP2 gene was performed on peripheral blood DNA by sequencing the coding region of the gene. RESULTS: Classical RS was seen in 68% of the patients. Pathogenic point mutations were found in 64.1% of all patients and in 70.42% of those with the classical phenotype. Four new sequence variations were found, and their nature suggests patogenicity. Genotype-phenotype correlations were performed. CONCLUSION: Detailed clinical descriptions and identification of the underlying genetic alterations of this Brazilian RS population add to our knowledge of genotype/phenotype correlations, guiding the implementation of mutation searching programs.

Genotype-phenotype correlation in Brazillian Rett syndrome patients / Correlação genótipo-fenótipo em pacientes brasileiras com síndrome de Rett

BACKGROUND: Rett syndrome (RS) is a severe neurodevelopmental X-linked dominant disorder caused by mutations in the MECP2 gene. PURPOSE: To search for point mutations on the MECP2 gene and to establish a correlation between the main point mutations found and the phenotype. METHOD: Clinical evaluation of 105 patients, following a standard protocol. Detection of point mutations on the MECP2 gene was performed on peripheral blood DNA by sequencing the coding region of the gene. RESULTS: Classical RS was seen in 68 percent of the patients. Pathogenic point mutations were found in 64.1 percent of all patients and in 70.42 percent of those with the classical phenotype. Four new sequence variations were found, and their nature suggests patogenicity. Genotype-phenotype correlations were performed. CONCLUSION: Detailed clinical descriptions and identification of the underlying genetic alterations of this Brazilian RS population add to our knowledge of genotype/phenotype correlations, guiding the implementation of mutation searching programs.

INTRODUÇÃO: A síndrome de Rett é uma grave doença do neurodesenvolvimento ligada ao X dominante, causada por mutações no gene MECP2. OBJETIVOS: Identificar mutações de ponto no gene MECP2 e estabelecer uma correlação entre as principais mutações encontradas e o fenótipo. MÉTODO: Avaliação clínica de 105 pacientes, seguindo um protocolo estabelecido. A identificação de mutações de ponto foi realizada em DNA de sangue periférico por sequenciamento da região codificante do gene amplificada por PCR. RESULTADOS: Em 68 por cento dos pacientes observou-se o quadro clássico da síndrome. Mutações de ponto patogênicas foram encontradas em 64,1 por cento dos pacientes e em 70,42 por cento das pacientes com o quadro clássico. Quatro novas variações de seqüência foram identificadas e sua natureza sugere patogenicidade. Correlações genótipo-fenótipo foram estabelecidas. CONCLUSÃO: Descrições clínicas detalhadas desta população brasileira de pacientes acrescenta conhecimento às correlações genótipo-fenótipo nesta grave condição, que podem auxiliar na implantação de programas de triagem de mutações.

[The importance of the use of stem cells for public health]. / A importância do uso das células tronco para a saúde pública.

Stem cells were elected 'Breakthrough of the Year' by Science(1) magazine in 1999, having shown that stem cells from adult tissues retained the ability to differentiate into other tissue types. During the previous year, the first human embryo stem cell lines were established. Since then, the number of scientific papers on stem cells has been increasing exponentially, establishing new paradigms that are rapidly challenged by subsequent experiments. This paper reviews the stem cell research field, divided into two groups: embryo and adult stem cells. While the differentiation potential of the former is well characterized in mice and humans, their use in cell therapy and research has been hampered by histocompatibility, safety and ethical issues. In contrast, adult stem cells do not present these problems. However, the extent of their plasticity is still under investigation. Nevertheless, numerous clinical trials in humans are under way, mainly with stem cells derived from bone marrow. This paper discusses discuss the importance of working with both classes of human stem cells in order to fulfill the promise of stem cell therapies.

