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Although atrial septal defects (ASD) can be subdivided based on their anatomical location, an essential aspect of human genetics and genetic counseling is distinguishing between isolated and familiar cases without extracardiac features and syndromic cases with the co-occurrence of extracardiac abnormalities, such as developmental delay. Isolated or familial cases tend to show genetic alterations in genes related to important cardiac transcription factors and genes encoding for sarcomeric proteins. By contrast, the spectrum of genes with genetic alterations observed in syndromic cases is diverse. Currently, it points to different pathways and gene networks relevant to the dysregulation of cardiomyogenesis and ASD pathogenesis. Therefore, this chapter reflects the current knowledge and highlights stable associations observed in human genetics studies. It gives an overview of the different types of genetic alterations in these subtypes, including common associations based on genome-wide association studies (GWAS), and it highlights the most frequently observed syndromes associated with ASD pathogenesis.
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Estudio de Asociación del Genoma Completo , Defectos del Tabique Interatrial , Humanos , Defectos del Tabique Interatrial/genética , Predisposición Genética a la Enfermedad/genética , MutaciónRESUMEN
Hydrocephalus is one of the most common congenital disorders of the central nervous system and often displays psychiatric co-morbidities, in particular autism spectrum disorder. The disease mechanisms behind hydrocephalus are complex and not well understood, but some association with dysfunctional cilia in the brain ventricles and subarachnoid space has been indicated. A better understanding of the genetic aetiology of hydrocephalus, including the role of ciliopathies, may bring insights into a potentially shared genetic aetiology. In this population-based case-cohort study, we, for the first time, investigated variants of postulated hydrocephalus candidate genes. Using these data, we aimed to investigate potential involvement of the ciliome in hydrocephalus and describe genotype-phenotype associations with an autism spectrum disorder. One-hundred and twenty-one hydrocephalus candidate genes were screened in a whole-exome-sequenced sub-cohort of the Lundbeck Foundation Initiative for Integrative Psychiatric Research study, comprising 72 hydrocephalus patients and 4181 background population controls. Candidate genes containing high-impact variants of interest were systematically evaluated for their involvement in ciliary function and an autism spectrum disorder. The median age at diagnosis for the hydrocephalus patients was 0 years (range 0-27 years), the median age at analysis was 22 years (11-35 years), and 70.5% were males. The median age for controls was 18 years (range 11-26 years) and 53.3% were males. Fifty-two putative hydrocephalus-associated variants in 34 genes were identified in 42 patients (58.3%). In hydrocephalus cases, we found increased, but not significant, enrichment of high-impact protein altering variants (odds ratio 1.51, 95% confidence interval 0.92-2.51, P = 0.096), which was driven by a significant enrichment of rare protein truncating variants (odds ratio 2.71, 95% confidence interval 1.17-5.58, P = 0.011). Fourteen of the genes with high-impact variants are part of the ciliome, whereas another six genes affect cilia-dependent processes during neurogenesis. Furthermore, 15 of the 34 genes with high-impact variants and three of eight genes with protein truncating variants were associated with an autism spectrum disorder. Because symptoms of other diseases may be neglected or masked by the hydrocephalus-associated symptoms, we suggest that patients with congenital hydrocephalus undergo clinical genetic assessment with respect to ciliopathies and an autism spectrum disorder. Our results point to the significance of hydrocephalus as a ciliary disease in some cases. Future studies in brain ciliopathies may not only reveal new insights into hydrocephalus but also, brain disease in the broadest sense, given the essential role of cilia in neurodevelopment.
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BACKGROUND: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD. METHODS: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model. RESULTS: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes. CONCLUSIONS: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.
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Señalización del Calcio , Calcio/metabolismo , Predisposición Genética a la Enfermedad , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Biología de Sistemas/métodos , Alelos , Animales , Biología Computacional/métodos , Bases de Datos Genéticas , Dinamarca , Femenino , Estudios de Asociación Genética/métodos , Variación Genética , Humanos , Masculino , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Sistema de Registros , Secuenciación del Exoma , Pez CebraRESUMEN
Genetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.
