Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

An Association between OXPHOS-Related Gene Expression and Malignant Hyperthermia Susceptibility in Human Skeletal Muscle Biopsies.

Malignant hyperthermia (MH) is a pharmacogenetic condition of skeletal muscle that manifests in hypermetabolic responses upon exposure to volatile anaesthetics. This condition is caused primarily by pathogenic variants in the calcium-release channel RYR1, which disrupts calcium signalling in skeletal muscle. However, our understanding of MH genetics is incomplete, with no variant identified in a significant number of cases and considerable phenotype diversity. In this study, we applied a transcriptomic approach to investigate the genome-wide gene expression in MH-susceptible cases using muscle biopsies taken for diagnostic testing. Baseline comparisons between muscle from MH-susceptible individuals (MHS, n = 8) and non-susceptible controls (MHN, n = 4) identified 822 differentially expressed genes (203 upregulated and 619 downregulated) with significant enrichment in genes associated with oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Investigations of 10 OXPHOS target genes in a larger cohort (MHN: n = 36; MHS: n = 36) validated the reduced expression of ATP5MD and COQ6 in MHS samples, but the remaining 8 selected were not statistically significant. Further analysis also identified evidence of a sex-linked effect in SDHB and UQCC3 expression, and a difference in ATP5MD expression across individuals with MH sub-phenotypes (trigger from in vitro halothane exposure only, MHSh (n = 4); trigger to both in vitro halothane and caffeine exposure, MHShc (n = 4)). Our data support a link between MH-susceptibility and dysregulated gene expression associated with mitochondrial bioenergetics, which we speculate plays a role in the phenotypic variability observed within MH.

RLIM Is a Candidate Dosage-Sensitive Gene for Individuals with Varying Duplications of Xq13, Intellectual Disability, and Distinct Facial Features.

Interpretation of the significance of maternally inherited X chromosome variants in males with neurocognitive phenotypes continues to present a challenge to clinical geneticists and diagnostic laboratories. Here we report 14 males from 9 families with duplications at the Xq13.2-q13.3 locus with a common facial phenotype, intellectual disability (ID), distinctive behavioral features, and a seizure disorder in two cases. All tested carrier mothers had normal intelligence. The duplication arose de novo in three mothers where grandparental testing was possible. In one family the duplication segregated with ID across three generations. RLIM is the only gene common to our duplications. However, flanking genes duplicated in some but not all the affected individuals included the brain-expressed genes NEXMIF, SLC16A2, and the long non-coding RNA gene FTX. The contribution of the RLIM-flanking genes to the phenotypes of individuals with different size duplications has not been fully resolved. Missense variants in RLIM have recently been identified to cause X-linked ID in males, with heterozygous females typically having normal intelligence and highly skewed X chromosome inactivation. We detected consistent and significant increase of RLIM mRNA and protein levels in cells derived from seven affected males from five families with the duplication. Subsequent analysis of MDM2, one of the targets of the RLIM E3 ligase activity, showed consistent downregulation in cells from the affected males. All the carrier mothers displayed normal RLIM mRNA levels and had highly skewed X chromosome inactivation. We propose that duplications at Xq13.2-13.3 including RLIM cause a recognizable but mild neurocognitive phenotype in hemizygous males.

Comparison of Transcriptomic Changes in Survivors of Exertional Heat Illness with Malignant Hyperthermia Susceptible Patients.

Exertional heat illness (EHI) is an occupational health hazard for athletes and military personnel-characterised by the inability to thermoregulate during exercise. The ability to thermoregulate can be studied using a standardised heat tolerance test (HTT) developed by The Institute of Naval Medicine. In this study, we investigated whole blood gene expression (at baseline, 2 h post-HTT and 24 h post-HTT) in male subjects with either a history of EHI or known susceptibility to malignant hyperthermia (MHS): a pharmacogenetic condition with similar clinical phenotype. Compared to healthy controls at baseline, 291 genes were differentially expressed in the EHI cohort, with functional enrichment in inflammatory response genes (up to a four-fold increase). In contrast, the MHS cohort featured 1019 differentially expressed genes with significant down-regulation of genes associated with oxidative phosphorylation (OXPHOS). A number of differentially expressed genes in the inflammation and OXPHOS pathways overlapped between the EHI and MHS subjects, indicating a common underlying pathophysiology. Transcriptome profiles between subjects who passed and failed the HTT (based on whether they achieved a plateau in core temperature or not, respectively) were not discernable at baseline, and HTT was shown to elevate inflammatory response gene expression across all clinical phenotypes.

