Expanding the Allelic Heterogeneity of ANO10-Associated Autosomal Recessive Cerebellar Ataxia.

Background and Objectives: The term autosomal recessive cerebellar ataxia (ARCA) encompasses a diverse group of heterogeneous degenerative disorders of the cerebellum. Spinocerebellar ataxia autosomal recessive 10 (SCAR10) is a distinct classification of cerebellar ataxia caused by variants in the ANO10 gene. Little is known about the molecular role of ANO10 or its role in disease. There is a wide phenotypic spectrum among patients, even among those with the same or similar genetic variants. This study aimed to characterize the molecular consequences of variants in ANO10 and determine their pathologic significance in patients diagnosed with SCAR10. Methods: We presented 4 patients from 4 families diagnosed with spinocerebellar ataxia with potential pathogenic variants in the ANO10 gene. Patients underwent either clinical whole-exome sequencing or screening of a panel of known neuromuscular disease genes. Effects on splicing were studied using reverse transcriptase PCR to analyze complementary DNA. Western blots were used to examine protein expression. Results: One individual who presented clinically at a much earlier age than typical was homozygous for an ANO10 variant (c.1864A > G [p.Met622Val]) that produces 2 transcription products by altering an exonic enhancer site. Two patients, both of Lebanese descent, had a homozygous intronic splicing variant in ANO10 (c.1163-9A > G) that introduced a cryptic splice site acceptor, producing 2 alternative transcription products and no detectable wild-type protein. Both these variants have not yet been associated with SCAR10. The remaining patient was found to have compound heterozygous variants in ANO10 previously associated with SCAR10 (c.132dupA [p.Asp45Argfs*9] and c.1537T > C [p.Cys513Arg]). Discussion: We presented rare pathogenic variants adding to the growing list of ANO10 variants associated with SCAR10. In addition, we described an individual with a much earlier age at onset than usually associated with ANO10 variants. This expands the phenotypic and allelic heterogeneity of ANO10-associated ARCA.

Functional analysis of RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia.

BACKGROUND: A major bottleneck to the introduction of noninvasive presymptomatic diagnostic tests for the pharmacogenetic disorder malignant hyperthermia is the lack of functional data for associated variants. METHODS: We screened 50 genes having a potential role in skeletal muscle calcium homeostasis using the HaloPlex™ (Agilent Technologies, Santa Clara, CA, USA) target enrichment system and next-generation sequencing. Twenty-one patients with a history of a clinical malignant hyperthermia reaction together with a positive in vitro contracture test were included. Eight variants in RYR1 were subsequently introduced into the cDNA for the human ryanodine receptor gene and tested in cultured human embryonic kidney (HEK293) cells for their effect on calcium release from intracellular stores in response to the ryanodine receptor-1 agonist 4-chloro-m-cresol using fura-2 as calcium indicator. Each variant was subjected to in silico curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 variant curation expert panel guidelines. RESULTS: Potentially causative RYR1 variants were identified in 15 patients. Of these, two families carried two RYR1 variants, five variants had been previously reported as 'pathogenic', two variants had been previously reported as 'likely benign', and eight were of 'uncertain significance'. Of these eight variants, four showed hypersensitivity to 4-chloro-m-cresol. Three variants were reclassified as either 'pathogenic' or 'likely pathogenic'. Two were classified as 'benign', whilst three remained of 'uncertain significance'. CONCLUSIONS: Three (p.Tyr1711Cys, p.Val2280Ile, and p.Arg4737Gln) additional variants can be added to the list of RYR1 disease-associated variants managed by the European Malignant Hyperthermia Group. These can therefore be used diagnostically in the future. Three variants (p.Glu2348Gly, p.Asn2634Lys, and p.Arg3629Trp) that remained classified as of uncertain significance require further family studies or a different functional test to determine clinical relevance in malignant hyperthermia.

CDKL5 deficiency disorder: molecular insights and mechanisms of pathogenicity to fast-track therapeutic development.

CDKL5 deficiency disorder (CDD) is an X-linked brain disorder of young children and is caused by pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene. Individuals with CDD suffer infantile onset, drug-resistant seizures, severe neurodevelopmental impairment and profound lifelong disability. The CDKL5 protein is a kinase that regulates key phosphorylation events vital to the development of the complex neuronal network of the brain. Pathogenic variants identified in patients may either result in loss of CDKL5 catalytic activity or are hypomorphic leading to partial loss of function. Whilst the progressive nature of CDD provides an excellent opportunity for disease intervention, we cannot develop effective therapeutics without in-depth knowledge of CDKL5 function in human neurons. In this mini review, we summarize new findings on the function of CDKL5. These include CDKL5 phosphorylation targets and the consequence of disruptions on signaling pathways in the human brain. This new knowledge of CDKL5 biology may be leveraged to advance targeted drug discovery and rapid development of treatments for CDD. Continued development of effective humanized models will further propel our understanding of CDD biology and may permit the development and testing of therapies that will significantly alter CDD disease trajectory in young children.