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Variaciones en el Número de Copia de ADN , Cardiopatías Congénitas/genética , Miocardio/citología , Proteínas de Neoplasias/genética , Proteínas Ribosómicas/genética , Animales , Células Cultivadas , Estudios de Cohortes , Modelos Animales de Enfermedad , Drosophila , Femenino , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/patología , Masculino , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/citología , Miocitos Cardíacos/patología , Estudios RetrospectivosRESUMEN
Family studies have established that the heritability of blood pressure is significant and genome-wide association studies (GWAS) have identified numerous susceptibility loci, including one within the non-coding part of Rho GTPase-activating protein 42 gene (ARHGAP42) on chromosome 11q22.1. Arhgap42-deficient mice have significantly elevated blood pressure, but the phenotypic effects of human variants in the coding part of the gene are unknown. In a Danish cohort of carriers with apparently balanced chromosomal rearrangements, we identified a family where a reciprocal translocation t(11;18)(q22.1;q12.2) segregated with hypertension and obesity. Clinical re-examination revealed that four carriers (age 50-77 years) have had hypertension for several years along with an increased body mass index (34-43 kg/m2). A younger carrier (age 23 years) had normal blood pressure and body mass index. Mapping of the chromosomal breakpoints with mate-pair and Sanger sequencing revealed truncation of ARHGAP42. A decreased expression level of ARHGAP42 mRNA in the blood was found in the translocation carriers relative to controls and allele-specific expression analysis showed monoallelic expression in the translocation carriers, confirming that the truncated allele of ARHGAP42 was not expressed. These findings support that haploinsufficiency of ARHGAP42 leads to an age-dependent hypertension. The other breakpoint truncated a regulatory domain of the CUGBP Elav-like family member 4 (CELF4) gene on chromosome 18q12.2 that harbours several GWAS signals for obesity. We thereby provide additional support for an obesity locus in the CELF4 domain.
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Proteínas Activadoras de GTPasa/genética , Predisposición Genética a la Enfermedad/genética , Haploinsuficiencia , Hipertensión/genética , Adulto , Anciano , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 18/genética , Dinamarca , Femenino , Proteínas Activadoras de GTPasa/sangre , Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Humanos , Hipertensión/sangre , Masculino , Persona de Mediana Edad , Obesidad/genética , Linaje , Translocación Genética , Adulto JovenRESUMEN
The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-ß1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.
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Proteínas Morfogenéticas Óseas/metabolismo , Centriolos/metabolismo , Cilios/metabolismo , Proteínas de Microtúbulos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Centrosoma/metabolismo , Humanos , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Aims: Congenital heart defects (CHD) affect almost 1% of all live born children and the number of adults with CHD is increasing. In families where CHD has occurred previously, estimates of recurrence risk, and the type of recurring malformation are important for counselling and clinical decision-making, but the recurrence patterns in families are poorly understood. We aimed to determine recurrence patterns, by investigating the co-occurrences of CHD in 1163 families with known malformations, comprising 3080 individuals with clinically confirmed diagnosis. Methods and results: We calculated rates of concordance and discordance for 41 specific types of malformations, observing a high variability in the rates of concordance and discordance. By calculating odds ratios for each of 1640 pairs of discordant lesions observed between affected family members, we were able to identify 178 pairs of malformations that co-occurred significantly more or less often than expected in families. The data show that distinct groups of cardiac malformations co-occur in families, suggesting influence from underlying developmental mechanisms. Analysis of human and mouse susceptibility genes showed that they were shared in 19% and 20% of pairs of co-occurring discordant malformations, respectively, but none of malformations that rarely co-occur, suggesting that a significant proportion of co-occurring lesions in families is caused by overlapping susceptibility genes. Conclusion: Familial CHD follow specific patterns of recurrence, suggesting a strong influence from genetically regulated developmental mechanisms. Co-occurrence of malformations in families is caused by shared susceptibility genes.
