Histone deacetylase inhibitors for the epigenetic therapy of proximal spinal muscular atrophy

Die proximal spinale Muskelatrophie (SMA) ist charakterisiert durch eine fortschreitende Degeneration der a-Motoneuronen in the Vorderhörnern des Rückenmarks. Sie wird durch den homozygoten funktionellen Verlust des survival motor neuron Gens 1 (SMN1) verursacht. Allerdings konnte gezeigt werden, dass alle SMA-Patienten zumindest über eine Kopie des SMN2-Genes verfügen. Während SMN1 ausschließlich Volllängen-Transkripte (FL-SMN) produziert, sind 90% aller SMN2 Transkripte alternativ gespleißt. Ihnen fehlt das Exon 7 (SMN2D7), was zu einem instabilen Protein führt. Zwar reicht die Menge and SMN2 Volllängenprotein nicht aus um für den Verlust von SMN1 zu kompensieren, andererseits beeinflusst es jedoch den SMA Phänotyp: Über je mehr SMN2 Kopien ein SMA Patient verfügt, desto milder ist der Krankheitsverlauf. Es konnte gezeigt werden, dass die Verwendung von Histon Deacetylase Inhibitoren (HDACi) einerseits das SMN2 Gen aktivieren und darüberhinaus auch sein Spleißmuster korregieren. Eine Pilotstudie mit SMA-Patienten, die mit dem HDACi VPA behandelt wurden, ergab jedoch ein recht unterschiedliches Bild. In knapp einem Drittel der Patienten ist die Menge an FL-SMN2 im Blut, wie erhofft, angestiegen. In einem weiteren Drittel jedoch konnte keine Änderung festgestellt werden, wohingegen im letzten Drittel die Menge an FL-SMN2 durch VPA-Gabe sogar reduziert war. Die vorliegende Arbeit beschäftigt sich mit der Suche nach den Ursachen, aufgrund derer SMA-Patienten nicht positiv auf VPA reagieren. Von allen SMA-Patienten, die an der Pilot-Studie teilgenommen hatten, wurden Fibroblasten Linien aus Hautstanzen etabliert. Es konnte gezeigt werden, dass in mehr als 60% der Fälle beide Gewebe, Blut wie Fibroblasten, gleichermaßen auf VPA ansprachen. Um zu verstehen, warum SMN2 nicht durch VPA in Non-Respondern aktiviert wird, wurden Chromatin-Immunopräzipitationen (ChIP) durchgeführt. Es zeigte sich, dass das SMN Protein in Non-Respondern unter VPA-Behandlung nicht ansteigt, da VPA nicht zu einer SMN2 Promotor Hyperacetylierung führt. Um die Frage nach den Gründen hierfür zu beantworten, wurde die Transkriptome von Pos- und Non-Responder Fibroblasten mittels Mikro-Array verglichen. Die Auswertung der Daten zeigte, dass in den Non-Respondern kein einziges Transkript differentiell unter VPA-Behandlung exprimiert wurde. Interessanterweise wurden lediglich neun Gene gefunden, die signifikant unterschiedlich zwischen unbehandelten Pos- and Non-Respondern exprimiert wurden. Basierend auf publizierten Daten wurden die Gene Cluster of Differentiation (CD36), IGF-binde Protein 5 (IGFBP5), Retinoic Acid Receptor b (RARb) und Transforming Growth Factor a (TGFa) für weitere Experimente ausgewählt. CD36 und RARb sind beide in Non-Respondern höher exprimiert, wohingegen höherer Menge von IGFBP5 und TGFa in Pos-Respondern gefunden wurden. Da CD36 ein Fettsäuretransporter ist, wurden ebenfalls massenspektroskopische Versuche zur Aufnahme und Metabolisierung von VPA durchgeführt. Eine differentielle Verstoffwechslung von VPA als Ursache für das Auftreten Non-Respondern konnte jedoch ausgeschlossen werden. Ausgehend von diesen Daten wurde die generelle Fettsäureaufnahme von Fibroblasten verglichen. Diese Daten lassen vermuten, dass CD36 zumindest in Fibroblasten eher als Fettsäureexporter denn als -importer fungiert. Ferner konnten wir den HDACi LBH589 und sein strukturell nah verwandtes Derivat JnJ-26481585 als potentielle SMA-Therapeutika identifizieren, die einem enormen Effekt auf die SMN-Protein Menge haben. So stieg die SMN Menge bis zu 10-fach bei LBH589 Konzentration von 400 nM beziehungsweise 1 uM an. Ein höherer Anstieg wurde bis dato noch nicht publiziert. ChIP-Analyse des SMN2 Promoters, sowie die Messung der SMN2 Promotoraktivität in einer Reporterzelllinie, zeigten, dass am SMN2 Promoter HDAC-Inhibition und Promoter-Aktivität in einer 1:1 Stöichiometrie korrelieren. Es wurde eine EC50 von 108 nM LBH589 bestimmt. Auf RNA-Ebene konnten wir zeigen, dass LBH589 einerseits die SMN Expression steigert, aber auch das Spleißmuster durch Hoch-Regulation des Spleißfaktors hTRA2-b1, welcher den Einbezug des SMN2 Exons 7 fördert, revertiert. Mittels Präzipitations-Experimenten konnten wir zeigen, dass die Ubiquitinylierung von SMN stark reduziert ist. Ursächlich hierfür ist vermutlich eine verstärkte SMN-Komplex Bildung, die dann zu einer Anreicherung von SMN im Laufe der Zeit führt. Wir konnten zeigen, dass LBH589 in humanen NSCs wie auch in MEFs von SMA-Mäusen SMN Protein hochreguliert. Abschließend wurden LBH589 subkutan in Mäuse injiziert um zu untersuchen, ob sich eine in vivo Untersuchung von LBH589 anbietet. Die Analyse von Gewebeextrakten aus dem ZNS zeigte, dass die Menge an SMN Protein im Gehirn steigt und gleichermaßen auch die Histonacetylierung zunahm. Daher sollten LBH589, bzw. auch JnJ-26481585, in einer größer angelegten Studie im SMA-Tiermodell untersucht werden