Just Say Yes? A Cluster Analytic Approach to Evaluating College Students' Understandings of Affirmative Consent.

Affirmative consent policies on college campuses establish more stringent standards for inferring consent to sex. Although these policies often permit nonverbal communication of consent, they rarely outline finer-grained distinctions about which specific behaviors can stand-in for verbal affirmation. It thus remains possible that students hold different understandings of this policy vis-à-vis the nonverbals used to convey and infer consent, which could undermine the purported utility of affirmative consent initiatives. We presently sampled 442 college undergraduates and asked them to rate whether specific behaviors often present during sexual interaction constitute affirmative indicators of consent. We hypothesized that students would separate into one of three groups depending on how restrictive (e.g., verbal communication only), inclusive (e.g., verbal and clear nonverbals) or potentially non-diagnostic (e.g., sexual arousal, passivity) their behavioral definitions were of affirmative consent. Using cluster analysis, we ultimately identified two groups adhering to a restrictive versus more inclusive operationalization. The former cluster understood affirmative consent as comprising verbal affirmation with variable endorsements of specific nonverbals, whereas the latter consistently endorsed a broader set of nonverbals along with variable ascription to behaviors that do not strongly imply consent. Students in the more inclusive group were more sexually experienced, less likely to use condoms, and viewed casual sex more favorably; as well as were likelier to have received sexual assault education from their parents before and during college, as well as from social media. These findings suggest that subgroups of college students construe affirmative consent policy differently and that these understandings may relate broadly to an individual's sexual experiences, attitudes, and/or education.

Biallelic Variants in PYROXD2 Cause a Severe Infantile Metabolic Disorder Affecting Mitochondrial Function.

Pyridine Nucleotide-Disulfide Oxidoreductase Domain 2 (PYROXD2; previously called YueF) is a mitochondrial inner membrane/matrix-residing protein and is reported to regulate mitochondrial function. The clinical importance of PYROXD2 has been unclear, and little is known of the protein's precise biological function. In the present paper, we report biallelic variants in PYROXD2 identified by genome sequencing in a patient with suspected mitochondrial disease. The child presented with acute neurological deterioration, unresponsive episodes, and extreme metabolic acidosis, and received rapid genomic testing. He died shortly after. Magnetic resonance imaging (MRI) brain imaging showed changes resembling Leigh syndrome, one of the more common childhood mitochondrial neurological diseases. Functional studies in patient fibroblasts showed a heightened sensitivity to mitochondrial metabolic stress and increased mitochondrial superoxide levels. Quantitative proteomic analysis demonstrated decreased levels of subunits of the mitochondrial respiratory chain complex I, and both the small and large subunits of the mitochondrial ribosome, suggesting a mitoribosomal defect. Our findings support the critical role of PYROXD2 in human cells, and suggest that the biallelic PYROXD2 variants are associated with mitochondrial dysfunction, and can plausibly explain the child's clinical presentation.

Pathogenic variants in nucleoporin TPR (translocated promoter region, nuclear basket protein) cause severe intellectual disability in humans.

The nuclear pore complex (NPC) is a multi-protein complex that regulates the trafficking of macromolecules between the nucleus and cytoplasm. Genetic variants in components of the NPC have been shown to cause a range of neurological disorders, including intellectual disability and microcephaly. Translocated promoter region, nuclear basket protein (TPR) is a critical scaffolding element of the nuclear facing interior of the NPC. Here, we present two siblings with biallelic variants in TPR who present with a phenotype of microcephaly, ataxia and severe intellectual disability. The variants result in a premature truncation variant, and a splice variant leading to a 12-amino acid deletion respectively. Functional analyses in patient fibroblasts demonstrate significantly reduced TPR levels, and decreased TPR-containing NPC density. A compensatory increase in total NPC levels was observed, and decreased global RNA intensity in the nucleus. The discovery of variants that partly disable TPR function provide valuable insight into this essential protein in human disease, and our findings suggest that TPR variants are the cause of the siblings' neurological disorder.

Abnormalities of mitochondrial dynamics and bioenergetics in neuronal cells from CDKL5 deficiency disorder.

CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by pathogenic variants in the Cyclin-dependent kinase-like 5 (CDKL5) gene, resulting in dysfunctional CDKL5 protein. It predominantly affects females and causes seizures in the first few months of life, ultimately resulting in severe intellectual disability. In the absence of targeted therapies, treatment is currently only symptomatic. CDKL5 is a serine/threonine kinase that is highly expressed in the brain, with a critical role in neuronal development. Evidence of mitochondrial dysfunction in CDD is gathering, but has not been studied extensively. We used human patient-derived induced pluripotent stem cells with a pathogenic truncating mutation (p.Arg59*) and CRISPR/Cas9 gene-corrected isogenic controls, differentiated into neurons, to investigate the impact of CDKL5 mutation on cellular function. Quantitative proteomics indicated mitochondrial defects in CDKL5 p.Arg59* neurons, and mitochondrial bioenergetics analysis confirmed decreased activity of mitochondrial respiratory chain complexes. Additionally, mitochondrial trafficking velocity was significantly impaired, and there was a higher percentage of stationary mitochondria. We propose mitochondrial dysfunction is contributing to CDD pathology, and should be a focus for development of targeted treatments for CDD.

Person- and Incident-Level Predictors of Blame, Disclosure, and Reporting to Authorities in Rape Scenarios.

Rapes perpetrated during college are both common and underreported. Research highlights that several person- and incident-level factors relating to gender and sexuality may diminish reporting, by themselves and as they pertain to attributions of blame for the assault. In this study, male and female college students (N = 916) read vignettes describing a rape perpetrated by a man against a woman, a man against a man, or a woman against a man. Participants rated the blameworthiness of both perpetrator and victim and rated the likelihood that they would disclose the rape to social ties or health services or report it to authorities if they were in the victim's position. We found that male gender and heterosexual orientation predicted higher victim blame, lower perpetrator blame, and lower likelihood of disclosure, although relative endorsement of masculine gender ideology seemed to be driving these associations, as well as predicted lower likelihood of reporting to authorities. Controlling for other factors, vignettes portraying a woman raping a man led to a lower likelihood of disclosing or reporting the assault, compared with a male-on-female rape. We also found that the effects of female-on-male rape and traditional masculine ideologies tied to rape disclosure partially by decreasing blame to the perpetrator, which itself carried a unique influence on decisions to report. Our findings overall indicate that factors related to gender, sexuality, and blame have myriad influences and may contribute to low rates of disclosing rape to important outlets.

Mutations in the exocyst component EXOC2 cause severe defects in human brain development.

The exocyst, an octameric protein complex, is an essential component of the membrane transport machinery required for tethering and fusion of vesicles at the plasma membrane. We report pathogenic variants in an exocyst subunit, EXOC2 (Sec5). Affected individuals have severe developmental delay, dysmorphism, and brain abnormalities; variability associated with epilepsy; and poor motor skills. Family 1 had two offspring with a homozygous truncating variant in EXOC2 that leads to nonsense-mediated decay of EXOC2 transcript, a severe reduction in exocytosis and vesicle fusion, and undetectable levels of EXOC2 protein. The patient from Family 2 had a milder clinical phenotype and reduced exocytosis. Cells from both patients showed defective Arl13b localization to the primary cilium. The discovery of mutations that partially disable exocyst function provides valuable insight into this essential protein complex in neural development. Since EXOC2 and other exocyst complex subunits are critical to neuronal function, our findings suggest that EXOC2 variants are the cause of the patients' neurological disorders.

Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A).

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron-specific ATP-dependent anterograde axonal transporter of synaptic cargo, are well-recognized to cause a spectrum of neurological conditions, commonly known as KIF1A-associated neurological disorders (KAND). Here, we report one mutation-negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH-SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.

Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability.

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

NAD(P)HX dehydratase (NAXD) deficiency: a novel neurodegenerative disorder exacerbated by febrile illnesses.

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.

Functional analysis of RYR1 variants linked to malignant hyperthermia.

