Single variant, yet "double trouble": TSC and KBG syndrome because of a large de novo inversion.

Despite the advances in high-throughput sequencing, many rare disease patients remain undiagnosed. In particular, the patients with well-defined clinical phenotypes and established clinical diagnosis, yet missing or partial genetic diagnosis, may hold a clue to more complex genetic mechanisms of a disease that could be missed by available clinical tests. Here, we report a patient with a clinical diagnosis of Tuberous sclerosis, combined with unusual secondary features, but negative clinical tests including TSC1 and TSC2 Short-read whole-genome sequencing combined with advanced bioinformatics analyses were successful in uncovering a de novo pericentric 87-Mb inversion with breakpoints in TSC2 and ANKRD11, which explains the TSC clinical diagnosis, and confirms a second underlying monogenic disorder, KBG syndrome. Our findings illustrate how complex variants, such as large inversions, may be missed by clinical tests and further highlight the importance of well-defined clinical diagnoses in uncovering complex molecular mechanisms of a disease, such as complex variants and "double trouble" effects.

A novel FAME1 repeat configuration in a European family identified using a combined genomics approach.

Familial adult myoclonic epilepsy (FAME) is an adult-onset neurological disease characterized by cortical tremor, myoclonus, and seizures due to a pentanucleotide repeat expansion: a combination of pathogenic TTTCA expansion associated with a TTTTA repeat in introns of six different genes. Repeat-primed PCR (RP-PCR) is an inexpensive test for expansions at known loci. The analysis of the SAMD12 locus revealed that the repeats have different size, configuration, and composition. The TTTCA repeats can be very long (>1000 repeats) but also very short (14 being the shortest identified). Here, we report siblings of European descent with the clinical diagnosis of FAME yet a negative RP-PCR test. Using short-read genome sequencing, we identified the pentanucleotide expansion in intron 4 of SAMD12, which was confirmed by CRIPSR-Cas9-mediated enrichment and long-read sequencing to be of (TTTTA)~879 (TTTCA)3 (TTTTA)7 (TTTCA)7 configuration. Our finding is the first to associate the SAMD12 locus in European patients with FAME and currently represents the shortest identified TTTCA expansion. Our results suggest that the SAMD12 locus should be tested in patients with suspected FAME independent of ethnicity. Furthermore, RP-PCR may miss the underlying mutation, and genome sequencing may be needed to confirm the pathogenic repeat.

Case Report: Biallelic Loss of Function ATM due to Pathogenic Synonymous and Novel Deep Intronic Variant c.1803-270T > G Identified by Genome Sequencing in a Child With Ataxia-Telangiectasia.

Ataxia-telangiectasia (AT) is a complex neurodegenerative disease with an increased risk for bone marrow failure and malignancy. AT is caused by biallelic loss of function variants in ATM, which encodes a phosphatidylinositol 3-kinase that responds to DNA damage. Herein, we report a child with progressive ataxia, chorea, and genome instability, highly suggestive of AT. The clinical ataxia gene panel identified a maternal heterozygous synonymous variant (NM_000051.3: c.2250G > A), previously described to result in exon 14 skipping. Subsequently, trio genome sequencing led to the identification of a novel deep intronic variant [NG_009830.1(NM_000051.3): c.1803-270T > G] inherited from the father. Transcript analyses revealed that c.1803-270T > G results in aberrant inclusion of 56 base pairs of intron 11. In silico tests predicted a premature stop codon as a consequence, suggesting non-functional ATM; and DNA repair analyses confirmed functional loss of ATM. Our findings highlight the power of genome sequencing, considering deep intronic variants in undiagnosed rare disease patients.

Whole genome sequencing facilitates intragenic variant interpretation following modifier screening in C. elegans.

BACKGROUND: Intragenic modifiers (in-phase, second-site variants) are known to have dramatic effects on clinical outcomes, affecting disease attributes such as severity or age of onset. However, despite their clinical importance, the focus of many genetic screens in model systems is on the discovery of extragenic variants, with many labs still relying upon more traditional methods to identify modifiers. However, traditional methods such as PCR and Sanger sequencing can be time-intensive and do not permit a thorough understanding of the intragenic modifier effects in the context of non-isogenic genomic backgrounds. RESULTS: Here, we apply high throughput approaches to identify and understand intragenic modifiers using Caenorhabditis elegans. Specifically, we applied whole genome sequencing (WGS) to a mutagen-induced forward genetic screen to identify intragenic suppressors of a temperature-sensitive zyg-1(it25) allele in C. elegans. ZYG-1 is a polo kinase that is important for centriole function and cell divisions, and mutations that truncate its human orthologue, PLK4, have been associated with microcephaly. Combining WGS and CRISPR/Cas9, we rapidly identify intragenic modifiers, show that these variants are distributed non-randomly throughout zyg-1 and that genomic context plays an important role on phenotypic outcomes. CONCLUSIONS: Ultimately, our work shows that WGS facilitates high-throughput identification of intragenic modifiers in clinically relevant genes by reducing hands-on research time and overall costs and by allowing thorough understanding of the intragenic phenotypic effects in the context of different genetic backgrounds.

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Unlike estrogens that increase PCSK9 levels post-menopause HSP27 vaccination lowers cholesterol levels and atherogenesis due to divergent effects on PCSK9 and LDLR.

AIMS: The estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti- HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, the rationale for this study is to compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis. METHODS AND RESULTS: ApoE-/- mice subjected to ovariectomy (OVX) showed a 65 % increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (-43 %), a subcutaneous E2 slow release pellet (-52 %) or a combination thereof (-82 %). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52 % higher in E2/OVX and 41 % lower in rHSP25/OVX mice (p < 0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148 % with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303 %, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64 %. CONCLUSION: The reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women - are there effects beyond cholesterol?

Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake.

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.

Transgenic mice overexpressing desmocollin-2 (DSC2) develop cardiomyopathy associated with myocardial inflammation and fibrotic remodeling.

BACKGROUND: Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology. METHODS AND RESULTS: We generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age. CONCLUSION: Cardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy.

High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach.

Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4',6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the microg/ml range. Here, we report that long-wavelength excitation (> or = 400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death.

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by beta-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells.

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.

The pore, not cytoplasmic domains, underlies inactivation in a prokaryotic sodium channel.

Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from approximately 50 ms at depolarized voltages to a maximum of approximately 100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by approximately 2.5-fold, and the midpoint of the steady-state inactivation curve was shifted approximately 20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction.

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.

The overexpression of Bcl-2 antagonizes the proapoptotic function of the kappa-opioid receptor.

Opioid receptors are G-protein-coupled cell-surface receptors that are mainly expressed in neuronal cells. Stimulation of the kappa-opioid receptor expressed by cultured human epithelial cancer cells promotes staurosporine-induced apoptosis. In this study, while Bcl-2 did not inhibit staurosporine-induced apoptosis, it did inhibit the kappa-opioid receptor-mediated potentiation of apoptosis. The results suggest that Bcl-2 targets a step that is specific to the signaling pathway of the kappa-opioid receptor.