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

Autosomal-Recessive Mutations in MESD Cause Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI

Exome sequencing identifies Laing distal myopathy MYH7 meutation in a Roma family previously diagnosed with distal neuronopathy

We describe a Hungarian Roma family originally investigated for autosomal dominant distal muscular atrophy. The mother started toe walking at 3 years and lost ambulation at age 27. Her three daughters presented with early steppage gait and showed variable progression. Muscle biopsies were nonspecific showing myogenic lesions in the mother and lesions resembling neurogenic atrophy in the two siblings. To identify the causative abnormality whole exome sequencing was performed in two affected girls and their unaffected father unexpectedly revealing the MYH7 mutation c.4849_4851delAAG (p.K1617del) in both girls reported to be causative for Laing distal myopathy. Sanger sequencing confirmed the mutation in the affected mother and third affected daughter. In line with variable severity in Laing distal myopathy our patients presented a more severe phenotype. Our case is the first demonstration of Laing distal myopathy in the Roma and the successful use of whole exome sequencing in obtaining a definitive diagnosis in ambiguous cases. (C) 2013 Elsevier B.V. All rights reserved

A Paucisymptomatic Neuromuscular Disease Mimicking Type III 5q-SMA With Complex Rearrangements in the SMN Gene

Spinal muscular atrophy is an autosomal-recessive neuromuscular disorder, causing progressive proximal weakness and atrophy of the voluntary muscles. More than 96% of the spinal muscular atrophy patients show a homozygous absence of exons 7 and 8, or exon 7 only, in SMN1, the telomeric copy of the SMN gene. We report a young male patient with neurogenic symptoms and sparse muscle fiber atrophy, suggestive of a mild form of type III spinal muscular atrophy. He was found to be a carrier of intragenic mutations in both copies of the SMN gene, exhibiting a homozygous duplication of exons 7 and 8 in SMN1 and a homozygous deletion of exon 8 as well as a heterozygous deletion of exon 7 in SMN2. However, an intact full-length SMN1 complementary deoxyribonucleic acid was identified, and SMN protein levels in a muscle specimen were identical to that of a healthy control, formally excluding the diagnosis of spinal muscular atrophy III

The phenotypic spectrum of duplication 5q35.2-q35.3 encompassing NSD1: Is it really a reversed sotos syndrome?

