A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and neuropsychiatric disorders

BACKGROUND: The recurrent ~600 kb 16p11.2 BP4-BP5 deletion is among the most frequent known genetic aetiologies of autism spectrum disorder (ASD) and related neurodevelopmental disorders. OBJECTIVE: To define the medical, neuropsychological, and behavioural phenotypes in carriers of this deletion. METHODS: We collected clinical data on 285 deletion carriers and performed detailed evaluations on 72 carriers and 68 intrafamilial non-carrier controls. RESULTS: When compared to intrafamilial controls, full scale intelligence quotient (FSIQ) is two standard deviations lower in carriers, and there is no difference between carriers referred for neurodevelopmental disorders and carriers identified through cascade family testing. Verbal IQ (mean 74) is lower than non-verbal IQ (mean 83) and a majority of carriers require speech therapy. Over 80% of individuals exhibit psychiatric disorders including ASD, which is present in 15% of the paediatric carriers. Increase in head circumference (HC) during infancy is similar to the HC and brain growth patterns observed in idiopathic ASD. Obesity, a major comorbidity present in 50% of the carriers by the age of 7 years, does not correlate with FSIQ or any behavioural trait. Seizures are present in 24% of carriers and occur independently of other symptoms. Malformations are infrequently found, confirming only a few of the previously reported associations. CONCLUSIONS: The 16p11.2 deletion impacts in a quantitative and independent manner FSIQ, behaviour and body mass index, possibly through direct influences on neural circuitry. Although non-specific, these features are clinically significant and reproducible. Lastly, this study demonstrates the necessity of studying large patient cohorts ascertained through multiple methods to characterise the clinical consequences of rare variants involved in common diseases.

16p11.2 600 kb Duplications confer risk for typical and atypical Rolandic epilepsy

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65,046 European population controls (5/393 cases versus 32/65,046 controls; Fisher's exact test P = 2.83 × 10(-6), odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10(-4)). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical RE.

Juvenile myoclonic epilepsy with photosensitivity in a female with Velocardiofacial syndrome (del(22)(q11.2))--causal relationship or coincidence?

We report on an adolescent female with Velocardiofacial syndrome (del(22)(q11.2)) and an epilepsy phenotype resembling juvenile myoclonic epilepsy (JME). Clinically, the patient has characteristic signs of both disorders. JME has been linked to several chromosomes, but has not been related to 22q11.2 and is rarely observed in other genetic syndromes. We discuss possible explanations for a relationship between the chromosomal aberration and epilepsy as well as the importance of precise delineation of both epilepsy phenotypes and genetic defects in chromosomal disorders.

Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice.

BACKGROUND The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Heterozygous deletion of the PU.1 locus in human AML

The transcription factor PU.1 is essential for myeloid development. Targeted disruption of an upstream regulatory element (URE) decreases PU.1 expression by 80% and leads to acute myeloid leukemia (AML) in mice. Here, we sequenced the URE sequences of PU.1 in 120 AML patients. Four polymorphisms (single nucleotide polymorphisms [SNPs]) in the URE were observed, with homozygosity in all SNPs in 37 patients. Among them, we compared samples at diagnosis and remission, and one patient with cytogenetically normal acute myeloid leukemia M2 was identified with heterozygosity in 3 of the SNPs in the URE at remission. Loss of heterozygosity was further found in this patient at 2 polymorphic sites in the 5' promoter region and in 2 intronic sites flanking exon 4, thus suggesting loss of heterozygosity covering at least 40 kb of the PU.1 locus. Consistently, PU.1 expression in this patient was markedly reduced. Our study suggests that heterozygous deletion of the PU.1 locus can be associated with human AML.

Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

Activation of sphingosine kinase 2 is an endogenous protective mechanism in cerebral ischemia

The two ubiquitously expressed sphingosine kinases (SphK) 1 and 2 are key regulators of the sphingolipid signaling pathway. Despite the formation of an identical messenger, i.e. sphingosine 1-phosphate (S1P), they exert strikingly different functions. Particularly, SphK2 is necessary for the phosphorylation of the sphingosine analog fingolimod (FTY720), which is protective in rodent stroke models. Using gene deficient mice lacking either SphK1 or SphK2, we investigated the role of the two lipid kinases in experimental stroke. We performed 2h transient middle cerebral artery occlusion (tMCAO) and analyzed lesion size and neurological function after 24h. Treatment groups received 1mg/kg FTY720. Neutrophil infiltration, microglia activation, mRNA and protein expression of SphK1, SphK2 and the S1P(1) receptor after tMCAO were studied. Genetic deletion of SphK2 but not SphK1 increased ischemic lesion size and worsened neurological function after tMCAO. The protective effect of FTY720 was conserved in SphK1(-/-) mice but not in SphK2(-/-) mice. This suggests that SphK2 activity is an important endogenous protective mechanism in cerebral ischemia and corroborates that the protective effect of FTY720 is mediated via phospho-FTY720.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Influence of growth hormone (GH) receptor deletion of exon 3 and full-length isoforms on GH response and final height in patients with severe GH deficiency

CONTEXT: A polymorphism of the GH receptor (GHR) gene resulting in genomic deletion of exon 3 (GHR-d3) has been associated with responsiveness to GH therapy. However, the data reported so far do vary according to the underlying condition, replacement dose, and duration of the treatment. OBJECTIVE, DESIGN: The aim of this study was to analyze the impact of the GHR genotypes in terms of the initial height velocity (HV) resulting from treatment and the impact upon adult height in patients suffering from severe isolated GH deficiency. CONTROLS, PATIENTS, SETTING: A total of 181 subjects (peak stimulated GH

Study of a Swiss dopa-responsive dystonia family with a deletion in GCH1: redefining DYT14 as DYT5

OBJECTIVE: To report the study of a multigenerational Swiss family with dopa-responsive dystonia (DRD). METHODS: Clinical investigation was made of available family members, including historical and chart reviews. Subject examinations were video recorded. Genetic analysis included a genome-wide linkage study with microsatellite markers (STR), GTP cyclohydrolase I (GCH1) gene sequencing, and dosage analysis. RESULTS: We evaluated 32 individuals, of whom 6 were clinically diagnosed with DRD, with childhood-onset progressive foot dystonia, later generalizing, followed by parkinsonism in the two older patients. The response to levodopa was very good. Two additional patients had late onset dopa-responsive parkinsonism. Three other subjects had DRD symptoms on historical grounds. We found suggestive linkage to the previously reported DYT14 locus, which excluded GCH1. However, further study with more stringent criteria for disease status attribution showed linkage to a larger region, which included GCH1. No mutation was found in GCH1 by gene sequencing but dosage methods identified a novel heterozygous deletion of exons 3 to 6 of GCH1. The mutation was found in seven subjects. One of the patients with dystonia represented a phenocopy. CONCLUSIONS: This study rules out the previously reported DYT14 locus as a cause of disease, as a novel multiexonic deletion was identified in GCH1. This work highlights the necessity of an accurate clinical diagnosis in linkage studies as well as the need for appropriate allele frequencies, penetrance, and phenocopy estimates. Comprehensive sequencing and dosage analysis of known genes is recommended prior to genome-wide linkage analysis.

Ventricular tachycardia in a Brugada syndrome patient caused by a novel deletion in SCN5A

The aim of the present study was to identify the molecular mechanism behind ventricular tachycardia in a patient with Brugada syndrome. Arrhythmias in patients with Brugada syndrome often occur during sleep. However, a 28-year-old man with no previously documented arrhythmia or syncope who experienced shortness of breath and chest pain during agitation is described. An electrocardiogram revealed monomorphic ventricular tachycardia; after he was converted to nodal rhythm, he spontaneously went into sinus rhythm, and showed classic Brugada changes with coved ST elevation in leads V(1) to V(2). Mutation analysis of SCN5A revealed a novel mutation, 3480 deletion T frame shift mutation, resulting in premature truncation of the protein. Heterologous expression of this truncated protein in human embryonic kidney 293 cells showed a markedly reduced protein expression level. By performing whole-cell patch clamp experiments using human embryonic kidney 293 cells transfected with the mutated SCN5A, no current could be recorded. Hence, the results suggest that the patient suffered from haploinsufficiency of Na(v)1.5, and that this mutation was the cause of his Brugada syndrome.

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.