From epidemiology to function

Arterial hypertension is the most prevalent risk factor for cardiovascular disorders. Most cases of hypertension are due to unknown etiology, with only 5-15 per cent being secondary effects. Primary aldosteronism (PA) is the single most prevalent form of secondary hypertension, and is defined by autonomous aldosterone secretion independent of the plasma renin activity. Routine usage of aldosterone to renin ratio (ARR) for screening PA has revealed greater prevalence of the disorder, especially in resistant or advanced forms of arterial hypertension. The two most common causes of PA are aldosterone producing adenomas (APA) and bilateral adrenal hyperplasia (BAH). Rare Mendelian forms of familial hyperaldosteronism are also described. Until recently, genetic background of only glucocorticoid-remediable familial PA was elucidated in detail. Utilization of the exome sequencing techniques since 2011 identified somatic mutations in the cation transporter genes KCNJ5, ATP1A1, ATP2B3 and CACNA1D as the causative factors for circa 50 % of APAs. The underlying genetic causes of BAH cases remain to be determined. Genome-wide association studies (GWAS) have been the predominant methodology in genetic epidemiological research in the past ten years, under the hypothesis of “common disease – common variant”. Its prevalent application identified many risk loci, containing targets for functional investigation. In this study ARR was used as a phenotypic parameter in a GWAS in the German KORA-F4 cohort of 1876 individuals, leading to genome-wide significance of a locus in chromosome 5q32. The four genes in this locus (SLC26A2, TIGD6, HMGXB3 and CSF1R) were evaluated by their known characteristics and functions, and functional studies investigating their relevance to aldosterone biosynthesis and function were carried out for SLC26A2 and CSF1R. SLC26A2, a ubiquitously expressed solute carrier with mainly sulfate, oxalate and chloride affinities, was found to be co-regulated with aldosterone production in vivo and in vitro. RNA interference in a model adrenocortical cell line resulted in significantly higher rate of aldosterone production and aldosterone synthase expression, as well as increased overall steroidogenic capacity. Subsequent studies identified calcium signaling dependent pathways as the mediator of this effect. A germline SLC26A2 knock-in mouse model also showed confirmatory endocrine and adrenal phenotype in a sex-specific manner, with elevated plasma aldosterone concentration and ARR in females. The evidence derived from these findings suggests a possible role of SLC26A2 function in the pathophysiology of PA, which requires further epidemiological and functional experiments to confirm and elucidate.Die arterielle Hypertonie gilt als wichtigster Risikofaktor für kardiovaskuläre Erkrankungen. Während in den meisten Fällen eine essentielle Hypertonie angenommen werden kann, liegt in 5-15 % der Patienten dem Bluthochdruck eine andere Erkrankung zugrunde. Mit dem Einsatz des Aldosteron-Renin-Quotienten (ARQ) konnte der primäre Hyperaldosteronismus als die häufigste Form des sekundären Bluthochdrucks eingeordnet werden. Die beiden häufigsten Ursachen des primären Hyperaldosteronismus sind das Aldosteron-produzierende Adenom (APA) und die beidseitige Nebennierenrindenhyperplasie (BAH). Monogenetische, familiäre Formen sind hingegen insgesamt sehr selten. Mit dem Einsatz moderner Sequenziertechniken konnten seit 2011 somatische Mutationen in Ionenkanälen und Transportern (KCNJ5, ATP1A1, ATP2B3 und CACNA1D) in etwa 50 % der APAs identifiziert werden. Die genetische Ursachen der BAH sind dagegen in der überwiegenden Mehrzahl der Fälle unbekannt. Unter der “common disease – common variant” Hypothese sind Genomweite Assoziationsstudien (GWAS) in den letzten zehn Jahren zur vorherrschenden Methode der genetischen epidemiologischen Forschung geworden. Ihr weit verbreiteter Einsatz hat zur Identifizierung vieler genetischer Risiko-Loci geführt, die dann funktionellen Untersuchungen zugeführt werden konnten. In der vorliegenden Arbeit wurde der ARQ als phänotypische Parameter in einer GWAS der deutschen KORA-F4 Kohorte von 1.876 Personen verwendet. Hierdurch fand sich eine genomweite Signifikanz eines Locus auf Chromosom 5q32. Die vier in diesem Locus enthaltenen Gene (SLC26A2, TIGD6, HMGXB3 und CSF1R) wurden anhand bekannter Eigenschaften und Funktionen eingeordnet und weitergehende funktionelle Studien für SLC26A2 und CSF1R durchgeführt. Für SLC26A2 – einem Transporter mit bekannten Affinitäten zu Sulfat, Oxalat und Chlorid - konnte in vivo und in vitro eine gemeinsame Regulation der adrenalen Expression mit Aldosteron gefunden werden. Ein knock-down von SCL26A2 in einem in vitro Modell durch siRNA führte zu einer relevanten Erhöhung der Aldosteron-Sekretion und transkriptionellen Veränderungen des Steroidbiosynthese-Apparats. Weitergehenden Untersuchungen identifizierten vor allem Kalzium-abhängige Signalkaskaden als für diesen Effekt ursächliche Mechanismen. In einem Slc26a2 knock-in Mausmodell konnten geschlechtsabhängig ein entsprechender endokriner Phänotyp mit einem erhöhten ARQ nachgewiesen werden. Zusammengenommen ergeben sich aus diesen Untersuchungen gute Hinweise für einen Einfluss von SLC26A2 in der Regulation der Aldosteronsekretion und in der Pathophysiologie des primären Hyperaldosteronismus. Weitere funktionelle, epidemiologische und genetische Untersuchungen werden notwendig sein, diese Ergebnisse weiter zu vertiefen und in ihrer potentiellen klinischen Wertigkeit einzuordnen