Loss-of-function mutations of NSD1 and 5q35 microdeletions encompassing NSD1 are a major cause of Sotos syndrome (Sos), which is characterized by overgrowth, macrocephaly, characteristic facies, and variable intellectual disability (ID). Microduplications of 5q35.2-q35.3 including NSD1 have been reported in only five patients so far and described clinically as a reversed Sos resulting from a hypothetical gene dosage effect of NSD1. Here, we report on nine patients from five families with interstitial duplication 5q35 including NSD1 detected by molecular karyotyping. The clinical features of all 14 individuals are reviewed. Patients with microduplications including NSD1 appear to have a consistent phenotype consisting of short stature, microcephaly, learning disability or mild to moderate ID, and distinctive facial features comprising periorbital fullness, short palpebral fissures, a long nose with broad or long nasal tip, a smooth philtrum and a thin upper lip vermilion. Behavioral problems, ocular and minor hand anomalies may be associated. Based on our findings, we discuss the possible etiology and conclude that it is possible, but so far unproven, that a gene dosage effect of NSD1 may be the major cause. © 2013 Wiley Periodicals, Inc.status: publishe

Identification and functional analysis of BICD2, a causal gene of autosomal dominant SMA

<p>Spinal muscular atrophies (SMAs) are characterized by degeneration of spinal motor neurons and muscle weakness. Autosomal recessive SMA is the most common form and is caused by homozygous deletions/mutations of the SMN1 gene. Additionally, dominant inheritance SMA families have been reported, for most of them the causal gene remains unknown. The starting point of the current study was a Dutch family (1) with non-progressive SMA, autosomal dominant (SMALED2; MIM #615290 AD) pattern of inheritance, unaltered SMN1, and congenital contractures. Linkage analysis and whole exome sequencing were performed and led to the identification of a heterozygous missense mutation (c.320C>T, p.Ser107Leu) in the BICD2 gene. BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D. BICD2 was sequenced in twenty additional families with SMALED2 finding additional mutations (p. Asn188Thr; p.Ala535Val; p.Thr703Met; p.Arg747Cys), and a rare variant p.90 Lys>Arg (VS frequency 0.4%) (5, 6). The patients differ in the severity of the symptoms, although some of those mutations lay on the same protein region. Overexpression of the mutant BICD2 cDNAs in HeLa cells was performed observing fragmentation of the Golgi Apparatus (GA). Fibroblast cell lines were derived from the patients carrying the mutations p.Thr703Met (severe phenotype) and p.Asn188Thr. A severe GA fragmentation was observed in the fibroblast cells from the patient p.Thr703Met suggesting a possible correlation between the grade of fragmentation and the severity of the disease. The integrity of the GA depends on the microtubule network and BICD2 is implicated in transport along microtubules. The microtubules of the fibroblasts carrying the p.Thr703Met were stained and alteration in their pattern was observed. BICD2 interacts with the small GTPase Rab6a (2); which plays an essential role in Golgi transport. Rab6a Pull Down analysis showed no alteration in the interaction of the BICD2 mutants and Rab6a. In order to understand the molecular mechanisms of the other mutations in BICD2, protein stability assays and additional interacting studies are being performed.</p> <p> </p

Mutations in SEC24D, Encoding a Component of the COPII Machinery, Cause a Syndromic Form of Osteogenesis Imperfecta

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205*) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish)