Whole-genome sequencing reveals a coding non-pathogenic variant tagging a non-coding pathogenic hexanucleotide repeat expansion in C9orf72 as cause of amyotrophic lateral sclerosis

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) has a familial cause in 10% of patients. Despite significant advances in the genetics of the disease, many families remain unexplained. We performed whole-genome sequencing in five family members from a pedigree with autosomal-dominant classical ALS. A family-based elimination approach was used to identify novel coding variants segregating with the disease. This list of variants was effectively shortened by genotyping these variants in 2 additional unaffected family members and 1500 unrelated population-specific controls. A novel rare coding variant in SPAG8 on chromosome 9p13.3 segregated with the disease and was not observed in controls. Mutations in SPAG8 were not encountered in 34 other unexplained ALS pedigrees, including 1 with linkage to chromosome 9p13.2–23.3. The shared haplotype containing the SPAG8 variant in this small pedigree was 22.7 Mb and overlapped with the core 9p21 linkage locus for ALS and frontotemporal dementia. Based on differences in coverage depth of known variable tandem repeat regions between affected and non-affected family members, the shared haplotype was found to contain an expanded hexanucleotide (GGGGCC)n repeat in C9orf72 in the affected members. Our results demonstrate that rare coding variants identified by whole-genome sequencing can tag a shared haplotype containing a non-coding pathogenic mutation and that changes in coverage depth can be used to reveal tandem repeat expansions. It also confirms (GGGGCC)n repeat expansions in C9orf72 as a cause of familial ALS

Adenylosuccinate lyase deficiency : study of mutations and pathophysiologic mechanisms