Different types of disease-causing noncoding variants revealed by genomic and gene expression analyses in families with X-linked intellectual disability.

The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide; however, approximately 30% of XLID families still remain unresolved. We postulated that noncoding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different noncoding variants. We used comprehensive structural, single-nucleotide, and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, reverse-transcription polymerase chain reactions, Western blots, and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic noncoding variants: a retrotransposon insertion, a novel intronic splice donor, and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favor of a regulatory noncoding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic noncoding variant discovery.

Bioenergetic defects in muscle fibers of RYR1 mutant knock-in mice associated with malignant hyperthermia.

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism, which has directed research interest into oxidative phosphorylation and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ∼16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression, and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA-Seq data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared with WT controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated oxidative phosphorylation in mutant muscle. Furthermore, we observed a gene-dose-specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively, these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

A recurrent missense variant in SLC9A7 causes nonsyndromic X-linked intellectual disability with alteration of Golgi acidification and aberrant glycosylation.

We report two unrelated families with multigenerational nonsyndromic intellectual disability (ID) segregating with a recurrent de novo missense variant (c.1543C>T:p.Leu515Phe) in the alkali cation/proton exchanger gene SLC9A7 (also commonly referred to as NHE7). SLC9A7 is located on human X chromosome at Xp11.3 and has not yet been associated with a human phenotype. The gene is widely transcribed, but especially abundant in brain, skeletal muscle and various secretory tissues. Within cells, SLC9A7 resides in the Golgi apparatus, with prominent enrichment in the trans-Golgi network (TGN) and post-Golgi vesicles. In transfected Chinese hamster ovary AP-1 cells, the Leu515Phe mutant protein was correctly targeted to the TGN/post-Golgi vesicles, but its N-linked oligosaccharide maturation as well as that of a co-transfected secretory membrane glycoprotein, vesicular stomatitis virus G (VSVG) glycoprotein, was reduced compared to cells co-expressing SLC9A7 wild-type and VSVG. This correlated with alkalinization of the TGN/post-Golgi compartments, suggestive of a gain-of-function. Membrane trafficking of glycosylation-deficient Leu515Phe and co-transfected VSVG to the cell surface, however, was relatively unaffected. Mass spectrometry analysis of patient sera also revealed an abnormal N-glycosylation profile for transferrin, a clinical diagnostic marker for congenital disorders of glycosylation. These data implicate a crucial role for SLC9A7 in the regulation of TGN/post-Golgi pH homeostasis and glycosylation of exported cargo, which may underlie the cellular pathophysiology and neurodevelopmental deficits associated with this particular nonsyndromic form of X-linked ID.

The Malaria-Protective Human Glycophorin Structural Variant DUP4 Shows Somatic Mosaicism and Association with Hemoglobin Levels.

Glycophorin A and glycophorin B are red blood cell surface proteins and are both receptors for the parasite Plasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A-glycophorin B fusion gene and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant and reveal somatic variation in copy number of the glycophorin B-glycophorin A fusion gene. By developing a simple, specific, PCR-based assay for DUP4, we show that the DUP4 variant reaches a frequency of 13% in the population of a malaria-endemic village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation and show that DUP4 is associated with a malarial-related phenotype in a longitudinally followed population.

Truncating Variants in NAA15 Are Associated with Variable Levels of Intellectual Disability, Autism Spectrum Disorder, and Congenital Anomalies.

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development.

Investigating the genetic susceptibility to exertional heat illness.

BACKGROUND: We aimed to identify rare (minor allele frequency ≤1%), potentially pathogenic non-synonymous variants in a well-characterised cohort with a clinical history of exertional heat illness (EHI) or exertional rhabdomyolysis (ER). The genetic link between malignant hyperthermia (MH) and EHI was investigated due to their phenotypic overlap. METHODS: The coding regions of 38 genes relating to skeletal muscle calcium homeostasis or exercise intolerance were sequenced in 64 patients (mostly military personnel) with a history of EHI, or ER and who were phenotyped using skeletal muscle in vitro contracture tests. We assessed the pathogenicity of variants using prevalence data, in silico analysis, phenotype and segregation evidence and by review of the literature. RESULTS: We found 51 non-polymorphic, potentially pathogenic variants in 20 genes in 38 patients. Our data indicate that RYR1 p.T3711M (previously shown to be likely pathogenic for MH susceptibility) and RYR1 p.I3253T are likely pathogenic for EHI. PYGM p.A193S was found in 3 patients with EHI, which is significantly greater than the control prevalence (p=0.000025). We report the second case of EHI in which a missense variant at CACNA1S p.R498 has been found. Combinations of rare variants in the same or different genes are implicated in EHI. CONCLUSION: We confirm a role of RYR1 in the heritability of EHI as well as ER but highlight the likely genetic heterogeneity of these complex conditions. We propose defects, or combinations of defects, in skeletal muscle calcium homeostasis, oxidative metabolism and membrane excitability are associated with EHI.

