Sudden Cardiac Death Due to Deficiency of the Mitochondrial Inorganic Pyrophosphatase PPA2.

We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.

Mutations in the planar cell polarity genes CELSR1 and SCRIB are associated with the severe neural tube defect craniorachischisis.

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.

Comparing the variants of takotsubo syndrome: an observational study of the ECG and structural changes from a New Zealand tertiary hospital.

OBJECTIVES: In takotsubo syndrome, QTc prolongation is a measure of risk of potentially fatal arrhythmia. It is not known how this risk, or derangement of other markers, differs across the echo variants of takotsubo syndrome. Therefore, we sought to explore whether apical takotsubo syndrome differs from the variants of the syndrome in more ways than just regional wall motion pattern. As the region of affected myocardium is usually larger, we hypothesised that patients with the classic apical ballooning form of takotsubo syndrome would have more severe derangement of their markers. DESIGN: Observational study of patients gathered from a prospective database (2010-2018) and by retrospective review (2006-2009). SETTING: The sole tertiary hospital from a New Zealand region in which case clusters of takotsubo syndrome were precipitated by large earthquakes in 2010, 2011 and 2016. PARTICIPANTS: A total of 222 patients who met a modified version of the Mayo criteria for takotsubo syndrome were included. All patients had digitally archived echocardiograms that were over-read by a second echocardiologist blinded to the clinical report. PRIMARY OUTCOME MEASURES: Ejection fraction, peak troponin and QTc interval. RESULTS: Patients with the apical form were older (p=0.011), had a lower initial left ventricular ejection fraction (35% vs 44%, p<0.0001) and a higher peak high-sensitivity troponin I (hsTnI) (p=0.01) than those with variant forms. There was no difference in the electrical abnormalities between the variants (QTc interval, heart rate, PR interval, QRS duration or T-wave axis). There was also no correlation between any of peak hsTnI, peak QTc and ejection fraction. QTc interval increased on day 2 and peaked on day 3 before falling steeply (p<0.0001). CONCLUSIONS: The variants of takotsubo syndrome differ in more ways than just their echo pattern but do not differ in their electrical abnormalities. There is a dissociation between the structural and electrical abnormalities. QTc peaks on day 3 and then falls steeply.

Abnormal folate metabolism in foetuses affected by neural tube defects.

Folic acid supplementation can prevent many cases of neural tube defects (NTDs), whereas suboptimal maternal folate status is a risk factor, suggesting that folate metabolism is a key determinant of susceptibility to NTDs. Despite extensive genetic analysis of folate cycle enzymes, and quantification of metabolites in maternal blood, neither the protective mechanism nor the relationship between maternal folate status and susceptibility are understood in most cases. In order to investigate potential abnormalities in folate metabolism in the embryo itself, we derived primary fibroblastic cell lines from foetuses affected by NTDs and subjected them to the dU suppression test, a sensitive metabolic test of folate metabolism. Significantly, a subset of NTD cases exhibited low scores in this test, indicative of abnormalities in folate cycling that may be causally linked to the defect. Susceptibility to NTDs may be increased by suppression of the methylation cycle, which is interlinked with the folate cycle. However, reduced efficacy in the dU suppression test was not associated with altered abundance of the methylation cycle intermediates, s-adenosylmethionine and s-adenosylhomocysteine, suggesting that a methylation cycle defect is unlikely to be responsible for the observed abnormality of folate metabolism. Genotyping of samples for known polymorphisms in genes encoding folate-associated enzymes did not reveal any correlation between specific genotypes and the observed abnormalities in folate metabolism. These data suggest that as yet unrecognized genetic variants result in embryonic abnormalities of folate cycling that may be causally related to NTDs.

Copy number variants implicate cardiac function and development pathways in earthquake-induced stress cardiomyopathy.

The pathophysiology of stress cardiomyopathy (SCM), also known as takotsubo syndrome, is poorly understood. SCM usually occurs sporadically, often in association with a stressful event, but clusters of cases are reported after major natural disasters. There is some evidence that this is a familial condition. We have examined three possible models for an underlying genetic predisposition to SCM. Our primary study cohort consists of 28 women who suffered SCM as a result of two devastating earthquakes that struck the city of Christchurch, New Zealand, in 2010 and 2011. To seek possible underlying genetic factors we carried out exome analysis, genotyping array analysis, and array comparative genomic hybridization on these subjects. The most striking finding was the observation of a markedly elevated rate of rare, heterogeneous copy number variants (CNV) of uncertain clinical significance (in 12/28 subjects). Several of these CNVs impacted on genes of cardiac relevance including RBFOX1, GPC5, KCNRG, CHODL, and GPBP1L1. There is no physical overlap between the CNVs, and the genes they impact do not appear to be functionally related. The recognition that SCM predisposition may be associated with a high rate of rare CNVs offers a novel perspective on this enigmatic condition.

