Emanuela V. Volpi

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3 , causes X-linked recessive hypoparathyroidism

X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.

Endothelial Nox2 overexpression potentiates vascular oxidative stress and hemodynamic response to angiotensin II: studies in endothelial-targeted Nox2 transgenic mice.

Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species (ROS) derived from vascular NADPH oxidases in both vascular smooth muscle cells (VSMCs) and endothelial cells. Recent evidence suggests an important role for VSMC NADPH oxidases in vascular ROS production. However, it is unclear whether increased NADPH oxidase activity in endothelial cells alone is sufficient to alter overall vascular ROS production and hemodynamics. We sought to address these questions using transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NADPH oxidase, Nox2. Aortas of Nox2 transgenic (Nox2-Tg) mice had increased total Nox2 mRNA and protein levels compared with wild-type littermates. Both p22phox mRNA and protein levels were also significantly elevated in Nox2-Tg aortas. Aortic superoxide production was significantly increased in Nox2-Tg mice compared with wild-type, but this difference was abolished by endothelial removal. Superoxide dismutase inhibition increased superoxide release and levels of Mn superoxide dismutase protein were significantly elevated in aortas from Nox2-Tg mice compared with wild type. Increased ROS production from endothelial Nox2 overexpression led to increased endothelial nitric oxide synthase protein and extracellular signal-regulated kinase 1/2 phosphorylation in transgenic aortas. Basal blood pressure was similar, however the pressor responses to both acute and chronic angiotensin II administration were significantly increased in Nox2-Tg mice compared with wild type. These results demonstrate that endothelial-targeted Nox2 overexpression is sufficient to increase vascular NADPH oxidase activity, activate downstream signaling pathways, and potentiate the hemodynamic response to angiotensin II, despite compensatory increases in vascular antioxidant enzymes. Endothelial cell Nox2-containing NADPH oxidase plays an important functional role in vascular redox signaling.

Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence.

Structures of Lysenin Reveal a Shared Evolutionary Origin for Pore-Forming Proteins And Its Mode of Sphingomyelin Recognition

Summary Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.

The leukocyte receptor complex in chicken is characterized by massive expansion and diversification of immunoglobulin-like loci

The innate and adaptive immune systems of vertebrates possess complementary, but intertwined functions within immune responses. Receptors of the mammalian innate immune system play an essential role in the detection of infected or transformed cells and are vital for the initiation and regulation of a full adaptive immune response. The genes for several of these receptors are clustered within the leukocyte receptor complex (LRC). The purpose of this study was to carry out a detailed analysis of the chicken (Gallus gallus domesticus) LRC. Bacterial artificial chromosomes containing genes related to mammalian leukocyte immunoglobulin-like receptors were identified in a chicken genomic library and shown to map to a single microchromosome. Sequencing revealed 103 chicken immunoglobulin-like receptor (CHIR) loci (22 inhibitory, 25 activating, 15 bifunctional, and 41 pseudogenes). A very complex splicing pattern was found using transcript analyses and seven hypervariable regions were detected in the external CHIR domains. Phylogenetic and genomic analysis showed that CHIR genes evolved mainly by block duplications from an ancestral inhibitory receptor locus, with transformation into activating receptors occurring more than once. Evolutionary selection pressure has led not only to an exceptional expansion of the CHIR cluster but also to a dramatic diversification of CHIR loci and haplotypes. This indicates that CHIRs have the potential to complement the adaptive immune system in fighting pathogens.

Copy number variation and association analysis of SHANK3 as a candidate gene for autism in the IMGSAC collection

SHANK3 is located on chromosome 22q13.3 and encodes a scaffold protein that is found in excitatory synapses opposite the pre-synaptic active zone. SHANK3 is a binding partner of neuroligins, some of whose genes contain mutations in a small subset of individuals with autism. In individuals with autism spectrum disorders (ASDs), several studies have found SHANK3 to be disrupted by deletions ranging from hundreds of kilobases to megabases, suggesting that 1% of individuals with ASDs may have these chromosomal aberrations. To further analyse the involvement of SHANK3 in ASD, we screened the International Molecular Genetic Study of Autism Consortium (IMGSAC) multiplex family sample, 330 families, for SNP association and copy number variants (CNVs) in SHANK3. A collection of 76 IMGSAC Italian probands from singleton families was also examined by multiplex ligation-dependent probe amplification for CNVs. No CNVs or SNP associations were found within the sample set, although sequencing of the gene was not performed. Our data suggest that SHANK3 deletions may be limited to lower functioning individuals with autism.

An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive ‘passaging’. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.

A family with autism and rare copy number variants disrupting the Duchenne/Becker muscular dystrophy gene DMD and TRPM3

Autism spectrum disorder is a genetically complex and clinically heterogeneous neurodevelopmental disorder. A recent study by the Autism Genome Project (AGP) used 1M single-nucleotide polymorphism arrays to show that rare genic copy number variants (CNVs), possibly acting in tandem, play a significant role in the genetic aetiology of this condition. In this study, we describe the phenotypic and genomic characterisation of a multiplex autism family from the AGP study that was found to harbour a duplication of exons 31–44 of the Duchenne/Becker muscular dystrophy gene DMD and also a rare deletion involving exons 1–9 of TRPM3. Further characterisation of these extremely rare CNVs was carried out using quantitative PCR, fluorescent in situ hybridisation, long-range PCR amplification and sequencing of junction fragments. The maternal chrX:32,097,213-32,321,945 tandem duplication and paternal chr9:72,480,413-73,064,196 deletion (NCBI build 36 coordinates) were transmitted to both affected boys, potentially signifying a multi-hit mechanism. The DMD reading frame rule predicts a Becker phenotype, characterised by later onset and milder symptoms. When last evaluated, neither child had developed signs of muscular dystrophy. These data are consistent with a degree of comorbidity between autism and muscular dystrophy and suggest that genomic background as well as the position of the mutation within the DMD gene may impact on the neurological correlates of Duchenne/Becker muscular dystrophy. Finally, communicating unexpected findings such as these back to families raises a number of ethical questions, which are discussed.

Perforin activity at membranes leads to invaginations and vesicle formation

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells.

Mapping of three translocation breakpoints associated with orofacial clefting within 6p24 and identification of new transcripts within the region

Orofacial clefting (OFC) is a common congenital malformation. Here we report the refinement of three translocation breakpoints of patients exhibiting OFC within the 6p24 region, and the isolation and characterisation of novel genes, one of which is directly disrupted by the translocation breakpoint of a patient. The gene has been characterized and orthologues identified in bovine, murine and pufferfish.