0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Original Contribution |

Mutations of the ANG Gene in French Patients With Sporadic Amyotrophic Lateral Sclerosis FREE

Agathe Paubel, MD; Jeremy Violette, MD; Maïté Amy, PhD; Julien Praline, MD; Vincent Meininger, MD, PhD; William Camu, MD, PhD; Philippe Corcia, MD, PhD; Christian R. Andres, MD, PhD; Patrick Vourc’h, PhD; French Amyotrophic Lateral Sclerosis (ALS) Study Group
[+] Author Affiliations

Author Affiliations: Laboratoire de Biochimie et Biologie Moléculaire (Drs Paubel, Violette, Andres, and Vourc’h) and Centre SLA (Drs Praline and Corcia), CHRU de Tours, and INSERM U930, Université François-Rabelais (Dr Amy, Praline, Corcia, Andres, and Vourc’h), Tours, Fédération des Maladies du Système Nerveux, Centre référent SLA (Dr Meininger), Paris, and Centre SLA, CHRU de Montpellier (Dr Camu), Montpellier, France.


Arch Neurol. 2008;65(10):1333-1336. doi:10.1001/archneur.65.10.1333.
Text Size: A A A
Published online

Background  Mutations in the angiogenin gene, ANG, have been associated recently with familial and sporadic forms of amyotrophic lateral sclerosis (ALS). However, the cellular and molecular mechanisms that link ANG, a multidomain protein, to ALS are still unknown.

Objective  To assess the frequency of ANG gene mutations in 855 French patients with sporadic ALS.

Design  We analyzed by direct sequencing the full coding region of the ANG gene in a cohort of French patients with sporadic ALS. The clinical characteristics of patients carrying ANG mutations are detailed.

Setting  French ALS Study Group.

Patients  A total of 855 patients with sporadic ALS.

Main Outcome Measures  Results of genetic analyses.

Results  We observed a previously identified mutation (pI46V) in 2 patients with ALS without a known family link and found a novel mutation (pR121H) in 1 patient who developed ALS with rapid progression. We did not observe an association between patients with ALS and the rs11701 polymorphism, as previously reported in certain ALS populations of other ethnic origins.

Conclusion  Overall, our findings support the implication of ANG gene mutations as a rare but widespread cause of ALS.

Figures in this Article

Several studies14 on the vascular endothelial growth factor gene have suggested a link between angiogenesis and the pathogenesis of amyotrophic lateral sclerosis (ALS). Recently, Greenway et al5 identified 7 missense mutations in a second hypoxia responsive gene, the ANG gene (OMIM 105850), in 15 Irish and Scottish patients with familial ALS or sporadic ALS. The ANG gene, located in 14q11.2, encodes angiogenin, a 14.1-kDa enzyme that belongs to the pancreatic ribonuclease A superfamily.6 Angiogenin is synthesized as a preprotein that contains a signal peptide of 24 amino acids. The mature angiogenin protein contains a putative receptor binding region, a domain implicated in immunomodulation, a nuclear localization sequence, and a catalytic site responsible for a low ribonuclease activity. It is expressed in several cells, including glial cells and motoneurons in the spinal cord and dorsal root ganglia.7 Angiogenin, whose expression is induced by hypoxia, mediates neovascularization and has been recently involved in neurite pathfinding.8,9 Thus, we analyzed the coding region of the ANG gene in a large French cohort of 855 patients with sporadic ALS.

Clinical data and blood samples were obtained from 855 patients with sporadic ALS. The ALS was diagnosed according to the El Escorial World Federation criteria by physicians in the French ALS Study Group centers. Clinical data comprised the sex, site of onset, age at onset, and duration of ALS. Control subjects were spouses of patients, who were matched with patients by age and sex. All patients and controls gave written informed consent.

DNA was extracted from peripheral blood samples using a standard procedure. The unique exon of the ANG gene was amplified by polymerase chain reaction using the following foward and reverse primers: 5′-tgttcttgggtctaccacacc-3′ and 5′-aatggaaggcaaggacagc-3′, respectively. Bidirectional sequencing (ABI 3130xl sequencer; Applied Biosystems, Courtaboeuf, France) and analysis (Seqscape software; Applied Biosystems) were then performed. Mutations were confirmed by reamplification and sequencing. We also analyzed by sequencing the ANG coding region in 234 controls. Modelization of the angiogenin variant was performed using the crystal structure of the normal angiogenin protein (PDB ID 1b1i), the Swiss model server (http://swissmodel.expasy.org/), and the molecular visualization system Pymol (http://pymol.sourceforge.net/).

We found coding mutations in the ANG gene in 3 patients with sporadic ALS (Figure, A-C) (estimated frequency, 0.35%; 95% confidence interval, 0.07%-1.02%). A novel mutation was found in 1 male patient with sporadic ALS. This mutation, which is a heterozygous substitution G434A, changes the amino acid (Arg121His) in the C-terminal region of the mature angiogenin protein (Figure, A). The modelization of the variant angiogenin suggests that histidine 121 may affect the environment of the catalytic center, in that a shorter and cyclic amino acid replaces a long and linear one (Figure, D and E). We also found the heterozygous substitution A208G (Ile46Val) in 2 patients with sporadic ALS without a known family link (Figure, B and C). No mutation was observed in a control population of 234 healthy individuals.

Place holder to copy figure label and caption
Figure.

ANG mutations and 3-dimensional structure of angiogenin. DNA sequences of the patients with amyotrophic lateral sclerosis with G434A (A) and A208G (B and C) mutations. Three-dimensional x-ray diffraction structure of the normal human angiogenin protein (PDB ID 1b1i) (D). Model of the angiogenin variant Arg121His (E).

Graphic Jump Location

We confirmed the presence of the rs11701 single- nucleotide polymorphism in the ANG gene in ALS and control populations. This polymorphism consists of a substitution T > G. We did not observe significant differences in allelic distributions between patients with sporadic ALS (T: n = 1538 [9.9%]; G: n = 170 [90.1%]) and controls (T: n = 408 [12.4%]; G: n = 58 [87.6%]) (P = .12; χ2 test = 2.42). Similarly, no variation in genotype distributions was found between patients with sporadic ALS (TT: 81%; TG: 18%; GG: 1.0%) and controls (TT: 76%; TG: 23.1%; GG: 0.9%) (P = .21; χ2 test = 3.13). The clinical characteristics of the 3 ANG mutations carriers are summarized in the Table.

Table Graphic Jump LocationTable. Clinical Characteristics of the Patients With Amyotrophic Lateral Sclerosis With Mutations in the ANG Gene

We report a novel mutation of the ANG gene in a patient who developed ALS with rapid progression and observed a previously known mutation in 2 patients with sporadic ALS. The novel mutation (G434A) changes the amino acid (R121H) in the C-terminal region of the mature angiogenin protein. This G434A mutation is rare; to date, it has been found in only 1 of 3155 patients with ALS, including our results and all published data.5,1012 The fact that it has not been observed in 2135 healthy individuals suggests that it is not a neutral polymorphism. However, functional studies will be necessary to confirm this theory. We observed that the R121H change may alter the conformation of the catalytic center. Wu et al12 recently identified a P112L mutation in 1 patient with ALS; therefore, this novel R121H mutation represents the second mutation identified in the C-terminal region of the ANG gene.

The A208G substitution that we observed in 2 unrelated patients with sporadic ALS already has been found in 2 Scottish patients with familial ALS and 1 Scottish patient with sporadic ALS.5 Patients described in the present study are not of Scottish descent. We did not observe this mutation in our 234 French controls. This substitution was observed in 2% of Italian controls, and thus it may represent a rare polymorphism.11 In our meta-analysis, we found a frequency of 0.2% for this polymorphism in apparently healthy individuals, which is in the range of the estimated life span incidence of ALS (1/800). It may be possible that this mutation is associated in some populations with other genetic variations, modulating its effect. The A208G substitution changes the amino acid in position 46 (I46V). A recent biochemical study13 of ANG mutations indicated that this I46V variation was associated with a 10-fold decrease in ribonuclease activity.

Greenway et al5 observed an association with the G allele of rs11701 in Irish or Scottish populations with ALS. A similar association was found in an Italian ALS population.11 However, no association was observed with another Italian,10 a US, an English, and a Swedish population.5 In our study, we did not observe a difference in allelic and genotypic distributions for rs11701 between French populations of controls and patients with ALS. These results suggest either that the observed associations were false-positive results or that linkage disequilibrium exists between this polymorphism and an unknown mutation in some populations.

The identification of new mutations in the ANG gene brings further support for a role for angiogenin in the physiopathologic mechanisms of ALS. The implication of the G434A and A208G substitutions in ALS will have to be assessed by biological assays on angiogenesis and neurite path finding and molecular assays on nuclear transport, ribonuclease activity, and ribosomal RNA synthesis. Indeed, angiogenin has been shown to enhance ribosomal RNA transcription via binding to DNA in ribosomal RNA genes.14 Interestingly, we and others15,16 have reported that the SMN1 gene, also presumed to act on ribosomal RNA synthesis (maturation), is a risk factor for ALS.

Correspondence: Christian R. Andres, MD, PhD, INSERM U930, Université François Rabelais Tours, CHRU de Tours, Faculté de Médecine, 10 boulevard Tonnellé, BP3223, 37032 Tours CEDEX, France (andres@med.univ-tours.fr).

Accepted for Publication: March 18, 2008.

Author Contributions:Study concept and design: Paubel, Violette, Praline, Corcia, Andres, and Vourc’h. Acquisition of data: Paubel, Violette, Amy, Meininger, Camu, Corcia, Andres, and Vourc’h. Analysis and interpretation of data: Paubel, Amy, Andres, and Vourc’h. Drafting of the manuscript: Paubel, Violette, Praline, and Vourc’h. Critical revision of the manuscript for important intellectual content: Amy, Meininger, Camu, Corcia, Andres, and Vourc’h. Statistical analysis: Andres and Vourc’h. Obtained funding: Violette, Camu, Andres, and Vourc’h. Administrative, technical, and material support: Paubel, Violette, Amy, Praline, Meininger, Camu, Andres, and Vourc’h. Study supervision: Corcia, Andres, and Vourc’h.

Financial Disclosure: None reported.

Funding/Support: The Association pour la Recherche sur la Sclérose Latérale Amyotrophique provided financial support for this study.

Group Information: The French ALS Study Group is composed of Fredric Dubas, MD, and Guillaume Nicolas, MD, PhD (Angers); Gwendal Lemasson, MD, PhD, and Emmanuelle Salort, MD (Bordeaux); Fausto Viader, MD, and Laurence Carluer, MD (Caen); Pierre Clavelou, MD, PhD, and Nathalie Guy, MD (Clermont-Ferrand), Maurice Giroud, MD, and Cecile Maugras, MD (Dijon); Gerard Besson, MD, PhD (Grenoble); Alain Destée, MD, PhD, and Veronique Danel, MD (Lille); Philippe Couratier, MD, PhD, and Matthieu Lacoste, MD (Limoges); Emmanuel Broussole, MD, Christophe Vial, MD, and Nadia Vandenberghe, MD (Lyon); Jean Pouget, MD, Dominique Lardiller, MD, and Annie Verschuren, MD (Marseille); William Camu, MD, PhD, Guillaume Garrigues, MD, and Nicholas Pageot, MD (Montpellier); Marc Debouverie, MD, PhD, and Sophie Pition, MD (Nancy); Claude Desnuelle, MD, PhD, and Marie Helene Soriani, MD (Nice); Vincent Meininger, MD, PhD, Francois Salachas, MD, Pierre-François Pradat, MD, PhD, Michel Dib, MD, Gaelle Bruneteau, MD, and Nadine Leforestier, MD (Paris); Christine Tranchant, MD, PhD, and Marie-Céline Fleury, MD (Strasbourg); Jean-Christophe Antoine, MD, PhD, and Jean Philippe Camdessanche, MD, PhD (Saint-Etienne); Marie-Christine Arne-Bes, MD and Pascal Cintas, MD (Toulouse); and Philippe Corcia, MD, PhD, and Julien Praline, MD (Tours).

Additional Contributions: Maryse Guillonneau, Jeanine Le Garrec, Isabelle Besson, Catherine Cherpi-Antar, and Rose-Anne Thépault provided technical help.

Oosthuyse  BMoons  LStorkebaum  E  et al.  Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001;28 (2) 131- 138
PubMed Link to Article
Lambrechts  DStorkebaum  EMorimoto  M  et al.  VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet 2003;34 (4) 383- 394
PubMed Link to Article
Storkebaum  ELambrechts  DDewerchin  M  et al.  Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat Neurosci 2005;8 (1) 85- 92
PubMed Link to Article
Azzouz  MRalph  GSStorkebaum  E  et al.  VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 2004;429 (6990) 413- 417
PubMed Link to Article
Greenway  MJAndersen  PMRuss  C  et al.  ANG mutations segregate with familial and “sporadic” amyotrophic lateral sclerosis. Nat Genet 2006;38 (4) 411- 413
PubMed Link to Article
Fett  JWStrydom  DJLobb  RR  et al.  Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 1985;24 (20) 5480- 5486
PubMed Link to Article
Kieran  DMGreenway  MJHardiman  OPrehn  J The effects of angiogenin on motor neuron degeneration [abstract]. Amyotroph Lateral Scler 2005;6 (6) ((suppl 1)) 42
Link to Article
Adams  SASubramanian  V The angiogenins: an emerging family of ribonuclease related proteins with diverse cellular functions. Angiogenesis 1999;3 (3) 189- 199
PubMed Link to Article
Subramanian  VFeng  Y A new role for angiogenin in neurite growth and pathfinding: implications for amyotrophic lateral sclerosis. Hum Mol Genet 2007;16 (12) 1445- 1453
PubMed Link to Article
Del Bo  RScarlato  MGhezzi  S  et al.  Absence of angiogenic genes modification in Italian ALS patients. Neurobiol Aging 2008;29 (2) 314- 316
PubMed Link to Article
Conforti  FLSprovieri  TMazzei  R  et al.  A novel angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern Italy. Neuromuscul Disord 2008;18 (1) 68- 70
PubMed Link to Article
Wu  DYu  WKishikawa  H  et al.  Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis. Ann Neurol 2007;62 (6) 609- 617
PubMed Link to Article
Crabtree  BThiyagarajan  NPrior  SH  et al.  Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis. Biochemistry 2007;46 (42) 11810- 11818
PubMed Link to Article
Xu  ZPTsuji  TRiordan  JFHu  GF The nuclear function of angiogenin in endothelial cells is related to rRNA production. Biochem Biophys Res Commun 2002;294 (2) 287- 292
PubMed Link to Article
Veldink  JHKalmijn  SVan der Hout  AH  et al.  SMN genotypes producing less SMN protein increase susceptibility to and severity of sporadic ALS. Neurology 2005;65 (6) 820- 825
PubMed Link to Article
Corcia  PCamu  WHalimi  JM  et al.  SMN1 gene, but not SMN2, is a risk factor for sporadic ALS. Neurology 2006;67 (7) 1147- 1150
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure.