Valproic acid exposure leads to upregulation and increased promoter histone acetylation of sepiapterin reductase in a serotonergic cell line.

Valproic acid (VPA) is a widely used antiepileptic drug and first-line treatment in bipolar disorder, although the mechanisms underlying its therapeutic effects are largely unknown. Recently, the recognition of VPA as an epigenetic drug offers new opportunities for understanding its therapeutic actions. In a rat serotonergic cell line (RN46A) we observed that VPA exposure has a strong upregulatory effect on the gene for sepiapterin reductase (SPR), a key enzyme involved in the tetrahydrobiopterin (BH4) synthetic pathway. BH4 is an essential cofactor in the biosynthesis of neurotransmitters like serotonin, dopamine and noradrenalin, and the BH4 pathway may thus be important in mood biology. Using real-time quantitative PCR we show that VPA, at therapeutically relevant doses, increases the expression of the Spr gene by about 8-fold in RN46A cells. In addition, Spr protein levels in VPA-exposed cells were elevated, as were the intracellular BH4 levels. HDAC inhibitors (HDACI) trichostatin A and sodium butyrate also upregulated Spr, but this was not observed using the VPA-analogue valpromide, which lacks HDAC inhibitory activity. Further examination of this effect revealed that exposure to VPA increased the acetylated histone mark H3K9/K14ac at the Spr promoter. The DNMT inhibitor 5'aza-dC also upregulated Spr by over 8-fold. However, DNA methylation status across the Spr promoter did not change in response to VPA. The BH4 pathway is fundamental to the regulation of neurotransmitters relevant to mood disorders, and this epigenetic effect of VPA at the Spr promoter may represent a novel mechanism through which VPA achieves its therapeutic action.

G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

TBX22 missense mutations found in patients with X-linked cleft palate affect DNA binding, sumoylation, and transcriptional repression.

The T-box transcription factor TBX22 is essential for normal craniofacial development, as demonstrated by the finding of nonsense, frameshift, splice-site, or missense mutations in patients with X-linked cleft palate (CPX) and ankyloglossia. To better understand the function of TBX22, we studied 10 different naturally occurring missense mutations that are phenotypically equivalent to loss-of-function alleles. Since all missense mutations are located in the DNA-binding T-box domain, we first investigated the preferred recognition sequence for TBX22. Typical of T-box proteins, the resulting sequence is a palindrome based around near-perfect copies of AGGTGTGA. DNA-binding assays indicate that missense mutations at or near predicted contact points with the DNA backbone compromise stable DNA-protein interactions. We show that TBX22 functions as a transcriptional repressor and that TBX22 missense mutations result in impaired repression activity. No effect on nuclear localization of TBX22 was observed. We find that TBX22 is a target for the small ubiquitin-like modifier SUMO-1 and that this modification is required for TBX22 repressor activity. Although the site of SUMO attachment at the lysine at position 63 is upstream of the T-box domain, loss of SUMO-1 modification is consistently found in all pathogenic CPX missense mutations. This implies a general mechanism linking the loss of SUMO conjugation to the loss of TBX22 function. Orofacial clefts are well known for their complex etiology and variable penetrance, involving both genetic and environmental risk factors. The sumoylation process is also subject to and profoundly affected by similar environmental stresses. Thus, we suggest that SUMO modification may represent a common pathway that regulates normal craniofacial development and is involved in the pathogenesis of both Mendelian and idiopathic forms of orofacial clefting.

Epithelial cell polarity genes are required for neural tube closure.

Human neural tube defects (NTD) are a heterogeneous group that exhibit complex inheritance, making it difficult to identify the underlying cause. Due to the uniform genetic background, inbred mouse strains are a more amenable target for genetic studies. We investigated the loop-tail (Lp) mouse as a model for the severe NTD, craniorachischisis. A homozygous point mutation was identified in the transmembrane protein Vangl2, which in Drosophila has been shown to function in the planar cell polarity (PCP) pathway. Morphological analysis of the Lp mice shows that the defect results from an abnormally broad floor plate, most likely through a failure in convergent extension. The elevated neural folds remain too far apart to contact, inhibiting neural tube closure. Recently, two other mouse mutants (crash and circletail) were described with a similar phenotype to Lp and were investigated as potentially new alleles. Mapping studies, however, showed that both mutants segregated to distinct loci. In the crash (Crsh) mouse, a mutation was identified in Celsr1, a seven pass transmembrane receptor that encodes a protein orthologous to Drosophila Flamingo. Like Vangl2, this gene also functions in the PCP pathway. While in circletail, a point mutation was identified introducing a premature stop codon into the apical-basal cell polarity gene scribble (Scrb1). We subsequently demonstrated a genetic interaction between all three genes, where double heterozygotes exhibit the same homozygous NTD phenotype. This strongly suggests both a candidate gene pathway and that interaction between independent recessive alleles may be a possible explanation for the complex inheritance in severe human NTD.