L’adénylosuccinante lyase (ADSL) est une enzyme qui intervient deux fois dans la synthèse des nucléotides puriques : une première fois pour scinder le succinylaminoimidazole carboxamide ribotide (SAICAR) en aminoimidazole carboxamide ribotide (AICAR) et fumarate ; une seconde fois pour scinder l’adénylosuccinate (S-AMP) en AMP et fumarate. Sa déficience se caractérise par l’accumulation, dans le liquide céphalo-rachidien et les urines, des produits de déphosphorylation des deux substrats de l’enzyme, le SAICARriboside et le succinyl-adénosone (S-Ado). Les enfants atteints de cette déficience présentent essentiellement une atteinte neurologique de sévérité variable. Vu cette importante variabilité, la première partie de mon travail visait à établir des corrélations génotype-phénotype. Pour ce faire, l’ADSL a été exprimée dans un modèle bactérien et les caractéristiques cinétiques des enzymes normales et mutées ont été étudiées. Il en ressort que les protéines mutées peuvent être subdivisées en deux groupes selon leur thermostabilité. D’une part, des mutations thermolabiles qui diminuent l’activité ADSL en parallèle avec les deux substrats, S-AMP et SAICAR. Les patients homozygotes pour ces mutations ont des rapports S-Ado/ SAICARriboside ~ 1 et sont profondément arriérés. D’autre part, des mutations thermostables qui, quant à elles, diminuent beaucoup plus l’activité avec le S-AMP qu’avec le SAICAR. Les patients homozygotes pour ces mutations ont des rapports S-Ado/SAICARriboside ~ 4 et sont beaucoup moins arriérés. Ces résultats suggèrent que la lésion génétique de l’ADSL détermine le rapport des activité S-AMP/SAICAR, qui à son tour détermine le rapport S-Ado- SAICARriboside et l’état mental des patients. La seconde partie de mon travail avait pour objectif d’élucider les mécanismes physiopathologiques à la base de cette maladie neurologiques. Deux hypothèses ont été principalement étudiées. La première est dérivée de l’observation d’une corrélation entre le rapport des succinylpurines et la gravité des symptômes cliniques et postule un effet neurotoxique du SAICARriboside qui serait contrecarré par la S-Ado. Dans ce cadre, trois approches ont été investiguées, à savoir (1) un effet des succinylpurines sur le récepteur ionotropique du glutamate de type N-méthyl-D-aspartate (NMDA), (2) une inhibition de l’hydrolyse de N-acétyl-aspartate (NAA), ce qui occasionnerait une accumulation de ce composé comme dans la maladie de Canavan, et (3) un effet des succinylpurines durant le développement neuronal. Ce travail a permis d’exclure les deux premières possibilités et laisse entrevoir une toxicité des succinylpurines sur des neurones non-différentiés, mais ce point doit être encore poursuivi. La seconde hypothèse postule la possibilité d’une carence des métabolites qui sont normalement formés en aval de l’ADSL, c’est-à-dire des nucléotides puriques et particulièrement ceux de l’adénine. Pour ce faire, j’ai mesuré la concentration des nucléotides puriques dans le seul modèle de cellules déficientes dont nous disposons, à savoir des fibroblastes de patients. Ce modèle n’est cependant pas optimal car la déficience n’y est que partielle. Pour tenter de rendre ces cellules plus dépendantes de leur synthèse de novo, elles ont été cultivées dans un milieu dépourvu de bases utilisées par la voie de récupération. Cependant, même dans ces conditions, aucune modification de la concentration des pruines n’a pu être mise en évidence. Cette étude n’a pas permis d’élucider le mécanisme à la base de la déficience mentale chez ces enfants, qui reste mystérieux comme dans de nombreuses maladies métaboliques. Néanmoins, comme décrit dans la dernière partie, il propose un nouveau concept concernant la déphosphorylation des substrats de l’ADSLAdenylosuccinate lyase (also termed adenylosuccinase, ADSL) catalyses two distinct reactions in purine nucleotide synthesis, and hence its deficiency is characterised by the accumulation in body fluids of SAICAriboside and succinyladenosine (S-Ado), the dephosphorylated derivatives of the two substrates of the enzyme SAICAR and S-AMP, respectively. Clinically, ADSL-deficient patients display mainly neurological symptoms, with a widely variable degree of psychomotor retardation that seems to correlate with the S-Ado : SAICAriboside ratio in their body fluids. In view of this wide variability, the first part of this work attempted to establish genotype-phenotype correlations in ADSL-deficient patients (Chapter 4). Recombinant mutated ADSL enzymes were expressed and their properties studied. A first group of mutations leads to the production of thermolabile proteins with activities decreased in parallel for both substrates, SAICAR and S-AMP, or more for SAICAR than for S-AMP. ADSL activities measured in fibroblasts of patients homozygous for one of these mutations, R426H, are in agreement with the activities of the corresponding recombinant mutated protein. These patients have a S-Ado to SAICAriboside ratio of ~1, and are profoundly retarded. The second group of mutations leads to the production of thermostable proteins, though with a much more pronounced decrease in the activity toward S-AMP than toward SAICAR. Fibroblasts of patients homozygous for one of these mutations, R303C, also display ADSL activities similar to those of the recombinant mutated protein. These patients have a S-Ado to SAICAriboside ratio between 3 and 4 and are mildly retarded. These results suggest that the nature of the genetic lesion of ADSL determines the ratio of its activities with S-AMP versus SAICAR, which in turn defines the S-Ado:SAICAriboside ratio and the patients mental status. The second part of this work was devoted to the elucidation of the pathophysiologic mechanisms of this neurological disease. Two main hypotheses were tested. Since the ratio of succinylpurines correlates with the severity of the phenotype, we first privileged the hypothesis that SAICAriboside is the neurotoxic compound and, that S-Ado counteracts its noxious effect (Chapter 5). Along this line, three different approaches were considered: succinylpurines could (1) interact with the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA), (2) inhibit the hydrolysis of N-acetyl-aspartate (NAA) and provoke an accumulation of this compound as in Canavan disease, (3) be toxic only during brain maturation. Results show that succinylpurines do not interfere with NMDA receptors or with NAA metabolism, but indicate that they could be toxic on undifferentiated neurons. The second pathophysiological hypothesis involves the possibility that the ADSL defect could lead to deficiency of purine, particularly adenine nucleotides (Chapter 6). Therefore, purine nucleotide concentrations were measured in ADSL-deficient patients fibroblasts with very low residual ADSL activity (less than 10% of control). In order to render these cells more dependent on purine de novo synthesis, they were also cultured in medium depleted in purine bases needed for the salvage pathway. Nevertheless, in comparison with control fibroblasts, no reduction of the nucleotide concentrations in the ADSL-deficient fibroblasts could be detected. Although this study failed to elucidate the cause of the neurologic symptoms in ADSL deficiency, it has led to new insights in the mechanisms involved in the dephosphorylation of the two substrates of ADSL. ...Thèse de doctorat en sciences biomédicales (SBIM 3)--UCL, 200