From epidemiology to function

Arterial hypertension is the most prevalent risk factor for cardiovascular disorders. Most cases of hypertension are due to unknown etiology, with only 5-15 per cent being secondary effects. Primary aldosteronism (PA) is the single most prevalent form of secondary hypertension, and is defined by autonomous aldosterone secretion independent of the plasma renin activity. Routine usage of aldosterone to renin ratio (ARR) for screening PA has revealed greater prevalence of the disorder, especially in resistant or advanced forms of arterial hypertension. The two most common causes of PA are aldosterone producing adenomas (APA) and bilateral adrenal hyperplasia (BAH). Rare Mendelian forms of familial hyperaldosteronism are also described. Until recently, genetic background of only glucocorticoid-remediable familial PA was elucidated in detail. Utilization of the exome sequencing techniques since 2011 identified somatic mutations in the cation transporter genes KCNJ5, ATP1A1, ATP2B3 and CACNA1D as the causative factors for circa 50 % of APAs. The underlying genetic causes of BAH cases remain to be determined. Genome-wide association studies (GWAS) have been the predominant methodology in genetic epidemiological research in the past ten years, under the hypothesis of “common disease – common variant”. Its prevalent application identified many risk loci, containing targets for functional investigation. In this study ARR was used as a phenotypic parameter in a GWAS in the German KORA-F4 cohort of 1876 individuals, leading to genome-wide significance of a locus in chromosome 5q32. The four genes in this locus (SLC26A2, TIGD6, HMGXB3 and CSF1R) were evaluated by their known characteristics and functions, and functional studies investigating their relevance to aldosterone biosynthesis and function were carried out for SLC26A2 and CSF1R. SLC26A2, a ubiquitously expressed solute carrier with mainly sulfate, oxalate and chloride affinities, was found to be co-regulated with aldosterone production in vivo and in vitro. RNA interference in a model adrenocortical cell line resulted in significantly higher rate of aldosterone production and aldosterone synthase expression, as well as increased overall steroidogenic capacity. Subsequent studies identified calcium signaling dependent pathways as the mediator of this effect. A germline SLC26A2 knock-in mouse model also showed confirmatory endocrine and adrenal phenotype in a sex-specific manner, with elevated plasma aldosterone concentration and ARR in females. The evidence derived from these findings suggests a possible role of SLC26A2 function in the pathophysiology of PA, which requires further epidemiological and functional experiments to confirm and elucidate.Die arterielle Hypertonie gilt als wichtigster Risikofaktor für kardiovaskuläre Erkrankungen. Während in den meisten Fällen eine essentielle Hypertonie angenommen werden kann, liegt in 5-15 % der Patienten dem Bluthochdruck eine andere Erkrankung zugrunde. Mit dem Einsatz des Aldosteron-Renin-Quotienten (ARQ) konnte der primäre Hyperaldosteronismus als die häufigste Form des sekundären Bluthochdrucks eingeordnet werden. Die beiden häufigsten Ursachen des primären Hyperaldosteronismus sind das Aldosteron-produzierende Adenom (APA) und die beidseitige Nebennierenrindenhyperplasie (BAH). Monogenetische, familiäre Formen sind hingegen insgesamt sehr selten. Mit dem Einsatz moderner Sequenziertechniken konnten seit 2011 somatische Mutationen in Ionenkanälen und Transportern (KCNJ5, ATP1A1, ATP2B3 und CACNA1D) in etwa 50 % der APAs identifiziert werden. Die genetische Ursachen der BAH sind dagegen in der überwiegenden Mehrzahl der Fälle unbekannt. Unter der “common disease – common variant” Hypothese sind Genomweite Assoziationsstudien (GWAS) in den letzten zehn Jahren zur vorherrschenden Methode der genetischen epidemiologischen Forschung geworden. Ihr weit verbreiteter Einsatz hat zur Identifizierung vieler genetischer Risiko-Loci geführt, die dann funktionellen Untersuchungen zugeführt werden konnten. In der vorliegenden Arbeit wurde der ARQ als phänotypische Parameter in einer GWAS der deutschen KORA-F4 Kohorte von 1.876 Personen verwendet. Hierdurch fand sich eine genomweite Signifikanz eines Locus auf Chromosom 5q32. Die vier in diesem Locus enthaltenen Gene (SLC26A2, TIGD6, HMGXB3 und CSF1R) wurden anhand bekannter Eigenschaften und Funktionen eingeordnet und weitergehende funktionelle Studien für SLC26A2 und CSF1R durchgeführt. Für SLC26A2 – einem Transporter mit bekannten Affinitäten zu Sulfat, Oxalat und Chlorid - konnte in vivo und in vitro eine gemeinsame Regulation der adrenalen Expression mit Aldosteron gefunden werden. Ein knock-down von SCL26A2 in einem in vitro Modell durch siRNA führte zu einer relevanten Erhöhung der Aldosteron-Sekretion und transkriptionellen Veränderungen des Steroidbiosynthese-Apparats. Weitergehenden Untersuchungen identifizierten vor allem Kalzium-abhängige Signalkaskaden als für diesen Effekt ursächliche Mechanismen. In einem Slc26a2 knock-in Mausmodell konnten geschlechtsabhängig ein entsprechender endokriner Phänotyp mit einem erhöhten ARQ nachgewiesen werden. Zusammengenommen ergeben sich aus diesen Untersuchungen gute Hinweise für einen Einfluss von SLC26A2 in der Regulation der Aldosteronsekretion und in der Pathophysiologie des primären Hyperaldosteronismus. Weitere funktionelle, epidemiologische und genetische Untersuchungen werden notwendig sein, diese Ergebnisse weiter zu vertiefen und in ihrer potentiellen klinischen Wertigkeit einzuordnen

Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages

Novel NDE1 homozygous mutation resulting in microhydranencephaly and not microlyssencephaly

Lissencephaly is characterized by deficient cortical lamination. Recently homozygous NDE1 mutations were reported in three kindred afflicted with extreme microcephaly with lissencephaly or microlissencephaly. Another severe developmental defect that involves the brain is microhydranencephaly which manifests with microcephaly, motor and mental retardation and brain malformations that include gross dilation of the ventricles with complete absence of the cerebral hemispheres or severe delay in their development. In the three related patients with microhydranencephaly that we had reported previously, we identified a homozygous deletion that encompasses NDE1 exon 2 containing the initiation codon. The mutation is predicted to result in a null allele. Herein we compare the clinical phenotypes of our research patients to those reported as microlissencephaly. The clinical findings in our patients having the fourth NDE1 mutation reported so far widen the spectrum of brain malformations resulting from mutations in NDE1

Familial microhydranencephaly, a family that does not map to 16p13.13-p12.2: relationship with hereditary fetal brain degeneration and fetal brain disruption sequence

Microhydranencephaly (MHAC) is a serious developmental brain anomaly characterized by microcephaly with severe reduction of brain hemispheres and intracranial space filled with cerebrospinal fluid without signs of intracranial hypertension. Clinical findings are very similar to fetal brain disruption sequence - severe microcephaly, scalp rugae, and profound developmental delay; however, although fetal brain disruption sequence is a sporadic condition caused by an external disruptive event, familial cases of MHAC presumably result from a process of progressive brain damage also termed as 'hereditary fetal brain degeneration'. Familial occurrence of this phenotype is very rare - only three reports on four families have been published so far. Here we present two new patients affected brothers from Slovakia - and provide an update on a previously described case from a Turkish Anatolian family. We also present data excluding linkage to an MHAC locus 16p13.13-p12.2 in the Slovak family. We compare clinical and imaging findings in all five families and suggest genetic heterogenity for this condition. In genetic counseling for this phenotype, especially in the absence of any known teratogenic factors in pregnancy, we suggest that the possibility of recurrence should be considered. Clin Dysmorphol 19: 107-118 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins

Diastrophic Dysplasia Sulfate Transporter (SLC26A2) Is Expressed in the Adrenal Cortex and Regulates Aldosterone Secretion

International audienceElucidation of the molecular mechanisms leading to autonomous aldosterone secretion is a prerequisite to define potential targets and biomarkers in the context of primary aldosteronism. After a genome-wide association study with subjects from the population-based Cooperative Health Research in the Region of Augsburg F4 survey, we observed a highly significant association (P=6.78x10(-11)) between the aldosterone to renin ratio and a locus at 5q32. Hypothesizing that this locus may contain genes of relevance for the pathogenesis of primary aldosteronism, we investigated solute carrier family 26 member 2 (SLC26A2), a protein with known transport activity for sulfate and other cations. Within murine tissues, adrenal glands showed the highest expression levels for SLC26A2, which was significantly downregulated on in vivo stimulation with angiotensin II and potassium. SLC26A2 expression was found to be significantly lower in aldosterone-producing adenomas in comparison with normal adrenal glands. In adrenocortical NCI-H295R cells, specific knockdown of SLC26A2 resulted in a highly significant increase in aldosterone secretion. Concomitantly, expression of steroidogenic enzymes, as well as upstream effectors including transcription factors such as NR4A1, CAMK1, and intracellular Ca2+ content, was upregulated in knockdown cells. To substantiate further these findings in an SLC26A2 mutant mouse model, aldosterone output proved to be increased in a sex-specific manner. In summary, these findings point toward a possible effect of SLC26A2 in the regulation of aldosterone secretion potentially involved in the pathogenesis of primary aldosteronism