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Anomalías Múltiples/genética , Predisposición Genética a la Enfermedad , Cardiopatías Congénitas/genética , Sistema de Registros , Anomalías Múltiples/epidemiología , Adulto , Europa (Continente)/epidemiología , Femenino , Cardiopatías Congénitas/epidemiología , Humanos , Recién Nacido , Masculino , Morbilidad/tendencias , Linaje , Factores de RiesgoRESUMEN
STUDY DESIGN: Mutation analysis of a candidate disease gene in a cohort of patients with moderate to severe Adolescent idiopathic scoliosis (AIS). OBJECTIVE: To investigate if damaging mutations in the planar cell polarity gene VANGL1 could be identified in AIS patients. SUMMARY OF BACKGROUND DATA: AIS is a spinal deformity which occurs in 1% to 3% of the population. The cause of AIS is often unknown, but genetic factors are important in the etiology. Rare variants in genes encoding regulators of WNT/planar cell polarity (PCP) signaling were recently identified in AIS patients. METHODS: We analyzed the coding region of the VANGL1 gene for mutations using Sanger sequencing in 157 unrelated patients with moderate to severe AIS. The frequency of mutations in the patient cohort was compared with their frequency in a large cohort of controls. Functional effect of mutations were predicted in silico and analyzed in vitro by transfection of normal and mutant recombinant VANGL1 protein in Madin-Darby Canine Kidney (MDCK) cells. Cellular localization of recombinant proteins was analyzed by immunofluorescence microscopy analysis. RESULTS: In the patient cohort, we identified two rare missense mutations in VANGL1, encoding a receptor involved in WNT/PCP signaling. The mutations, p.I136N and p.F440âV, are very rare in the normal population. Both mutations are predicted to be damaging, and to affect evolutionary conserved amino acid residues of VANGL1. Functional analysis in MDCK cells showed that the mutations abolished the normal translocation of VANGL1 to the cell membrane. CONCLUSION: Our data support that mutations in genes involved in WNT/PCP signaling may be associated with AIS, but replication in other patient cohorts and further analysis of the role of WNT/PCP signaling in AIS is needed. LEVEL OF EVIDENCE: 4.
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Proteínas Portadoras/genética , Polaridad Celular/genética , Proteínas de la Membrana/genética , Escoliosis/genética , Adolescente , Adulto , Anciano , Células Cultivadas , Heterocigoto , Humanos , Mutación Missense , Vía de Señalización Wnt/genéticaRESUMEN
YKL-40, a glycoprotein involved in cell differentiation, has been associated with neurodevelopmental disorders, angiogenesis, neuroinflammation and glioblastomas. We evaluated YKL-40 protein distribution in the early human forebrain using double-labeling immunofluorescence and immunohistochemistry. Immunoreactivity was detected in neuroepithelial cells, radial glial end feet, leptomeningeal cells and choroid plexus epithelial cells. The subpial marginal zone was YKL-40-positive, particularly in the hippocampus, from an early beginning stage in its development. Blood vessels in the intermediate and subventricular zones showed specific YKL-40 reactivity confined to pericytes. Furthermore, a population of YKL-40-positive, small, rounded cells was identified in the ventricular and subventricular zones. Real-time quantitative RT-PCR analysis showed strong YKL-40 mRNA expression in the leptomeninges and the choroid plexuses, and weaker expression in the telencephalic wall. Immunohistochemistry revealed a differential distribution of YKL-40 across the zones of the developing telencephalic wall. We show that YKL-40 is associated with sites of the brain barrier systems and propose that it is involved in controlling local angiogenesis and access of peripheral cells to the forebrain via secretion from leptomeningeal cells, choroid plexus epithelium and pericytes. Furthermore, we suggest that the small, rounded, YKL-40-positive cells represent a subpopulation of astroglial progenitors, and that YKL-40 could be involved in the differentiation of a particular astrocytic lineage.
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Adipoquinas/metabolismo , Astrocitos/metabolismo , Lectinas/metabolismo , Células-Madre Neurales/metabolismo , Prosencéfalo/metabolismo , Adipoquinas/genética , Secuencia de Bases , Proteína 1 Similar a Quitinasa-3 , Cartilla de ADN , Humanos , Inmunohistoquímica , Lectinas/genética , Microscopía Confocal , Prosencéfalo/citología , Prosencéfalo/embriología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Genome instability plays fundamental roles in human evolution and phenotypic variation within our population. This instability leads to genomic rearrangements that are involved in a wide variety of human disorders, including congenital and neurodevelopmental disorders, and cancers. Insight into the molecular mechanisms governing such genomic rearrangements may increase our understanding of disease pathology and evolutionary processes. Here we analyse 17 carriers of non-recurrent deletions in the NRXN1 gene, which have been associated with neurodevelopmental disorders, e.g. schizophrenia, autism and epilepsies. METHODS: 17 non-recurrent NRXN1 deletions identified by GWA were sequenced to map the breakpoints of each. Meme etc. was used to identify shared patterns between the deletions and compare these were previously studies on non-recurrent deletions. RESULTS: We discovered two novel sequence motifs shared between all 17 NRXN1 deletions and a significantly higher AT nucleotide content at the breakpoints, compared to the overall nucleotide content on chromosome 2. We found different alteration of sequence at the breakpoint; small insertions and duplications giving rise to short microhomology sequences. CONCLUSIONS: No single mechanism seems to be implicated in the deletion events, but the results suggest that NHEJ, FoSTeS or MMBIR is implicated. The two novel sequence motifs together with a high AT content in all in NRXN1 deletions may lead to increased instability leading to a increase susceptibility to a single stranded structures. This favours potentially repaired by NHEJ mechanism of double strand breaks or may leading to replication errors. © 2013 Wiley Periodicals, Inc.
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Trastorno Autístico/genética , Moléculas de Adhesión Celular Neuronal/genética , Epilepsia/genética , Eliminación de Gen , Proteínas del Tejido Nervioso/genética , Esquizofrenia/genética , Composición de Base/genética , Secuencia de Bases , Proteínas de Unión al Calcio , Variaciones en el Número de Copia de ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Inestabilidad Genómica , Humanos , Moléculas de Adhesión de Célula Nerviosa , Análisis de Secuencia de ADNRESUMEN
Congenital heart disease (CHD) occurs in â¼1% of newborns. CHD arises from many distinct etiologies, ranging from genetic or genomic variation to exposure to teratogens, which elicit diverse cell and molecular responses during cardiac development. To systematically explore the relationships between CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation and environmental exposures that modulate critical biological systems during heart development.
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Ambiente , Predisposición Genética a la Enfermedad/epidemiología , Deformidades Congénitas de la Mano/epidemiología , Deformidades Congénitas de la Mano/genética , Mapas de Interacción de Proteínas/genética , Bases de Datos Genéticas , Corazón/embriología , Humanos , Recién Nacido , Factores de Riesgo , Estadísticas no Paramétricas , TranscriptomaRESUMEN
The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3ß, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3ß-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.
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Adipoquinas/metabolismo , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismo , Lectinas/metabolismo , Adipoquinas/genética , Animales , Biomarcadores/análisis , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula , Línea Celular , Proliferación Celular , Proteína 1 Similar a Quitinasa-3 , Células Madre Embrionarias/citología , Ensayo de Inmunoadsorción Enzimática , Células Nutrientes , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Estratos Germinativos/citología , Estratos Germinativos/crecimiento & desarrollo , Humanos , Lectinas/genética , Ratones , Especificidad de Órganos , Oxígeno/farmacología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Aberrant organ development is associated with a wide spectrum of disorders, from schizophrenia to congenital heart disease, but systems-level insight into the underlying processes is very limited. Using heart morphogenesis as general model for dissecting the functional architecture of organ development, we combined detailed phenotype information from deleterious mutations in 255 genes with high-confidence experimental interactome data, and coupled the results to thorough experimental validation. Hereby, we made the first systematic analysis of spatio-temporal protein networks driving many stages of a developing organ identifying several novel signaling modules. Our results show that organ development relies on surprisingly few, extensively recycled, protein modules that integrate into complex higher-order networks. This design allows the formation of a complicated organ using simple building blocks, and suggests how mutations in the same genes can lead to diverse phenotypes. We observe a striking temporal correlation between organ complexity and the number of discrete functional modules coordinating morphogenesis. Our analysis elucidates the organization and composition of spatio-temporal protein networks that drive the formation of organs, which in the future may lay the foundation of novel approaches in treatments, diagnostics, and regenerative medicine.
