Neonatal Form Research Articles

0205 : Cause of death in Marfan syndrome

the last 30 years have been associated with increased survival of 30 years in patients with Marfan syndrome. AWe thought to assess cause of death in patients with Marfan syndrome who were seen in the CNMR Syndrome de Marfan and related patients who came at least once in CNMR, had a diagnosis of MFS with a FBN1 gene mutation were identified; patients who died where selected. 53 deaths were reported among the 1253 patients. Median age at death was 41 years and 35% were women. Three deaths occurred in children affected by a neonatal form of MFS: cause of death was 1) acute respiratory failure in a 2 years old boy with severe mitral regurgitation and severe restrictive respiratory failure, 2) post-operative death after planned surgery for MR and aortic aneurysm in a 4 years old boy and 3) sudden death in a 6 years old boy with aortic root diameter at 42mm;Thirteen deaths occurred after planned aortic surgery.Surgery has been performed for isolated aortic root dilatation or with mitral valve repair, Redo surgery, aortic root dilatation after supracoronary tube implantation, false aneurysm after a Bentall, dilatation of dissected thoracic descending aorta or mitral valve repair. Ten deaths were related to acute aortic events: Dissection of ascending aorta in 8, descending aorta in 1 and abdominal aorta in 1. Six patients died after myocardial infarction, Stroke, heart failure, aortic mechanical prosthesis thrombosis, ruptured mitral. Nine sudden cardiac deaths without autopsy performed were reported.Three deaths were related a mechanical aortic valve endocarditis in 2 patients and one patient died of a hemorrhagic shock complicating anticoagulation. Lastly, 9 deaths were non cardiac. In MFS patients with a FBN1 gene mutation, cardiovascular deaths remains the leading cause of death despite increase in life expectancy associated with the modern care of the patients. Notably, post-operative mortality is high in this population. The author hereby declares no conflict of interest

Maladies peroxysomales

Peroxisomes are small intracellular organelles that catalyse key metabolic reactions such as the beta-oxidation of some straight-chain or branched-chain fatty acids and the alpha-oxidation of phytanic acid. These enzyme reactions produce hydrogen peroxide, which is subsequently neutralized by the peroxisomal catalase. Peroxisomes also metabolize glyoxylate to glycine, and catalyze the first steps of plasmalogen biosynthesis. There are more than a dozen inherited peroxisomal disorders in humans. These metabolic diseases are due to monogenic defects that affect either a single function (such as enzyme or a transporter) or more than two distinct functions because of the impairment of several aspects of peroxisome biogenesis. With the notable exception of X-linked adrenoleucodystrophy, these inborn disorders are transmitted as autosomal recessive traits. Their clinical presentation can be very heterogeneous, and include neonatal, infantile or adult forms. The present review describes the symptomatology of these genetic diseases, the underlying genetic and biochemical alterations, and summarizes their diagnostic approach.

Hydroxysteroid 17-Beta Dehydrogenase Type 10 Disease in Siblings.

Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) deficiency (HSD10 disease) is a rare X-linked neurodegenerative condition caused by abnormalities in the HSD17B10 gene. A total of 10 mutations have been reported in the literature since 2000. Described phenotypes include a severe neonatal or progressive infantile form with hypotonia, choreoathetosis, seizures, cardiomyopathy, neurodegeneration, and death, as well as an attenuated form with variable regression. Here we present the second report of a c.194T>C (p.V65A) mutation in two half-brothers with a clinical phenotype characterized by neurodevelopmental delay, choreoathetosis, visual loss, cardiac findings, and behavioral abnormalities, with regressions now noted in the older sibling. Neither has experienced a metabolic crisis. Both of the siblings had normal tandem mass spectroscopy analysis of their newborn screening samples. The older brother's phenotype may be complicated by the presence of a 3q29 microduplication. Diagnosis requires a high index of suspicion, as the characteristic urine organic acid pattern may escape detection. The exact pathogenic mechanism of disease remains to be elucidated, but may involve the non-dehydrogenase functionalities of the HSD10 protein. Our report highlights clinical features of two patients with the less fulminant phenotype associated with a V65A mutation, compares the reported phenotypes to date, and reviews recent findings regarding the potential pathophysiology of this condition.Summary Sentence Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) disease (HSD10 disease) is a rare X-linked neurodegenerative condition with a variable clinical phenotype; diagnosis requires a high index of suspicion.