Constraint and conservation of paired-type homeodomains predicts the clinical outcome of missense variants of uncertain significance.

The need to interpret the pathogenicity of novel missense variants of unknown significance identified in the homeodomain of X-chromosome aristaless-related homeobox (ARX) gene prompted us to assess the utility of conservation and constraint across these domains in multiple genes compared to conventional in vitro functional analysis. Pathogenic missense variants clustered in the homeodomain of ARX contribute to intellectual disability (ID) and epilepsy, with and without brain malformation in affected males. Here we report novel c.1112G>A, p.Arg371Gln and c.1150C>T, p.Arg384Cys variants in male patients with ID and severe seizures. The third case of a male patient with a c.1109C>T, p.Ala370Val variant is perhaps the first example of ID and autism spectrum disorder (ASD), without seizures or brain malformation. We compiled data sets of pathogenic variants from ClinVar and presumed benign variation from gnomAD and demonstrated that the high levels of sequence conservation and constraint of benign variation within the homeodomain impacts upon the ability of publicly available in silico prediction tools to accurately discern likely benign from likely pathogenic variants in these data sets. Despite this, considering the inheritance patterns of the genes and disease variants with the conservation and constraint of disease variants affecting the homeodomain in conjunction with current clinical assessments may assist in predicting the pathogenicity of missense variants, particularly for genes with autosomal recessive and X-linked patterns of disease inheritance, such as ARX. In vitro functional analysis demonstrates that the transcriptional activity of all three variants was diminished compared to ARX-Wt. We review the associated phenotypes of the published cases of patients with ARX homeodomain variants and propose expansion of the ARX-related phenotype to include severe ID and ASD without brain malformations or seizures. We propose that the use of the constraint and conservation data in conjunction with consideration of the patient phenotype and inheritance pattern may negate the need for the experimental functional validation currently required to achieve a diagnosis.

A Recurrent De Novo Nonsense Variant in ZSWIM6 Results in Severe Intellectual Disability without Frontonasal or Limb Malformations.

A recurrent de novo missense variant within the C-terminal Sin3-like domain of ZSWIM6 was previously reported to cause acromelic frontonasal dysostosis (AFND), an autosomal-dominant severe frontonasal and limb malformation syndrome, associated with neurocognitive and motor delay, via a proposed gain-of-function effect. We present detailed phenotypic information on seven unrelated individuals with a recurrent de novo nonsense variant (c.2737C>T [p.Arg913Ter]) in the penultimate exon of ZSWIM6 who have severe-profound intellectual disability and additional central and peripheral nervous system symptoms but an absence of frontonasal or limb malformations. We show that the c.2737C>T variant does not trigger nonsense-mediated decay of the ZSWIM6 mRNA in affected individual-derived cells. This finding supports the existence of a truncated ZSWIM6 protein lacking the Sin3-like domain, which could have a dominant-negative effect. This study builds support for a key role for ZSWIM6 in neuronal development and function, in addition to its putative roles in limb and craniofacial development, and provides a striking example of different variants in the same gene leading to distinct phenotypes.

X-linked intellectual disability: Phenotypic expression in carrier females.

To better understand the landscape of female phenotypic expression in X-linked intellectual disability (XLID), we surveyed the literature for female carriers of XLID gene alterations (n = 1098) and combined this with experience evaluating XLID kindreds at the Greenwood Genetic Center (n = 341) and at the University of Adelaide (n = 157). One-hundred forty-four XLID genes were grouped into nine categories based on the level of female phenotypic expression, ranging from no expression to female only expression. For each gene, the clinical presentation, gene expression in blood, X-inactivation (XI) pattern, biological pathway involved, and whether the gene escapes XI were noted. Among the XLID conditions, 88 (61.1%) exhibited female cognitive phenotypic expression only, while 56 (38.9%) had no female phenotypic expression (n = 45), phenotype expression with normal cognition in females (n = 8), or unknown status for female phenotypic expression (n = 3). In twenty-four (16.6%) XLID genes, XI was consistently skewed in female carriers, in 54 (37.5%) XI showed variable skewing, and in 33 (22.9%) XI was consistently random. The XI pattern was unknown in 33 (22.9%) XLID conditions. Therefore, there is evidence of a female carrier phenotype in the majority of XLID conditions although how exactly XI patterns influence the female phenotype in XLID conditions remains unclear.

Pathogenic variants in E3 ubiquitin ligase RLIM/RNF12 lead to a syndromic X-linked intellectual disability and behavior disorder.

RLIM, also known as RNF12, is an X-linked E3 ubiquitin ligase acting as a negative regulator of LIM-domain containing transcription factors and participates in X-chromosome inactivation (XCI) in mice. We report the genetic and clinical findings of 84 individuals from nine unrelated families, eight of whom who have pathogenic variants in RLIM (RING finger LIM domain-interacting protein). A total of 40 affected males have X-linked intellectual disability (XLID) and variable behavioral anomalies with or without congenital malformations. In contrast, 44 heterozygous female carriers have normal cognition and behavior, but eight showed mild physical features. All RLIM variants identified are missense changes co-segregating with the phenotype and predicted to affect protein function. Eight of the nine altered amino acids are conserved and lie either within a domain essential for binding interacting proteins or in the C-terminal RING finger catalytic domain. In vitro experiments revealed that these amino acid changes in the RLIM RING finger impaired RLIM ubiquitin ligase activity. In vivo experiments in rlim mutant zebrafish showed that wild type RLIM rescued the zebrafish rlim phenotype, whereas the patient-specific missense RLIM variants failed to rescue the phenotype and thus represent likely severe loss-of-function mutations. In summary, we identified a spectrum of RLIM missense variants causing syndromic XLID and affecting the ubiquitin ligase activity of RLIM, suggesting that enzymatic activity of RLIM is required for normal development, cognition and behavior.

Novel mutations in NLGN3 causing autism spectrum disorder and cognitive impairment.

The X-linked NLGN3 gene, encoding a postsynaptic cell adhesion molecule, was involved in a nonsyndromic monogenic form of autism spectrum disorder (ASD) by the description of one unique missense variant, p.Arg451Cys (Jamain et al. 2003). We investigated here the pathogenicity of additional missense variants identified in two multiplex families with intellectual disability (ID) and ASD: c.1789C>T, p.Arg597Trp, previously reported by our group (Redin et al. 2014) and present in three affected cousins and c.1540C>T, p.Pro514Ser, identified in two affected brothers. Overexpression experiments in HEK293 and HeLa cell lines revealed that both variants affect the level of the mature NLGN3 protein, its localization at the plasma membrane and its presence as a cleaved form in the extracellular environment, even more drastically than what was reported for the initial p.Arg451Cys mutation. The variants also induced an unfolded protein response, probably due to the retention of immature NLGN3 proteins in the endoplasmic reticulum. In comparison, the c.1894A>G, p.Ala632Thr and c.1022T>C, p.Val341Ala variants, present in males from the general population, have no effect. Our report of two missense variants affecting the normal localization of NLGN3 in a total of five affected individuals reinforces the involvement of the NLGN3 gene in a neurodevelopmental disorder characterized by ID and ASD.

O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling.

It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant-expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.

Disruption of POGZ Is Associated with Intellectual Disability and Autism Spectrum Disorders.

Intellectual disability (ID) and autism spectrum disorders (ASD) are genetically heterogeneous, and a significant number of genes have been associated with both conditions. A few mutations in POGZ have been reported in recent exome studies; however, these studies do not provide detailed clinical information. We collected the clinical and molecular data of 25 individuals with disruptive mutations in POGZ by diagnostic whole-exome, whole-genome, or targeted sequencing of 5,223 individuals with neurodevelopmental disorders (ID primarily) or by targeted resequencing of this locus in 12,041 individuals with ASD and/or ID. The rarity of disruptive mutations among unaffected individuals (2/49,401) highlights the significance (p = 4.19 × 10(-13); odds ratio = 35.8) and penetrance (65.9%) of this genetic subtype with respect to ASD and ID. By studying the entire cohort, we defined common phenotypic features of POGZ individuals, including variable levels of developmental delay (DD) and more severe speech and language delay in comparison to the severity of motor delay and coordination issues. We also identified significant associations with vision problems, microcephaly, hyperactivity, a tendency to obesity, and feeding difficulties. Some features might be explained by the high expression of POGZ, particularly in the cerebellum and pituitary, early in fetal brain development. We conducted parallel studies in Drosophila by inducing conditional knockdown of the POGZ ortholog row, further confirming that dosage of POGZ, specifically in neurons, is essential for normal learning in a habituation paradigm. Combined, the data underscore the pathogenicity of loss-of-function mutations in POGZ and define a POGZ-related phenotype enriched in specific features.