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Enfermedades Cardiovasculares/embriología , Enfermedades Cardiovasculares/metabolismo , Corazón/embriología , Proteínas/metabolismo , Transducción de Señal , Corazón/anatomía & histología , Humanos , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.
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The 22q11 deletion syndrome, which is caused by a 1.5- to 3.0-megabase hemizygous deletion in chromosome 22q11.2, has a prevalence of 1/2000 to 1/4000. However, the syndrome presents with highly variable phenotypes and thus may be underestimated among Danish newborns. To establish a true incidence of 22q11.2 deletions among certain manifestations, eg, congenital heart disease, on selected Danes, a multiplex ligation-dependant probe amplification (MLPA) analysis was designed. The analysis was planned to be performed on DNA extracted from dried blood spot samples (DBSS) obtained from Guthrie cards collected during neonatal screening programs. However, the DNA concentration necessary for a standard MLPA analysis (20 ng) could not be attained from DBSS, and a novel MLPA design was developed to permit for analysis on limited amounts of DNA (2 ng). A pilot study is reported here that validates the new MLPA design using nine patients diagnosed with the 22q11.2 deletion and 101 controls. All deletions were identified using DNA extracted from DBSS, and no copy number variations were detected in the controls, resulting in a specificity and sensitivity of 100%. It is thereby concluded that the novel MLPA probe design is successful and reliable using minimal amounts of DNA. This allows for use of DBSS samples in a retrospective study of 22q11.2 deletion among certain manifestations associated with DiGeorge Syndrome.
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Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Recién Nacido , Técnicas de Amplificación de Ácido Nucleico/métodos , Niño , ADN/análisis , ADN/genética , Variaciones en el Número de Copia de ADN , Dinamarca , Humanos , Recién Nacido/sangre , Tamizaje Masivo/métodosRESUMEN
Defects in the assembly or function of primary cilia, which are sensory organelles, are tightly coupled to developmental defects and diseases in mammals. Here, we investigated the function of the primary cilium in regulating hedgehog signaling and early cardiogenesis. We report that the pluripotent P19.CL6 mouse stem cell line, which can differentiate into beating cardiomyocytes, forms primary cilia that contain essential components of the hedgehog pathway, including Smoothened, Patched-1 and Gli2. Knockdown of the primary cilium by Ift88 and Ift20 siRNA or treatment with cyclopamine, an inhibitor of Smoothened, blocks hedgehog signaling in P19.CL6 cells, as well as differentiation of the cells into beating cardiomyocytes. E11.5 embryos of the Ift88(tm1Rpw) (Ift88-null) mice, which form no cilia, have ventricular dilation, decreased myocardial trabeculation and abnormal outflow tract development. These data support the conclusion that cardiac primary cilia are crucial in early heart development, where they partly coordinate hedgehog signaling.
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Diferenciación Celular , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Miocitos Cardíacos/citología , Transducción de Señal , Animales , Cilios/genética , Corazón/embriología , Proteínas Hedgehog/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Receptores Patched , Receptor Patched-1 , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Proteína Gli2 con Dedos de ZincRESUMEN
Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed. Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend that future gene expression in CL/P studies should use tissue from the correct embryonic time and place if possible, to overcome the biases in the presented study.
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Labio Leporino/genética , Labio Leporino/inmunología , Fisura del Paladar/genética , Fisura del Paladar/inmunología , Osteopontina/genética , Fisura del Paladar/embriología , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Lactante , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteopontina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Primary cilia assemble as solitary organelles in most mammalian cells during growth arrest and are thought to coordinate a series of signal transduction pathways required for cell cycle control, cell migration, and cell differentiation during development and in tissue homeostasis. Recently, primary cilia were suggested to control pluripotency, proliferation, and/or differentiation of stem cells, which may comprise an important source in regenerative biology. We here provide a method using a P19.CL6 embryonic carcinoma (EC) stem cell line to study the function of the primary cilium in early cardiogenesis. By knocking down the formation of the primary cilium by nucleofection of plasmid DNA with siRNA sequences against genes essential in ciliogenesis (IFT88 and IFT20) we block hedgehog (Hh) signaling in P19.CL6 cells as well as the differentiation of the cells into beating cardiomyocytes (Clement et al., 2009). Immunofluorescence microscopy, western blotting, and quantitative PCR analysis were employed to delineate the molecular and cellular events in cilia-dependent cardiogenesis. We optimized the nucleofection procedure to generate strong reduction in the frequency of ciliated cells in the P19.CL6 culture.
