Sudden cardiac death due to deficiency of the mitochondrial inorganic pyrophosphatase PPA2

We have used whole exome sequencing to identify biallelic missense mutations in the nuclearencoded mitochondrial inorganic pyrophosphatase (PPA2) in ten individuals from four unrelated pedigrees 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 and 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 mutated PPA2 containing mitochondria from fibroblasts showed the activity of inorganic pyrophosphatase 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 new cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized

Disruption of scribble (Scrib1) 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

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

10.1086/521033AMERICAN JOURNAL OF HUMAN GENETICS814700-71

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

Sequence of MEST promoter region indicating key features.

<p>This figure encompasses the hg19 coordinates chr7:130131340-130132187. Features indicated are the three G4s (grey shading, with the extended G4MEST1L region indicated by a dashed underline), the three SNPs (arrowheads with rs IDs as indicated), CpG dinucleotides within the G4 regions (underlined), and the transcription start site (TSS, arrowed) and start codon (double underline) for the imprinted isoform of <i>MEST</i> (NM_001253900). The most 5′ base of the 381bp region studied by Bunyan <i>et al</i>. (2011)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113955#pone.0113955-Bunyan1" target="_blank">[37]</a> is indicated by an asterisk.</p

Comparison of G4 conformation in different buffers.

<p>CD spectra for G4MEST1L (A), G4MEST2 (B), and G4MEST3 (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). (D) CD spectra for G4MEST3 in NaPi, 50 mM KCl (solid line) or NaPi containing 50 mM KCl and 1.5 mM Mg²⁺ (dashed line). Molar ellipticity (x10⁵ deg.cm².dmol⁻¹) is on the vertical axis and wavelength (nm) is on the horizontal axis.</p

Comparison of methylated G4 conformation in different buffers.

<p>CD spectra for methylated oligonucleotides G4MEST1LM (A), G4MEST2M (B), and G4MEST3M (C) in NaPi, 50 mM KCl (solid line) or PCR buffer (dashed line). Molar ellipticity (x10⁵ deg.cm².dmol⁻¹) is on the vertical axis and wavelength (nm) is on the horizontal axis.</p

PAGE analysis of G4 oligonucleotides.

<p>(A). Non-denaturing 15% PAGE of G4 forming oligonucleotides run in the presence of 100 mM KCl, and visualised using UV shadowing. Lane 1: Oligo-dT size markers (size in bases indicated to left). Lane 2: G4MEST1A (mutant). Lane 3: G4MEST1 (wild-type). Lane 4: G4MEST2A (mutant). Lane 5: G4MEST2 (wild-type). Lane 6: G4MEST3A (mutant). Lane 7: G4MEST3 (wild-type). Lane 8: G4MEST2M (methylated). Lane 9: G4MEST3M (methylated). (B). Same gel stained with SYBR Safe, a dsDNA specific, intercalating dye. Lane numbering as above. Note the oligo-dT size markers are single stranded and therefore not visible.</p

Synthetic MEST template mixing experiments using marked templates.

<p>PCR with primers MESTPF1/MESTPR3C or MESTPF1A/MESTPR3C on the two gBlock constructs (wild-type and mutant) generated synthetic templates that were identical except for the presence of one variant base introduced with the mismatch primer MESTPF1A. Methylated and unmethylated forms of these amplicons were diluted and mixed, subjected to PCR, and then genotyped by Sanger sequencing. Products derived from the synthetic templates could be distinguished due to the presence of either an A or T at this position. (A) wild-type templates for which the “T” allele was methylated, and the “A” allele was unmethylated, showing apparent “A” homozygosity; (B) mutant (non-G4 forming) templates for which the “T” allele was methylated, and the “A” allele was unmethylated, showing apparent heterozygosity (W).</p