Permeabilised skeletal muscle reveals mitochondrial deficiency in malignant hyperthermia-susceptible individuals.

BACKGROUND: Individuals genetically susceptible to malignant hyperthermia (MH) exhibit hypermetabolic reactions when exposed to volatile anaesthetics. Mitochondrial dysfunction has previously been associated with the MH-susceptible (MHS) phenotype in animal models, but evidence of this in human MH is limited. METHODS: We used high resolution respirometry to compare oxygen consumption rates (oxygen flux) between permeabilised human MHS and MH-negative (MHN) skeletal muscle fibres with or without prior exposure to halothane. A substrate-uncoupler-inhibitor titration protocol was used to measure the following components of the electron transport chain under conditions of oxidative phosphorylation (OXPHOS) or after uncoupling the electron transport system (ETS): complex I (CI), complex II (CII), CI+CII and, as a measure of mitochondrial mass, complex IV (CIV). RESULTS: Baseline comparisons without halothane exposure showed significantly increased mitochondrial mass (CIV, P=0.021) but lower flux control ratios in CI+CII(OXPHOS) and CII(ETS) of MHS mitochondria compared with MHN (P=0.033 and 0.005, respectively) showing that human MHS mitochondria have a functional deficiency. Exposure to halothane triggered a hypermetabolic response in MHS mitochondria, significantly increasing mass-specific oxygen flux in CI(OXPHOS), CI+CII(OXPHOS), CI+CII(ETS), and CII(ETS) (P=0.001-0.012), while the rates in MHN samples were unaltered by halothane exposure. CONCLUSIONS: We present evidence of mitochondrial dysfunction in human MHS skeletal muscle both at baseline and after halothane exposure.

Identification and characterization of a missense mutation in the O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase gene that segregates with X-linked intellectual disability.

O-GlcNAc is a regulatory post-translational modification of nucleocytoplasmic proteins that has been implicated in multiple biological processes, including transcription. In humans, single genes encode enzymes for its attachment (O-GlcNAc transferase (OGT)) and removal (O-GlcNAcase (OGA)). An X-chromosome exome screen identified a missense mutation, which encodes an amino acid in the tetratricopeptide repeat, in OGT (759G>T (p.L254F)) that segregates with X-linked intellectual disability (XLID) in an affected family. A decrease in steady-state OGT protein levels was observed in isolated lymphoblastoid cell lines from affected individuals, consistent with molecular modeling experiments. Recombinant expression of L254F-OGT demonstrated that the enzyme is active as both a glycosyltransferase and an HCF-1 protease. Despite the reduction in OGT levels seen in the L254F-OGT individual cells, we observed that steady-state global O-GlcNAc levels remained grossly unaltered. Surprisingly, lymphoblastoids from affected individuals displayed a marked decrease in steady-state OGA protein and mRNA levels. We observed an enrichment of the OGT-containing transcriptional repressor complex mSin3A-HDAC1 at the proximal promoter region of OGA and correspondingly decreased OGA promoter activity in affected cells. Global transcriptome analysis of L254F-OGT lymphoblastoids compared with controls revealed a small subset of genes that are differentially expressed. Thus, we have begun to unravel the molecular consequences of the 759G>T (p.L254F) mutation in OGT that uncovered a compensation mechanism, albeit imperfect, given the phenotype of affected individuals, to maintain steady-state O-GlcNAc levels. Thus, a single amino acid substitution in the regulatory domain (the tetratricopeptide repeat domain) of OGT, which catalyzes the O-GlcNAc post-translational modification of nuclear and cytosolic proteins, appears causal for XLID.

THOC2 Mutations Implicate mRNA-Export Pathway in X-Linked Intellectual Disability.

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.