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Diferenciación Celular/fisiología , Cilios/fisiología , Técnicas de Silenciamiento del Gen , Miocitos Cardíacos/fisiología , Células Madre Neoplásicas , ARN Interferente Pequeño , Transfección , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen/instrumentación , Técnicas de Silenciamiento del Gen/métodos , Proteínas Hedgehog/metabolismo , Miocitos Cardíacos/citología , Células Madre Neoplásicas/fisiología , Células Madre Neoplásicas/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transducción de Señal/fisiología , Transfección/instrumentación , Transfección/métodosRESUMEN
YKL-40 is a growth factor for chondrocytes and fibroblasts. The aim was to evaluate YKL-40 expression in the musculoskeletal system during early human development. We studied sections from 15 human embryos [weeks 5.5-8; 7- to 31-mm crown-rump length (CRL)] and 68 fetuses (weeks 9-14; 33- to 105-mm CRL) for YKL-40 protein expression by immunohistochemistry. YKL-40 mRNA expression was evaluated in two human embryos (days 41 and 51). Initially YKL-40 is expressed in all germ layers: ecto-, meso-, and endoderm. YKL-40 mRNA and protein expression are found in tissues of the ecto-, meso-, and endoderm, and YKL-40 protein expression is present during development of cartilage, bone, joints, and muscles. At the cellular level, YKL-40 protein expression is high in tissues characterized by rapid proliferation, marked differentiation, and undergoing morphogenetic changes. Examples of rapid cell proliferation include the chondrogenic inner layer of perichondrium and the osteogenic inner layer of periosteum. Differences in YKL-40 expression during differentiation are found in the chondrogenic and osteogenic cell lineages. The initial shaping of cartilage and bone models and joints is concomitant with a strong outline of YKL-40-positive cells. This indicates that YKL-40 is associated with cell proliferation, differentiation, and tissue morphogenesis during development of the human musculoskeletal system.
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Glicoproteínas/biosíntesis , Sistema Musculoesquelético/metabolismo , Adipoquinas , Proteína 1 Similar a Quitinasa-3 , Edad Gestacional , Glicoproteínas/genética , Humanos , Inmunohistoquímica , Lectinas , Sistema Musculoesquelético/embriología , Especificidad de Órganos , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The Pierre Robin sequence (PRS), consisting of cleft palate, micrognathia and glossoptosis, can be seen as part of the phenotype in other Mendelian syndromes--for instance, campomelic dysplasia (CD) which is caused by SOX9 mutations--but the aetiology of non-syndromic PRS has not yet been unravelled. OBJECTIVE: To gain more insight into the aetiology of PRS by studying patients with PRS using genetic and cytogenetic methods. METHODS: 10 unrelated patients with PRS were investigated by chromosome analyses and bacterial artificial chromosome arrays. A balanced translocation was found in one patient, and the breakpoints were mapped with fluorescence in situ hybridisation and Southern blot analysis. All patients were screened for SOX9 and KCNJ2 mutations, and in five of the patients expression analysis of SOX9 and KCNJ2 was carried out by quantitative real-time PCR. RESULTS: An abnormal balanced karyotype 46,XX, t(2;17)(q23.3;q24.3) was identified in one patient with PRS and the 17q breakpoint was mapped to 1.13 Mb upstream of the transcription factor SOX9 and 800 kb downstream of the gene KCNJ2. Furthermore, a significantly reduced SOX9 and KCNJ2 mRNA expression was observed in patients with PRS. CONCLUSION: Our findings suggest that non-syndromic PRS may be caused by both SOX9 and KCNJ2 dysregulation.