Observational cohort study of the natural history of Niemann-Pick disease type C in the UK: a 5-year update from the UK clinical database.

BackgroundNiemann-Pick disease type C (NP-C) is a rare neurovisceral lipid storage disorder characterised by progressive, disabling neurological symptoms and premature death in most patients. During the last decade, national cohort studies have accrued a great deal of data on the symptomatology and natural history of NP-C.MethodsIn an observational cohort study, we present a substantial update based on the clinical presentation and follow-up of all known UK-based patients with a confirmed diagnosis of NP-C who have been tracked on an electronic database at the Department of Genetic Medicine, University of Manchester, UK. Patients were stratified according to accepted age-at-neurological-onset categories. Data on patients’ clinical signs and symptoms, medical history and genetic studies are summarised using descriptive methods.ResultsA total of 146 patients with NP-C were included, representing the full known UK NP-C cohort, as observed from database information between 1999 and the end of 2011: 72 patients (49 %) were alive at the end of the observation period. Among a total of 116 patients (79 %) who possessed at least one identified, disease-causing NP-C gene mutation, 114 (98 %) had NPC1 and two (2 %) had NPC2 mutations. Overall, 53/194 (27 %) identified mutations were novel. Six patients (4 %) had an early, non-neurological neonatal onset form of NP-C. The numbers (%) of patients with accepted age-at-neurological onset forms were: 8 (5 %) early-infantile onset, 51 (35 %) late-infantile onset, 42 (29 %) juvenile onset, and 25 (17 %) adolescent/adult onset. Fourteen patients diagnosed based on visceral symptoms and/or sibling history, confirmed in most cases by genetic analysis, did not have any neurological manifestations at last follow up (11 patients with mean [SD] age at last follow up 2.5 [1.8] years: 3 with mean [SD] age at death 20.8 [15.9] years). A total of 51 patients (35 %) received miglustat therapy. The mean (SD) overall treatment duration up to the end of the observation period was 2.6 (2.3) years.ConclusionsThis UK cohort is the largest national NP-C cohort reported to date, and confirms the wide phenotypic variability of the disease, as reported in other countries. Further analyses are required to assess the impact of miglustat therapy on neurological disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12883-015-0511-1) contains supplementary material, which is available to authorized users.

Révélation néonatale d’une mucolipidose de type II

Mucolipidosis type II (ML II, OMIM 252,500) is an autosomal recessive disorder clinically characterized by facial dysmorphia similar to Hurler syndrome and pronounced gingival hypertrophy. The disorder is caused by a defect in targeting acid hydrolases on the surface of lysosomes, which impede their entry and lead to accumulation of undigested substrates in lysosomes. The onset of the symptoms is usually in infancy, beginning in the 6th month of life. Early onset, at birth or even in utero, is a sign of severity and involves the specific dysmorphia as well as skeletal dysplasia related to hyperparathyroidism. We report on a severe neonatal form of this disorder revealed by respiratory distress with severe chest deformity. The dysmorphic syndrome, combining coarse features, pronounced gingival hypertrophy, with diffuse bone demineralization and secondary hyperparathyroidism associating significant elevation of parathyroid hormone and alkaline phosphatase with normal levels of vitamin D and calcium were characteristics of mucolipidosis type II. Recognizing this specific association of anomalies helps eliminate the differential diagnosis and establish appropriate diagnosis and care.

In vitro effects of phenytoin and DAPT on MDA-MB-231 breast cancer cells.

Voltage-gated sodium channel (VGSC) activity enhances cell behaviors related to metastasis, such as motility, invasion, and oncogene expression. Neonatal alternative splice form of Nav1.5 isoform is expressed in metastatic breast cancers. Furthermore, aberrant Notch signaling pathway can induce oncogenesis and may promote the progression of breast cancers. In this study, we aimed to analyze the effect of the nNav1.5 inhibitor phenytoin and Notch signal inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester (DAPT) on triple negative breast cancer cell line (MDA-MB-231) via inhibition of nNav1.5 VGSC activity and Notch signaling, respectively. In order to determine the individual and combined effects of these inhibitors, the 4-[3-(4-iyodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) test, wound healing assay, and zymography were performed to detect the proliferation, lateral motility, and matrix metalloproteinase-9 (MMP9) activity, respectively. The expressions of nNav1.5, Notch4, MMP9, and tissue inhibitor of metalloproteinases-1 (TIMP1) were also detected by quantitative real-time reverse transcriptase-polymerase chain reaction. DAPT caused an antiproliferative effect when the doses were higher than 10 µM, whereas phenytoin showed no inhibitory action either alone or in combination with DAPT on the MDA-MB-231 cells. Furthermore, it was found that the lateral motility was inhibited by both inhibitors; however, this inhibitory effect was partially rescued when they were used in combination. Meanwhile, the results showed that the MMP9 activity and the ratio of MMP9 mRNA to TIMP1 mRNA were only decreased by DAPT. Thus, we conclude that the combined effect of DAPT and phenytoin is not as beneficial as using DAPT alone on MDA-MB-231 breast cancer cells.

A case of Bartter's syndrome associated with nephrocalcinosis presenting with tetany

A 10-year-old boy was admitted to hospital with a 3 month history of intermittent spasms of the wrists and ankles, and twitching of the eyelids. He also had polyuria, polydipsia, nocturnal enuresis, fatigue and constipation since he was a toddler. Physical examination revealed normal blood pressure, myokymia on the right eyelid and bilateral carpopedal spasms. Laboratory investigation revealed hypocalcemia, hypokalemia, increased plasma renin and aldosterone , hypercalciuria, metabolic alkalosis, and bilateral medullary nephrocalcinosis. Cranial computed tomography was normal. Based on the clinical and laboratory findings he was diagnosed as having Bartter’s syndrome, which is characterized by hypochloremia, hypokalaemia and metabolic alkalosis associated with potassium renal leakage, with normal blood pressure despite increased plasma renin activity. It is well known that tetany is not uncommon in the neonatal form of Bartter’s syndrome and nephrocalcinosis is usually not present in the classic form. Interestingly, our patient had both the clinical manifestations of the neonatal form and of the classic form of Bartter’s syndrome. In conclusion, we would like to emphasize that both the clinical manifestations of neonatal and classic forms of Bartter’s syndrome (as an overlapping syndrome) might be seen in children and that Bartter’s syndrome should also be considered in children with tetany as in our case. (J Pediatr Neurol 2004; 2 (1): 45-47).

Novel perspectives in cancer therapy: Targeting ion channels.

By controlling ion fluxes at multiple time scales, ion channels shape rapid cell signals, such as action potential and synaptic transmission, as well as much slower processes, such as mitosis and cell migration. As is currently increasingly recognized, a variety of channel types are involved in cancer hallmarks, and regulate specific stages of neoplastic progression. Long-term in vitro work has established that inhibition of these ion channels impairs the growth of cancer cells. Recently, these studies have been followed up in vivo, hence revealing that ion channels constitute promising pharmacological targets in oncology. The channel proteins can be often accessed from the extracellular milieu, which allows use of lower drug doses and decrease untoward toxicity. However, because of the central physiological roles exerted by ion channels in excitable cells, other types of side effects may arise, the gravest of which is cardiac arrhythmia. A paradigmatic case is offered by Kv11.1 (hERG1) channels. HERG1 blockers attenuate the progression of both hematologic malignancies and solid tumors, but may also lead to the lengthening of the electrocardiographic QT interval, thus predisposing the patient to ventricular arrhythmias. These side effects can be avoided by specifically inhibiting the channel isoforms which are highly expressed in certain tumors, such as Kv11.1B and the neonatal forms of voltage-gated Na(+) channels. Preclinical studies are also being explored in breast and prostate cancer (targeting voltage-gated Na(+) channels), and gliomas (targeting CLC-3). Overall, the possible approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1) the development of specific inhibitors for the channel subtypes expressed in specific tumors; (2) drug delivery into the tumor by using antibodies or nanotechnology-based approaches; (3) combination regimen therapy and (4) blocking specific conformational states of the ion channel. We believe that expanding this relatively neglected field of oncology research might lead to unforeseen therapeutic benefits for cancer patients.

