title: Validation of genetic modifiers for Duchenne muscular dystrophy: a multicentre study assessing SPP1 and LTBP4 variants.
abstract: OBJECTIVE
  Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials.
  
  
  METHODS
  Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates.
  
  
  RESULTS
  We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss.
  
  
  CONCLUSIONS
  This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.
authors.affiliation: Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Janneke C
authors.initials: JC
authors.lastname: van den Bergen
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Monika
authors.initials: M
authors.lastname: Hiller
authors.affiliation: Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Stefan
authors.initials: S
authors.lastname: Böhringer
authors.affiliation: Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: Vijfhuizen
authors.affiliation: Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Hendrika B
authors.initials: HB
authors.lastname: Ginjaar
authors.affiliation: Institute of Human Genetics, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK.
authors.forename: Amina
authors.initials: A
authors.lastname: Chaouch
authors.affiliation: Institute of Human Genetics, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK.
authors.forename: Kate
authors.initials: K
authors.lastname: Bushby
authors.affiliation: Institute of Human Genetics, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK.
authors.forename: Volker
authors.initials: V
authors.lastname: Straub
authors.affiliation: Dubowitz Neuromuscular Centre, University College London, London, UK.
authors.forename: Mariacristina
authors.initials: M
authors.lastname: Scoto
authors.affiliation: Dubowitz Neuromuscular Centre, University College London, London, UK Children's National Medical Center, Research Center for Genetic Medicine, Washington DC, USA.
authors.forename: Sebahattin
authors.initials: S
authors.lastname: Cirak
authors.affiliation: Laboratoire de Génétique de Maladies Rares, CHU Montpellier, Université Montpellier 1, UFR Médecine, and Inserm U827, Montpellier, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Humbertclaude
authors.affiliation: Laboratoire de Génétique de Maladies Rares, CHU Montpellier, Université Montpellier 1, UFR Médecine, and Inserm U827, Montpellier, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Claustres
authors.affiliation: Department of Medical Sciences, Section of Microbiology and Medical Genetics, University of Ferrara, Ferrara, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Scotton
authors.affiliation: Paediatric Hospital Bambino Gesù, Rome, Italy.
authors.forename: Chiara
authors.identifier: http://orcid.org/0000-0001-7919-4626
authors.initials: C
authors.lastname: Passarelli
authors.affiliation: Institute of Human Genetics, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK.
authors.forename: Hanns
authors.initials: H
authors.lastname: Lochmüller
authors.affiliation: Dubowitz Neuromuscular Centre, University College London, London, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: Muntoni
authors.affiliation: Laboratoire de Génétique de Maladies Rares, CHU Montpellier, Université Montpellier 1, UFR Médecine, and Inserm U827, Montpellier, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Tuffery-Giraud
authors.affiliation: Department of Medical Sciences, Section of Microbiology and Medical Genetics, University of Ferrara, Ferrara, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ferlini
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Annemieke M
authors.initials: AM
authors.lastname: Aartsma-Rus
authors.affiliation: Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jan J G M
authors.initials: JJ
authors.lastname: Verschuuren
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Peter Ac
authors.initials: PA
authors.lastname: 't Hoen
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Pietro
authors.initials: P
authors.lastname: Spitali
mesh_terms: D000367:Age Factors; D002648:Child; D015331:Cohort Studies; D018450:Disease Progression; D005060:Europe; D005260:Female; D005838:Genotype; D006801:Humans; D053779:Latent TGF-beta Binding Proteins / Q000235:genetics*; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D053495:Osteopontin / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011379:Prognosis; D015203:Reproducibility of Results; D013256:Steroids / Q000627:therapeutic use; D016138:Walking; D014910:Wheelchairs
chemical_list: C509040:LTBP4 protein, human; D053779:Latent TGF-beta Binding Proteins; C506895:SPP1 protein, human; D013256:Steroids; D053495:Osteopontin
keywords: DYSTROPHIN; GENETICS; MUSCLE DISEASE; MUSCULAR DYSTROPHY; NEUROMUSCULAR