Malignant hyperthermia manifests as a rapid and sustained rise in temperature in response to pharmacological triggering agents, e.g. inhalational anesthetics and the muscle relaxant suxamethonium. Other clinical signs include an increase in end-tidal CO2, increased O2 consumption, as well as tachycardia, and if untreated a malignant hyperthermia episode can result in death. The metabolic changes are caused by dysregulation of skeletal muscle Ca2+ homeostasis, resulting from a defective ryanodine receptor Ca2+ channel, which resides in the sarcoplasmic reticulum and controls the flux of Ca2+ ions from intracellular stores to the cytoplasm. Most genetic variants associated with susceptibility to malignant hyperthermia occur in the RYR1 gene encoding the ryanodine receptor type 1. While malignant hyperthermia susceptibility can be diagnosed by in vitro contracture testing of skeletal muscle biopsy tissue, it is advantageous to use DNA testing. Currently only 35 of over 400 potential variants in RYR1 have been classed as functionally causative of malignant hyperthermia and thus can be used for DNA diagnostic tests. Here we describe functional analysis of 2 RYR1 variants (c. 7042_7044delCAG, p.ΔGlu2348 and c.641C>T, p.Thr214Met) that occur in the same malignant hyperthermia susceptible family. The p.Glu2348 deletion, causes hypersensitivity to ryanodine receptor agonists using in vitro analysis of cloned human RYR1 cDNA expressed in HEK293T cells, while the Thr214Met substitution, does not appear to significantly alter sensitivity to agonist in the same system. We suggest that the c. 7042_7044delCAG, p.ΔGlu2348 RYR1 variant could be added to the list of diagnostic mutations for susceptibility to malignant hyperthermia.

Modern Prejudice and Same-Sex Parenting: Shifting Judgments in Positive and Negative Parenting Situations.

The current study compares the effects of traditional and modern anti-homosexual prejudice on evaluations of parenting practices of same-sex and opposite-sex couples. Undergraduate university student participants (N = 436) completed measures of traditional and modern anti-homosexual prejudice and responded to a vignette describing a restaurant scene in which parents react to their child's undesirable behavior. The parents' sexual orientation and the quality of their parenting (positive or negative quality) were varied randomly. It was predicted that participants who score higher in modern prejudice would rate the negative parenting behaviors of same-sex parents more negatively than similar behaviors in opposite-sex parents. It was also predicted that this modern prejudice effect would be most pronounced for male participants. Both hypotheses were supported.

Sexual Hookup Culture: A Review.

"Hookups," or uncommitted sexual encounters, are becoming progressively more engrained in popular culture, reflecting both evolved sexual predilections and changing social and sexual scripts. Hook-up activities may include a wide range of sexual behaviors, such as kissing, oral sex, and penetrative intercourse. However, these encounters often transpire without any promise of, or desire for, a more traditional romantic relationship. A review of the literature suggests that these encounters are becoming increasingly normative among adolescents and young adults in North America, representing a marked shift in openness and acceptance of uncommitted sex. We reviewed the current literature on sexual hookups and considered the multiple forces influencing hookup culture, using examples from popular culture to place hooking up in context. We argue that contemporary hookup culture is best understood as the convergence of evolutionary and social forces during the developmental period of emerging adulthood. We suggest that researchers must consider both evolutionary mechanisms and social processes, and be considerate of the contemporary popular cultural climate in which hookups occur, in order to provide a comprehensive and synergistic biopsychosocial view of "casual sex" among emerging adults today.

Polymorphous prejudice: liberating the measurement of heterosexuals' attitudes toward lesbians and gay men.

A multidimensional measure of sexual prejudice was developed to assess the increasing complexity of heterosexuals' attitudes toward gay men and lesbians. Exploratory and confirmatory factor analyses revealed a valid and reliable 7-factor measure that assessed: 1) traditional heterosexism; 2) tendency to deny anti-gay discrimination continues; 3) aversion toward gay men; 4) aversion to lesbians; 5) judgments regarding the value of the gay and lesbian movement; 6) resistance to heteronormative expectations; and 7) endorsement of positive beliefs about gay people. A modern heterosexism theory was supported and queer/liberationist notions of anti-heteronormativity and positive beliefs were found to be related to pro-homosexual attitudes.

Anti-homosexual prejudice . . . as opposed to what? Queer theory and the social psychology of anti-homosexual attitudes.

This article uses Sedgwick's distinction between minoritizing and universalizing theories of sexuality to analyze variability in social psychologists' studies of anti-homosexual prejudice, focusing on studies of attitudes. Anti-homosexual prejudice was initially defined in conversation with gay liberationists and presumed, among other things, that fear of homoerotic potential was present in all persons. Later social psychologists theorized anti-homosexual prejudice in strict minoritizing terms: as prejudice towards a distinct out-group. In the first section of this paper we discuss corresponding shifts in the conceptualization of anti-homosexual attitudes. Next, using a universalizing framework, we re-interpret experiments on behavioral aspects of anti-homosexual attitudes which were originally conceptualized using a minoritizing framework, and suggest avenues for future research. Finally, we examine how queer theory might enrich this area of social psychological inquiry by challenging assumptions about the politics of doing scientific work and the utility of identity-based sexual politics.