Antenatal diagnosis of cardio-facio-cutaneous syndrome: Prenatal characteristics and contribution of fetal facial dysmorphic signs in utero. About a case and review of literature.

Antenatal diagnosis of cardio-facio-cutaneous syndrome: prenatal characteristics and contribution of fetal facial dysmorphic signs in utero. This paper is a case study and review of literature. "RASopathies" is the term coined for a group of genetic diseases that share modulation inside the MAPKinase pathway. Mutations inside the coding sequence of any of these genes may be responsible for the upregulation of the RAS pathway, leading on the clinical level to Type 1 Neurofibromatosis (NF1), Noonan syndrome (NS), Costello syndrome (CS), Multiple Lentigines, Loose Anagen Hair syndrome, Cardio-Facio-Cutaneous syndrome (CFCS), and, more recently, Legius syndrome. While the postnatal presentation of this group is well-known, prenatal findings are less well recognized. The presence of a RASopathy during the prenatal period can be suspected on account of non-specific abnormalities: polyhydramnios, cystic hygroma or high nuchal translucency, macrosomia with proportionate short long bones, macrocephaly, renal, lymphatic, or cardiac defects. The current case report underlines the characteristic dysmorphic facial features on 3D-ultrasound (hypertelorism, down-slanting palpebral fissures, a long and marked philtrum, and low-set posteriorly rotated ears) that allow for a "RASopathy" to be postulated. After detecting a copy number variation (CNV) absence on a CGH array, we performed a RASopathy gene panel analysis, which identified a so-far unreported heterozygous de novo mutation in the BRAF gene (namely NM_004333.4 : c.1396 G > C ; p.Gly466Arg). Genetic counseling has, therefore, focused on the diagnosis of a RASopathy and predictable phenotype of CFCS, a distinct entity characterized by an increased risk of intellectual disability and early-onset feeding problems. We suggest that a more detailed prenatal facial evaluation should be performed in fetuses presenting high nuchal thickness, heart defects, or unusual findings, along with the absence of a CNV on a CGH array. Due to the dysmorphic facial features, targeted RASopathy genes are presumed to likely to be responsible for NS, CFCS, and CS

Approaches to homozygosity mapping and exome sequencing for the identification of novel types of CDG

In the past decade, the identification of most genes involved in Congenital Disorders of Glycosylation (CDG) (type I) was achieved by a combination of biochemical, cell biological and glycobiological investigations. This has been truly successful for CDG-I, because the candidate genes could be selected on the basis of the homology of the synthetic pathway of the dolichol linked oligosaccharide in human and yeast. On the contrary, only a few CDG-II defects were elucidated, be it that some of the discoveries represent wonderful breakthroughs, like e.g, the identification of the COG defects. In general, many rare genetic defects have been identified by positional cloning. However, only a few types of CDG have effectively been elucidated by linkage analysis and so-called reverse genetics. The reason is that the families were relatively small and could—except for CDG-PMM2— not be pooled for analysis. Hence, a large number of CDG cases has long remained unsolved because the search for the culprit gene was very laborious, due to the heterogeneous phenotype and the myriad of candidate defects. This has changed when homozygosity mapping came of age, because it could be applied to small (consanguineous) families. Many novel CDG genes have been discovered in this way. But the best has yet to come: what we are currently witnessing, is an explosion of novel CDG defects, thanks to exome sequencing: seven novel types were published over a period of only two years. It is expected that exome sequencing will soon become a diagnostic tool, that will continuously uncover new facets of this fascinating group of diseases.Fil: Matthij, Gert. Katholikie Universiteit Leuven; BélgicaFil: Rymen, Daisy. Katholikie Universiteit Leuven; BélgicaFil: Bistue Millon, Maria Beatriz. Katholikie Universiteit Leuven; Bélgica. Universidad Nacional de Cordoba. Facultad de Medicina. Centro de Estudios de las Metabolopatías Congénitas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Souche, Erika. Katholikie Universiteit Leuven; BélgicaFil: Race, Valérie. Katholikie Universiteit Leuven; Bélgic

Abnormal Cartilage Development and Altered N-Glycosylation in Tmem165-Deficient Zebrafish Mirrors the Phenotypes Associated with TMEM165-CDG

The Congenital Disorders of Glycosylation (CDG), a group of inherited diseases characterized by defective glycosylation, encompass a wide range of defects including glycosyltransferases, glycosidases, nucleotide-sugar transporters, as well as proteins involved in maintaining Golgi architecture, pH, and vesicular trafficking. Mutations in a previously undescribed protein, TMEM165, were recently shown to cause a new form of CDG, termed TMEM165-CDG. TMEM165-CDG patients exhibit cartilage and bone dysplasia and altered glycosylation of serum glycoproteins. We utilized a morpholino knockdown strategy in zebrafish to investigate the physiologic and pathogenic functions of TMEM165. Inhibition of tmem165 expression in developing zebrafish embryos caused craniofacial abnormalities, largely attributable to fewer chondrocytes. Decreased expression of several markers of cartilage and bone development suggests that Tmem165 deficiency alters both chondrocyte and osteoblast differentiation. Glycomic analysis of tmem165 morphants also revealed altered initiation, processing, and extension of N-glycans, paralleling some of the glycosylation changes noted in human patients. Collectively, these findings highlight the utility of zebrafish to elucidate pathogenic mechanisms associated with glycosylation disorders and suggest that the cartilage and bone dysplasia manifested in TMEM165-CDG patients may stem from abnormal development of chondrocytes and osteoblasts.status: publishe

Frequency of C9orf72 repeat expansions in amyotrophic lateral sclerosis: a Belgian cohort study

We determined the frequency of C9orf72 repeat expansions in a large cohort of Belgian patients with familial (fALS) and sporadic (sALS) amyotrophic lateral sclerosis (ALS). In total, 119 patients with fALS from 62 kindreds, 471 patients with sALS, and 384 control subjects were included. A C9orf72 repeat expansion was found in 32 of 62 fALS pedigrees (51.6%), in 45 of 471 patients with sALS (9.6%), but in none of the control subjects. Compared with fALS of unknown etiology or fALS caused by mutations in other ALS-causing genes, C9orf72 repeat expansion carriers had a later age at onset (57.3 vs. 51.4 years; p = 0.0061), a higher proportion of bulbar onset (31.9% vs. 12.5%, p < 0.0001), and a reduced survival (29.4 vs. 67.7 months, p = 0.0003). In the sALS cohort, there were no significant differences in these disease characteristics between the C9orf72 repeat expansion carriers and the noncarriers. C9orf72 repeat expansions are a frequent cause of ALS in Belgium, and also in sALS patients. These results might justify genetic testing of C9orf72 in all ALS patients.status: publishe

COG5-CDG: Expanding the clinical spectrum

Background: The Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350-6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia. Methods. CDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis. Results and conclusion. We identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level. © 2012 Rymen et al. licensee BioMed Central Ltd.SCOPUS: ar.jinfo:eu-repo/semantics/publishe