Vera Mayo

Exome sequencing of extended families with autism reveals genes shared across neurodevelopmental and neuropsychiatric disorders

BackgroundAutism spectrum disorders (ASDs) comprise a range of neurodevelopmental conditions of varying severity, characterized by marked qualitative difficulties in social relatedness, communication, and behavior. Despite overwhelming evidence of high heritability, results from genetic studies to date show that ASD etiology is extremely heterogeneous and only a fraction of autism genes have been discovered.MethodsTo help unravel this genetic complexity, we performed whole exome sequencing on 100 ASD individuals from 40 families with multiple distantly related affected individuals. All families contained a minimum of one pair of ASD cousins. Each individual was captured with the Agilent SureSelect Human All Exon kit, sequenced on the Illumina Hiseq 2000, and the resulting data processed and annotated with Burrows-Wheeler Aligner (BWA), Genome Analysis Toolkit (GATK), and SeattleSeq. Genotyping information on each family was utilized in order to determine genomic regions that were identical by descent (IBD). Variants identified by exome sequencing which occurred in IBD regions and present in all affected individuals within each family were then evaluated to determine which may potentially be disease related. Nucleotide alterations that were novel and rare (minor allele frequency, MAF, less than 0.05) and predicted to be detrimental, either by altering amino acids or splicing patterns, were prioritized.ResultsWe identified numerous potentially damaging, ASD associated risk variants in genes previously unrelated to autism. A subset of these genes has been implicated in other neurobehavioral disorders including depression (SLIT3), epilepsy (CLCN2, PRICKLE1), intellectual disability (AP4M1), schizophrenia (WDR60), and Tourette syndrome (OFCC1). Additional alterations were found in previously reported autism candidate genes, including three genes with alterations in multiple families (CEP290, CSMD1, FAT1, and STXBP5). Compiling a list of ASD candidate genes from the literature, we determined that variants occurred in ASD candidate genes 1.65 times more frequently than in random genes captured by exome sequencing (P = 8.55 × 10-5).ConclusionsBy studying these unique pedigrees, we have identified novel DNA variations related to ASD, demonstrated that exome sequencing in extended families is a powerful tool for ASD candidate gene discovery, and provided further evidence of an underlying genetic component to a wide range of neurodevelopmental and neuropsychiatric diseases.

The Expanding Role of MBD Genes in Autism: Identification of a MECP2 Duplication and Novel Alterations in MBD5, MBD6, and SETDB1

The methyl‐CpG‐binding domain (MBD) gene family was first linked to autism over a decade ago when Rett syndrome, which falls under the umbrella of autism spectrum disorders (ASDs), was revealed to be predominantly caused by MECP2 mutations. Since that time, MECP2 alterations have been recognized in idiopathic ASD patients by us and others. Individuals with deletions across the MBD5 gene also present with ASDs, impaired speech, intellectual difficulties, repetitive behaviors, and epilepsy. These findings suggest that further investigations of the MBD gene family may reveal additional associations related to autism. We now describe the first study evaluating individuals with ASD for rare variants in four autosomal MBD family members, MBD5, MBD6, SETDB1, and SETDB2, and expand our initial screening in the MECP2 gene. Each gene was sequenced over all coding exons and evaluated for copy number variations in 287 patients with ASD and an equal number of ethnically matched control individuals. We identified 186 alterations through sequencing, approximately half of which were novel (96 variants, 51.6%). We identified 17 ASD specific, nonsynonymous variants, four of which were concordant in multiplex families: MBD5 Tyr1269Cys, MBD6 Arg883Trp, MECP2 Thr240Ser, and SETDB1 Pro1067del. Furthermore, a complex duplication spanning of the MECP2 gene was identified in two brothers who presented with developmental delay and intellectual disability. From our studies, we provide the first examples of autistic patients carrying potentially detrimental alterations in MBD6 and SETDB1, thereby demonstrating that the MBD gene family potentially plays a significant role in rare and private genetic causes of autism. Autism Res 2012, 5: 385–397.

Neural crest-derived dental stem cells—Where we are and where we are going

OBJECTIVES There are five types of post-natal human dental stem cells that have been identified, isolated and characterized. Here, we review the information available on dental stem cells as well as their potential applications in dentistry, regenerative medicine and the development of other therapeutic approaches. DATA Data pertinent to dental stem cells and their applications, published in peer-reviewed journals from 1982 to 2013 in English were reviewed. SOURCES Sources were retrieved from PubMed databases as well as related references that the electronic search yielded. STUDY SELECTION Manuscripts describing the origin, retrieval, characterization and application of dental stem cells were obtained and reviewed. CONCLUSIONS Dental stem cell populations present properties similar to those of mesenchymal stem cells, such as the ability to self-renew and the potential for multilineage differentiation. While they have greater capacity to give rise to odontogenic cells and regenerate dental pulp and periodontal tissue, they have the capacity to differentiate into all three germ line cells, proving that a population of pluripotent stem cells exists in the dental tissues. CLINICAL SIGNIFICANCE Dental stem cells have the capacity to differentiate into endoderm, mesoderm and ectoderm tissues. Consequently they do not only have applications in dentistry, but also neurodegenerative and ischemic diseases, diabetes research, bone repair, and other applications in the field of tissue regeneration.

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca(2+) concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca(2+) content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy.

Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis

Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1-2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium Leak

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium Leak Role of Nitroso–Redox Balance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Engineered microenvironments for maturation of stem cell derived cardiac myocytes

Through the use of stem cell-derived cardiac myocytes, tissue-engineered human myocardial constructs are poised for modeling normal and diseased physiology of the heart, as well as discovery of novel drugs and therapeutic targets in a human relevant manner. This review highlights the recent bioengineering efforts to recapitulate microenvironmental cues to further the maturation state of newly differentiated cardiac myocytes. These techniques include long-term culture, co-culture, exposure to mechanical stimuli, 3D culture, cell-matrix interactions, and electrical stimulation. Each of these methods has produced various degrees of maturation; however, a standardized measure for cardiomyocyte maturation is not yet widely accepted by the scientific community.

Interaction Between Neuronal NOS Signaling and Temperature Influences SR Ca2+ Leak: Role of Nitroso-Redox Balance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.

Interaction Between Neuronal Nitric Oxide Synthase Signaling and Temperature Influences Sarcoplasmic Reticulum Calcium LeakNovelty and Significance

Rationale: Although nitric oxide (NO) signaling modulates cardiac function and excitation–contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. Objective: To test the hypothesis that temperature profoundly affects nitroso–redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca2+) leak. Methods and Results: We measured SR Ca2+ leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase–deficient mice-1 [NOS1−/−]), and hyper S-nitrosoglutathione reductase–deficient (GSNOR−/−) mice. In WT cardiomyocytes, SR Ca2+ leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1−/− cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR−/− cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1−/− cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1−/− cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1−/− cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca2+ content in NOS1−/− cells but had no effect in WT or GSNOR−/−. Conclusions: Ca2+ leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca2+.