ANG mutations and 3-dimensional structure of angiogenin. DNA sequences of the patients with amyotrophic lateral sclerosis with G434A (A) and A208G (B and C) mutations. Three-dimensional x-ray diffraction structure of the normal human angiogenin protein (PDB ID 1b1i) (D). Model of the angiogenin variant Arg121His (E).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable. Clinical Characteristics of the Patients With Amyotrophic Lateral Sclerosis With Mutations in the ANG Gene

References

Oosthuyse  BMoons  LStorkebaum  E  et al.  Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001;28 (2) 131- 138
PubMed Link to Article
Lambrechts  DStorkebaum  EMorimoto  M  et al.  VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet 2003;34 (4) 383- 394
PubMed Link to Article
Storkebaum  ELambrechts  DDewerchin  M  et al.  Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat Neurosci 2005;8 (1) 85- 92
PubMed Link to Article
Azzouz  MRalph  GSStorkebaum  E  et al.  VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 2004;429 (6990) 413- 417
PubMed Link to Article
Greenway  MJAndersen  PMRuss  C  et al.  ANG mutations segregate with familial and “sporadic” amyotrophic lateral sclerosis. Nat Genet 2006;38 (4) 411- 413
PubMed Link to Article
Fett  JWStrydom  DJLobb  RR  et al.  Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 1985;24 (20) 5480- 5486
PubMed Link to Article
Kieran  DMGreenway  MJHardiman  OPrehn  J The effects of angiogenin on motor neuron degeneration [abstract]. Amyotroph Lateral Scler 2005;6 (6) ((suppl 1)) 42
Link to Article
Adams  SASubramanian  V The angiogenins: an emerging family of ribonuclease related proteins with diverse cellular functions. Angiogenesis 1999;3 (3) 189- 199
PubMed Link to Article
Subramanian  VFeng  Y A new role for angiogenin in neurite growth and pathfinding: implications for amyotrophic lateral sclerosis. Hum Mol Genet 2007;16 (12) 1445- 1453
PubMed Link to Article
Del Bo  RScarlato  MGhezzi  S  et al.  Absence of angiogenic genes modification in Italian ALS patients. Neurobiol Aging 2008;29 (2) 314- 316
PubMed Link to Article
Conforti  FLSprovieri  TMazzei  R  et al.  A novel angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern Italy. Neuromuscul Disord 2008;18 (1) 68- 70
PubMed Link to Article
Wu  DYu  WKishikawa  H  et al.  Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis. Ann Neurol 2007;62 (6) 609- 617
PubMed Link to Article
Crabtree  BThiyagarajan  NPrior  SH  et al.  Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis. Biochemistry 2007;46 (42) 11810- 11818
PubMed Link to Article
Xu  ZPTsuji  TRiordan  JFHu  GF The nuclear function of angiogenin in endothelial cells is related to rRNA production. Biochem Biophys Res Commun 2002;294 (2) 287- 292
PubMed Link to Article
Veldink  JHKalmijn  SVan der Hout  AH  et al.  SMN genotypes producing less SMN protein increase susceptibility to and severity of sporadic ALS. Neurology 2005;65 (6) 820- 825
PubMed Link to Article
Corcia  PCamu  WHalimi  JM  et al.  SMN1 gene, but not SMN2, is a risk factor for sporadic ALS. Neurology 2006;67 (7) 1147- 1150
PubMed Link to Article

Correspondence

CME
Also Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
Please click the checkbox indicating that you have read the full article in order to submit your answers.
Your answers have been saved for later.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.

Multimedia

Some tools below are only available to our subscribers or users with an online account.

1,090 Views
34 Citations
×

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections
PubMed Articles
Jobs