Collaborating to Create a Comprehensive Neonatal Resuscitation Form

Poster Presentation Purpose for the Program High‐quality care of the newborn at birth is critically important in obstetrics. To assess response to newborns who require extra assistance at birth, a newborn code review committee was formed in July 2012. Newborn resuscitation documentation was audited for 3 months, and it was determined the current form was a barrier to accurate documentation and care. Of the 16 indicators audited, documentation was found to be 0% to 67% complete in seven categories. The code committee searched the literature, Internet, and area hospitals to find a better documentation form. Forms found were either equal to or less comprehensive than the current form. Proposed Change To create a new form that follows the guidelines of the American Academy of Pediatrics (AAP) Neonatal Resuscitation Program to increase documentation adherence and provide documentation prompts to perform correct actions during a newborn code. The American Heart Association (AHA) Get with the Guidelines neonatal resuscitation form was used as a starting point. Content was added and removed based on team experience with newborn codes and AAP guidelines. Implementation, Outcomes, and Evaluation The new form, completed in October 2012, was taken to delivery team nurses and neonatologists for review. After suggested changes were made, the code team approved the final form, and staff members involved in newborn resuscitation were taught to use it. Health care providers began using the new form in May 2013. Neonatal resuscitation records from July through October 2013 were reviewed to identify trends and improvements after implementation of the new form. Of 16 categories audited, adherence scores increased from 88% to 100%. Nursing and medical staff expressed satisfaction and acceptance of the new form, noting ease of use and guidance through code documentation and prompts for needed actions. Audits continue to show improvement in documentation after the implementation of the form. Implications for Nursing Practice Teams should review current neonatal resuscitation forms for documentation accuracy and adherence. Revisions may be necessary to help guide practitioners through resuscitation and assist with accurate documentation.

Assessment of Efficiency of the Use of Transcutaneous Electrical Nerve Stimulation in Labor Pain Relief

Objective To determine the efficiency of low‐level electrical stimulation applied by a transcutaneous electrical nerve stimulation (TENS) unit on labor pain relief. Design A prospective randomized controlled trial study. Setting The study was conducted at the obstetrics and gynecology clinic of a university hospital. Sample The sample included women who volunteered and met necessary criteria for each group: 64 women in the intervention group and 60 women in the control group. Methods A demographic data collection form, visual analog scale (VAS), labor observation form, neonatal observation form, and satisfaction form on TENS application were used as data collection tools. All data were analyzed using a statistical program. Chi‐squared test was used for the descriptive data, t test was used for the quantitative data, and regression analysis was used to determine the effect of independent variables on dependent variables. Results The VAS score means were calculated as 5.49 ± 0.98 for the intervention group and 7.71 ± 0.64 for the control group. VAS mean of the intervention group were less than the control group, which was statistically significant (p < .05). The duration of labor was 6.54 ± 2.34 hours for the TENS applied group and 8.11 ± 0.27 hours for the control group, and the difference was statistically significant (p < .05). As a result, it was determined that TENS reduces VAS scores regarding labor pain, shortens duration of labor, its efficiency is independent of other variables, and it has no side effects. Conclusion/Implications for Nursing Practice The low level electrical stimulation TENS unit reduces the VAS scores relating to labor pain, shortens the period of delivery, has no adverse effects, and its efficiency is independent from other variables. With these features, TENS application may reduce negative effects of severe labor pain on the health of the mother and infant and help pregnant women have satisfactory birth experiences. To determine the efficiency of low‐level electrical stimulation applied by a transcutaneous electrical nerve stimulation (TENS) unit on labor pain relief. A prospective randomized controlled trial study. The study was conducted at the obstetrics and gynecology clinic of a university hospital. The sample included women who volunteered and met necessary criteria for each group: 64 women in the intervention group and 60 women in the control group. A demographic data collection form, visual analog scale (VAS), labor observation form, neonatal observation form, and satisfaction form on TENS application were used as data collection tools. All data were analyzed using a statistical program. Chi‐squared test was used for the descriptive data, t test was used for the quantitative data, and regression analysis was used to determine the effect of independent variables on dependent variables. The VAS score means were calculated as 5.49 ± 0.98 for the intervention group and 7.71 ± 0.64 for the control group. VAS mean of the intervention group were less than the control group, which was statistically significant (p < .05). The duration of labor was 6.54 ± 2.34 hours for the TENS applied group and 8.11 ± 0.27 hours for the control group, and the difference was statistically significant (p < .05). As a result, it was determined that TENS reduces VAS scores regarding labor pain, shortens duration of labor, its efficiency is independent of other variables, and it has no side effects. The low level electrical stimulation TENS unit reduces the VAS scores relating to labor pain, shortens the period of delivery, has no adverse effects, and its efficiency is independent from other variables. With these features, TENS application may reduce negative effects of severe labor pain on the health of the mother and infant and help pregnant women have satisfactory birth experiences.

Long-term outcomes in Ornithine Transcarbamylase deficiency: a series of 90 patients.

BackgroundThe principal aim of this study was to investigate the long-term outcomes of a large cohort of patients with ornithine transcarbamylase deficiency (OTCD) who were followed up at a single medical center.MethodsWe analyzed clinical, biochemical and genetic parameters of 90 patients (84 families, 48 males and 42 females) with OTCD between 1971 and 2011.ResultsTwenty-seven patients (22 boys, 5 girls) had a neonatal presentation; 52 patients had an “intermediate” late-onset form of the disease (21 boys, 31 girls) that was revealed between 1 month and 16 years; and 11 patients (5 boys, 6 girls) presented in adulthood (16 to 55 years). Patients with a neonatal presentation had increased mortality (90% versus 13% in late-onset forms) and peak plasma ammonium (mean value: 960 μmol/L versus 500 μmol/L) and glutamine (mean value: 4110 μmol/L versus 1000 μmol/L) levels at diagnosis. All of the neonatal forms displayed a greater number of acute decompensations (mean value: 6.2/patient versus 2.5 and 1.4 in infants and adults, respectively). In the adult group, some patients even recently died at the time of presentation during their first episode of coma. Molecular analyses identified a deleterious mutation in 59/68 patients investigated. Single base substitutions were detected more frequently than deletions (69% and 12%, respectively), with a recurrent mutation identified in the late-onset groups (pArg40 His; 13% in infants, 57% in adults); inherited mutations represented half of the cases. The neurological score did not differ significantly between the patients who were alive in the neonatal or late-onset groups and did not correlate with the peak ammonia and plasma glutamine concentrations at diagnosis. However, in late-onset forms of the disease, ammonia levels adjusted according to the glutamine/citrulline ratio at diagnosis were borderline predictors of low IQ (p = 0.12 by logistic regression; area under the receiver operating characteristic curve of 76%, p <0.05).ConclusionsOTCD remains a severe disease, even in adult-onset patients for whom the prevention of metabolic decompensations is crucial. The combination of biochemical markers warrants further investigations to provide additional prognostic information regarding the neurological outcomes of patients with OTCD.

174. Liver Fibrosis in Aged OTC-KO Heterozygotes and Successful Correction by AAV8-Mediated Gene Therapy

Ornithine transcarbamylase deficiency (OTCD) is an X-linked disorder of the urea cycle. Affected males are at risk of life threatening elevation of ammonia that can lead to irreversible cognitive impairment, coma and death. Female carriers show variable clinical manifestations depending on the level of X chromosome inactivation skewing. Histological analyses on liver samples from patients with urea cycle disorders found diffuse microvesicular steatosis, nuclear glycogen, and variable portal-to-portal bridging fibrosis. The OTC knockout (KO) model generated in our laboratory through the deletion of exons 2-3 closely mimics the severe neonatal onset form of OTCD in humans. Neonatal male OTC KO pups have elevated plasma ammonia levels due to the absence of OTC expression in the liver, and they inevitably die within 24 hours after birth. Heterozygous females breed normally, have normal plasma ammonia levels, reduced liver OTC enzyme activity, elevated urine orotic acid levels, and in some cases lower body weight compared to wild type (WT) littermates. Sirius red staining on liver samples from heterozygous mice of different ages (6, 12, and 18 months old) showed liver fibrosis in aged (18-month old) OTC-KO heterozygous female mice, similar to a liver sample from a 11-year-old OTCD patient. We then tested if gene therapy could prevent the liver fibrosis. Two-month old OTC-KO heterozygous received a single tail vein injection of a self-complementary AAV8 vector encoding a codon-optimized human OTC gene (hOTCco) at 1×10^10, 3×10^10, and 1×10^11 vector genome copies per mouse. One week following vector treatment, mice in all three vector dose groups had normal urine orotic acid levels which were maintained throughout the study (16 months). Liver samples were harvested from 18 month old treated mice for pathology analysis and compared to age-matched untreated heterozygous mice and WT littermates. All treated mice showed normal liver histology similar to WT, in contrast to the untreated heterozygous animals which had fibrosis throughout the liver. In conclusion, a single injection of AAV8sc-hOTCco vector can prevent liver fibrosis in OTC-KO heterozygous and has great potential for correction of liver fibrosis in OTCD patients.

Clinical features and ACADVL gene mutation spectrum analysis of 11 Chinese patients with very long chain acyl-CoA dehydrogenase deficiency

To investigate the clinical and laboratory features of very long chain acyl-CoA dehydrogenase deficiency ( VLCADD ) and the correlations between its genotype and phenotype. Eleven patients diagnosed as VLCADD of Shanghai Jiaotong University School of Medicine seen from September 2006 to May 2014 were included. There were 9 boys and 2 girls, whose age was 2 d-17 years. Analysis was performed on clinical features, routine laboratory examination, and tandem mass spectrometry (MS-MS) , gas chromatography mass spectrometry (GC-MS) and genetic analysis were conducted. All cases had elevated levels of blood tetradecanoylcarnitine (C14:1) recognized as the characteristic biomarker for VLCADD. The eleven patients were classified into three groups: six cases in neonatal onset group, three in infancy onset group form patients and two in late onset group. Neonatal onset patients were characterized by hypoactivity, hypoglycemia shortly after birth. Infancy onset patients presented hepatomegaly and hypoglycemia in infancy. The two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Six of the eleven patients died at the age of 2-8 months, including four neonatal onset and two infant onset patients, with one or two null mutations. The other two neonatal onset patients were diagnosed since early birth through neonatal screening and their clinical manifestation are almost normal after treatments. Among 11 patients, seventeen different mutations in the ACADVL gene were identified, with a total mutation detection rate of 95.45% (21/22 alleles), including eleven reported mutations ( p. S22X, p. G43D, p. R511Q, p. W427X, p. A213T, p. C215R, p. G222R, p. R450H, p. R456H, c. 296-297delCA, c. 1605 + 1G > T) and six novel mutations (p. S72F, p. Q100X, p. M437T, p. D466Y, c. 1315delG insAC, IVS7 + 4 A > G). The p. R450H was the most frequent mutation identified in three alleles (13.63%, 3/22 alleles), followed by p. S22X and p. D466Y mutations which were detected in two alleles (9.09%, 2/22 alleles). The ACADVL gene mutations were heterozygous in our patients. The mortality of neonatal onset form and infant onset form is much higher than the late onset form patients, suggesting a certain correlation between the genotype and phenotype was found. The earlier diagnosis and treatment of VLCADD are of vital importance for the improvement of the prognosis of the patients.

