title: A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver-Russell and Beckwith-Wiedemann syndromes.
abstract: BACKGROUND
  Multiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver-Russell (SRS) and Beckwith-Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate.
  
  
  RESULTS
  Molecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as "borderline." A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between "easy to diagnose" and "borderline" cases, which were characterized by values ≤mean -3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean -2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia.
  
  
  CONCLUSIONS
  A conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.
authors.affiliation: Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Russo
authors.affiliation: Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Calzari
authors.affiliation: Department of Pediatric and Public Health Sciences, University of Turin, Torino, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Mussa
authors.affiliation: Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
authors.forename: Ester
authors.initials: E
authors.lastname: Mainini
authors.affiliation: Clinical Genetics Unit, Department of Women's and Children's Health, University of Padua, Padova, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Cassina
authors.affiliation: Department of Pediatrics, San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Di Candia
authors.affiliation: Clinical Genetics Unit, Department of Women's and Children's Health, University of Padua, Padova, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Clementi
authors.affiliation: Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Guzzetti
authors.affiliation: Division of Pathology - Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Department of Pathophysiology and Transplantation, University of Milan, Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Tabano
authors.affiliation: Division of Pathology - Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Department of Pathophysiology and Transplantation, University of Milan, Milano, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Miozzo
authors.affiliation: Department of Health Sciences, University of Milan, Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sirchia
authors.affiliation: Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
authors.forename: Palma
authors.initials: P
authors.lastname: Finelli
authors.affiliation: Medical Genetics Unit, Department of Surgical and Biomedical Sciences, University of Perugia, Hospital "S. M. della Misericordia", Perugia, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Prontera
authors.affiliation: Clinical Pediatric Genetics Unit, Pediatrics Clinics, MBBM Foundation, S. Gerardo Hospital, Monza, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Maitz
authors.affiliation: Department of Pediatrics and Medical Sciences, AO "Policlinico Vittorio Emanuele", Catania, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Sorge
authors.affiliation: Pediatric Endocrine Unit, Department of Pediatrics, IRCCS, Children's Hospital Giannina Gaslini, Genova, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Calcagno
authors.affiliation: Pediatric Endocrine Unit, Department of Pediatrics, IRCCS, Children's Hospital Giannina Gaslini, Genova, Italy.
authors.forename: Mohamad
authors.initials: M
authors.lastname: Maghnie
authors.affiliation: Department of Medical Genetics, IRCCS, Children's Hospital Giannina Gaslini, Genova, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Divizia
authors.affiliation: Clinical Pediatric Genetics, Department of Pediatrics, University "Federico II", Napoli, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Melis
authors.affiliation: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca' Granda Hospital, Milano, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Manfredini
authors.affiliation: Department of Pediatric and Public Health Sciences, University of Turin, Torino, Italy.
authors.forename: Giovanni Battista
authors.initials: GB
authors.lastname: Ferrero
authors.affiliation: Institute for Maternal and Child Health, Foundation IRCCS Burlo Garofolo Institute, Trieste, Italy.
authors.forename: Vanna
authors.initials: V
authors.lastname: Pecile
authors.affiliation: Human Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy.
authors.forename: Lidia
authors.initials: L
authors.lastname: Larizza
mesh_terms: D001506:Beckwith-Wiedemann Syndrome / Q000175:diagnosis* / Q000235:genetics; D015139:Blotting, Southern / Q000379:methods; D002648:Child; D002675:Child, Preschool; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D009030:Mosaicism; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D056730:Silver-Russell Syndrome / Q000175:diagnosis* / Q000235:genetics
keywords: Beckwith–Wiedemann syndrome; Borderline cases; MS-MLPA; Molecular diagnosis; Mosaic (epi)genetic alterations; Multi-method approach; Pyrosequencing; SNP array; Silver–Russell syndrome; Southern blot