Cloning and characterization of Igsf9 in mouse and human: a new member of the immunoglobulin superfamily expressed in the developing nervous system.

We describe the cloning and characterization of a novel member of the immunoglobulin superfamily, Igsf9. The predicted protein structure of IGSF9 closely matches that of the neural cell-adhesion molecule (NCAM) subfamily, consisting of an extracellular region containing five immunoglobulin domains and two fibronectin type III (FnIII) repeats, a transmembrane region, and a cytoplasmic tail. We have also characterized the orthologous human IGSF9 gene at 1q22-q23, revealing a highly conserved sequence and genomic organization. Expression of Igsf9 was detected by RT-PCR in mouse embryonic RNA from embryonic day (E) 7.5 to E16.5, while whole-mount in situ hybridization at E10.5 shows intense expression within the dorsal root ganglia, trigeminal ganglia, and olfactory epithelium, and less intense expression in the neuroepithelium, retina, and hindgut. In the human, transcription was detected in a wide variety of fetal tissues at both 8 and 14 weeks. Protein homology of IGSF9 is most similar to the Drosophila melanogaster Turtle protein that functions in coordinated motor output in complex behaviors.

Genomic organization and embryonic expression of Igsf8, an immunoglobulin superfamily member implicated in development of the nervous system and organ epithelia.

Igsf8 is an immunoglobulin protein that binds to the tetraspanin molecules, CD81 and CD9. We describe the genomic organization of mouse and human Igsf8, and reveal a dynamic expression pattern during embryonic and fetal development. Igsf8 is first expressed at E9.5 in a ventral domain of the neural tube, with dorsal expression apparent at E10.5. We show that the ventral, but not the dorsal, domain of neural tube expression is dependent on Shh signaling. From E11.5, Igsf8 is expressed at the lateral edge of the ventricular zone, in early postmitotic neuroblasts, and in dorsal root and cranial ganglia. Igsf8 is also expressed in the branchial arches, dorsal pancreatic primordium, neural retina, olfactory epithelium, gut, kidney, and lung.

Disruption of scribble (Scrb1) causes severe neural tube defects in the circletail mouse.

Circletail is one of only two mouse mutants that exhibit the most severe form of neural tube defect (NTD), termed craniorachischisis. In this disorder, almost the entire brain and spinal cord is affected, owing to a failure to initiate neural tube closure. Craniorachischisis is a significant cause of lethality in humans, yet the molecular mechanisms involved remain poorly understood. Here, we report the identification of the gene mutated in circletail (Crc), using a positional cloning approach. This gene, Scrb1, encodes a member of the LAP protein family related to Drosophila scribble, with 16 leucine rich repeats and four PDZ domains. The Crc mutant contains a single base insertion that creates a frame shift and leads to premature termination of the Scrb1 protein. We report the expression pattern of Scrb1 during embryonic and fetal development, and show that Scrb1 expression closely mirrors the phenotypic defects observed in Crc/Crc mutants. In addition, circletail genetically interacts with the loop-tail mutant, and we reveal overlapping expression of Scrb1 with Vangl2, the gene mutated in loop-tail. The identification of the Crc gene further defines the nature of the genetic pathway required for the initiation of neural tube closure and provides an important new candidate that may be implicated in the aetiology of human NTDs.

Craniofacial expression of human and murine TBX22 correlates with the cleft palate and ankyloglossia phenotype observed in CPX patients.

Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a Mendelian, semidominant X-linked disorder and has been described in several large families from different ethnic origins. It is a useful genetic model for non-syndromic cleft palate, a common congenital disorder. Recently, the underlying genetic defect in CPX was identified, where unique mutations were found in the T-box-containing transcription factor TBX22. Here we report two new familial cases with novel missense and insertion mutations, each occurring within the T-box domain and highlighting the functional significance of this DNA-binding motif. We describe TBX22 expression in early human development, where expression is found in the palatal shelves and is highest prior to elevation to a horizontal position above the tongue. mRNA is also detected in the base of the tongue in the region of the frenulum that corresponds to the ankyloglossia seen in CPX patients. Other sites of expression include the inferior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop, and the tooth buds themselves. We have also identified the orthologous mouse Tbx22 gene and performed expression analysis in E12.5-E17.5 mouse embryos. The location of mRNA expression closely correlates between mouse and human, while at later stages of development, we also detected expression in mouse lung and whisker follicles. We conclude that expression of TBX22 is entirely consistent with the CPX phenotype and that the mouse should provide a useful model for elucidating its role in craniofacial development.