MYBPC3 Gene Therapy for Neonatal Sarcomeric Cardiomyopathies

Hypertrophic cardiomyopathy (HCM) is mainly characterized by left ventricular hypertrophy (LVH), diastolic dysfunction and increased interstitial fibrosis. HCM has an estimated prevalence in a young adult population of 1:500 and is the leading cause of sudden cardiac death in young athletes. HCM is transmitted in an autosomal-dominant fashion and often caused by mutations in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Most MYBPC3 mutations result in truncated proteins. Findings in humans and in cat/mouse models indicate that haploinsufficiency is the most prevalent HCM mechanism. The presence of double truncating MYBPC3 mutations in infants has been shown to result in severe heart failure and death within 1 year. One of our goals is to prevent the development of the severe cardiac disease phenotype by gene therapy. We developed a mouse model that genetically mimics some human neonatal forms of HCM. The Mybpc3-targeted knock-in (KI) mice carry a homozygous G>A transition in exon 6, resulting in 3 different aberrant mRNAs and proteins.I will present the results of the different gene therapy approaches we performed in this mouse model, which include 5′-trans-splicing, exon skipping and overexpression. The different strategies were evaluated in isolated cardiac myocytes, engineered heart tissue or in vivo in the homozygous KI mice. With the 3 approaches, we provided proof-of-concept studies that paved the way to evaluate the concept in larger animal models and then in severe forms of human neonatal cardioymyopathies.ReferencesGedicke-Hornung, Behrens-Gawlik et al., EMBO Mol Med 2013Mearini, Stimpel et al., Mol Therapy - Nucl Acids 2013Behrens-Gawlik et al., Pflugers Arch - Eur J Physiol 2014Mearini, Stimpel et al., Nat Commun, in press

X-linked adrenoleucodystrophy: diagnosis and progression quantification.

Radiol Bras. 2014 Nov/Dez;47(6):VII–VIII Adrenoleukodystrophy (ALD) is a white matter disease whose bone marrow transplant (BMT) as a therapeutic option is allowed for early diagnosis. Because of the high sensitivity of magnetic resonance imaging (MRI) for the diagnosis of WM lesions, the radiologist is frequently asked to give his/her opinion on the differentiation between a case of hypoxia-related sequelae and evolutive lesions such as leukodystrophy. In general, the clinician can distinguish both groups using anamnesis and neurologic evaluation. However, not always the disease presentation is so clear, white or black, most frequently presenting with different shades of gray, with a very confusing history of loss of abilities, behavior disorders, questionable psychomotor involution and different degrees of disorders potentially attributable to psychiatric diseases acting as confounding factors. In such a situation, the opinion of the radiologist about a brain MRI study is critical for the clinical management. The typical presentation of X-linked ALD in association with adrenal insufficiency symptoms is relatively easy to be diagnosed by a radiologist with experience in central nervous system (CNS) diseases, which increases the responsibility of the professional. Recently, the relevance of the role played by diagnostic imaging was enhanced by good outcomes achieved with the treatment by BMT in early diagnosed cases. Thus, the reading of the excellent article published in the present issue of Radiologia Brasileira, approaching the utilization of diffusion tensor imaging and developed by researchers at Hospital das Clinicas of Universidade Federal do Parana, becomes even more interesting. ALD is a genetic disease classified in the group of peroxisomal disorders whose most frequent presentation is that of a recessive X-linked disease. Thus, women, although being carriers of the gene which fundamentally affects men, are uncommonly affected, in such cases presenting with a variant of the disease. Despite the existence of neonatal and adult-onset forms of the disease, classic childhood adrenoleukodystrophy is more frequent and severe. Such a disease presentation was made widely known by the movie “Lorenzo’s Oil” (USA, 1992), directed by George Miller, where it was brightly described with emphasis not only on the patient’s suffering but also on the repercussion of the progressive deterioration of the child on the family. Additionally, one should consider the current availability of access to information through the me-

Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain.

Although crystal structures of various voltage-gated K(+) (Kv) and Na(+) channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K(+) channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the IM current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd(2+) profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd(2+) bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4-S1 Cd(2+) bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.

Adult-onset autosomal dominant centronuclear myopathy due to BIN1 mutations.

Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.

G.P.38: Adult-onset autosomal dominant centronuclear myopathy due to BIN1 mutations

Centronuclear myopathies (CNM) are congenital muscle disorders characterized by an increased number of muscle fibers with central nuclei. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive CNM with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). However, the genetic basis of the disease is not known for all cases. Here we characterize a novel CNM entity. We established a homogeneous cohort of nine patients from five families with dominant CNM without mutations in DNM2. All patients presented with a mildly progressive adult-onset myopathy and had similar histological and ultrastructural features involving type I fiber predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all five families: two missense mutations in the N-terminal amphipathic helix, and three single nucleotide deletions involving the loss of the stop codon. Amphiphysin 2 (encoded by BIN1) induces membrane tubulation and is implicated in excitation-contraction coupling (ECC) at the T-tubules. We confirmed mutation segregation with the disease in the dominant families, and we complemented the molecular diagnosis by functional analyses. Both missense mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Additional immunolabeling experiments revealed abnormal central accumulation of dynamin 2 and caveolin-3, and general membrane alterations of the triad, the sarcolemma, and the basal lamina. In conclusion, we identified BIN1 as a novel gene for dominant adult-onset CNM. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant CNM and that both disorders involve different pathomechanisms.