A importância do uso das células tronco para a saúde pública / The importance of the use of stem cells for public health

Em 1999, as células-tronco foram eleitas "Scientific Breakthrough of the Year" (avanço científico do ano) pela revista Science¹. Naquele ano, foi demonstrado que células-tronco de tecidos adultos mantinham a capacidade de se diferenciar em outros tipos de tecidos. No ano anterior, as primeiras linhagens de células-tronco embrionárias humanas foram estabelecidas. Desde então, o número de artigos científicos sobre células-tronco vem crescendo exponencialmente, onde novos paradigmas são estabelecidos. Neste artigo, farei uma revisão da área de células-tronco com um foco especial em seu uso como agente terapêutico em doenças comuns como diabetes e cardiopatias. As células-tronco serão tratadas em dois grupos distintos: as embrionárias e as adultas. Enquanto o potencial de diferenciação das primeiras está bem caracterizado em camundongos e em humanos, seu uso em terapia celular e em pesquisa tem sido dificultado por questões de histocompatibilidade, segurança e ética. Em contraste, células-tronco adultas não apresentam estes empecilhos, apesar da extensão de sua plasticidade ainda estar sob investigação. Mesmo assim, diversos testes clínicos em humanos estão em andamento utilizando células-tronco adultas, principalmente derivadas da medula óssea. Discutirei ainda a importância de se trabalhar com as duas classes de células-tronco humanas de forma a se cumprir suas promessas terapêuticas.

Stem cells were elected 'Breakthrough of the Year' by Science¹ magazine in 1999, having shown that stem cells from adult tissues retained the ability to differentiate into other tissue types. During the previous year, the first human embryo stem cell lines were established. Since then, the number of scientific papers on stem cells has been increasing exponentially, establishing new paradigms that are rapidly challenged by subsequent experiments. This paper reviews the stem cell research field, divided into two groups: embryo and adult stem cells. While the differentiation potential of the former is well characterized in mice and humans, their use in cell therapy and research has been hampered by histocompatibility, safety and ethical issues. In contrast, adult stem cells do not present these problems. However, the extent of their plasticity is still under investigation. Nevertheless, numerous clinical trials in humans are under way, mainly with stem cells derived from bone marrow. This paper discusses discuss the importance of working with both classes of human stem cells in order to fulfill the promise of stem cell therapies.

Association between Parkinson's disease and glucocerebrosidase mutations in Brazil.

OBJECTIVE: To evaluate the association between parkinsonism and mutations in the glucocerebrosidase gene (GBA) in Brazilian patients. METHODS: We searched for three GBA common mutations (N370S, L444P and G377S) in 65 Brazilian patients affected by PD with disease onset before the age of 55 and compared the results to 267 age- and sex-matched controls. RESULTS: GBA mutations were detected at a significantly higher frequency among Parkinson's disease patients (2/65=3%), when compared to the control group (0/267): P=0.0379. CONCLUSION: These results provide further evidence for GBA mutations being a possible hereditary risk factor for PD.

The frequency of Tay-Sachs disease causing mutations in the Brazilian Jewish population justifies a carrier screening program

CONTEXT: Tay-Sachs disease is an autosomal recessive disease characterized by progressive neurologic degeneration, fatal in early childhood. In the Ashkenazi Jewish population the disease incidence is about 1 in every 3,500 newborns and the carrier frequency is 1 in every 29 individuals. Carrier screening programs for Tay-Sachs disease have reduced disease incidence by 90 percent in high-risk populations in several countries. The Brazilian Jewish population is estimated at 90,000 individuals. Currently, there is no screening program for Tay-Sachs disease in this population. OBJECTIVE: To evaluate the importance of a Tay-Sachs disease carrier screening program in the Brazilian Jewish population by determining the frequency of heterozygotes and the acceptance of the program by the community. SETTING: Laboratory of Molecular Genetics - Institute of Biosciences - Universidade de Säo Paulo. PARTICIPANTS: 581 senior students from selected Jewish high schools. PROCEDURE: Molecular analysis of Tay-Sachs disease causing mutations by PCR amplification of genomic DNA, followed by restriction enzyme digestion. RESULTS: Among 581 students that attended educational classes, 404 (70 percent) elected to be tested for Tay-Sachs disease mutations. Of these, approximately 65 percent were of Ashkenazi Jewish origin. Eight carriers were detected corresponding to a carrier frequency of 1 in every 33 individuals in the Ashkenazi Jewish fraction of the sample. CONCLUSION: The frequency of Tay-Sachs disease carriers among the Ashkenazi Jewish population of Brazil is similar to that of other countries where carrier screening programs have led to a significant decrease in disease incidence. Therefore, it is justifiable to implement a Tay-Sachs disease carrier screening program for the Brazilian Jewish population