G.P.183: TK2 mutation: An expanding clinical phenotype

Background Thymidine kinase 2 (TK2) mutation, a rare mitochondrial DNA depletion syndrome, was first described as an aggressive infantile myopathy with hypotonia, developmental regression, and respiratory failure leading to early death. Aim We describe 3 patients who presented to us with distinct clinical phenotypes. Patients Patient 1 had a classical infantile onset with poor head control, feeding difficulties and lethargy. An older sister presented with similar symptoms and died at the age of 15 months after having a muscle biopsy which showed respiratory chain abnormalities. His symptoms progressed rapidly and he subsequently died at the age of 18 months. His CK was raised and his muscle biopsy also showed evidence of respiratory chain enzyme abnormalities. Genetic testing confirmed a diagnosis of TK2 mutation. Patient 2 presented with a severe neonatal form, requiring ventilatory support from birth. She had no respiratory effort and was significantly hypotonic. Her CK was elevated and a muscle biopsy showed mitochondrial cytopathy. She died at the age of two months when her care was withdrawn. Further investigations done confirmed a common mutation in the TK2 gene. Patient 3 presented at the age of 4 with a less aggressive onset, consisting of mild facial and neck flexion weakness with proximal myopathy. Her CK was slightly raised and a muscle biopsy showed mitochondrial myopathy. She is now predominantly wheelchair bound at the age of 17, has a gastrostomy in situ, and is established on nocturnal non-invasive ventilation. She has been found to be homozygous for a novel variant in the TK2 gene, which has not been described previously in literature. Conclusion We would like to highlight that the clinical phenotype for TK2 mutation is variable and can range from mild to very severe. In milder forms of this disorder, early multidisciplinary input should be sought to ensure that mobility, feeding and respiratory difficulties are all managed appropriately.

G.P.265: Nebulin-related nemaline myopathy: Clinical and histopathological spectrum in Argentinean patients

Nebulin (NEB) is a sarcomeric structural protein. Mutations in NEB genes particularly cause autosomal recessive nemaline myopathy (NM) and core-rod myopathy. Clinical presentations range from a severe neonatal form to a mild adult-onset form as part of the different diagnosis of distal myopathy. We describe the clinical, histopathological, and molecular findings of three patients with different phenotypes of NEB gene mutations. Case 1: A 5-year-old boy with distal finger retractions, hypotonia, and motor development delay since birth with mainly distal general weakness and hyperlaxity. At age 1 he developed progressive respiratory difficulties needing ongoing invasive ventilation by tracheostomy. He was never able to walk. Spine surgery was performed at 4years. Cognitive development was normal. Quadriceps muscle biopsy showed NM consisting of rod structures, type 1 predominance and atrophy. Exome sequencing showed mutations in exon 139 and 172. Case 2: A 2.5-year-old girl presenting with motor developmental delay, hypomimia, and hypotonia since birth. At 18months of age she walked independently and steppage gait was noticed. She is still improving. Deltoid biopsy showed NM consisting of rod structures and type 1 predominance. Exome sequencing showed mutations in intron 12 and exon 150. Case 3: A 22-year-old girl had frequent falls since she started to walk at 1year, linked to distal hyperlaxity, as well as distal weakness, arreflexia, and steppagge gait. Slow symptom progression was seen. EMG ruled out neuropathic disease and muscle biopsy showed rod and core structures with type 1 predominance. Exome sequencing showed mutations in exon 129 and exon 74 to exon144. Conclusion: NEB-related NM has a broad clinical spectrum particularly including congenital and distal myopathy forms that may mimic a neurogenic process. Our three patients presented with predominantly distal weakness and muscle biopsies showed rod or core-rod myopathies.