A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver–Russell and Beckwith–Wiedemann syndromes

Silvia Russo, Luciano Calzari, Alessandro Mussa, Ester Mainini, Matteo Cassina, Stefania Di Candia, M. Clementi, Sara Guzzetti, Silvia Tabano, Monica Rosa Miozzo, Silvia Maria Sirchia, Palma Finelli, P. Prontera, Silvia Maitz, G. Sorge, Annalisa Calcagno, Mohamad Maghnie, Maria Teresa Divizia, Daniela Melis, Emanuela ManfrediniG. B. Ferrero, Vanna Pecile, Lidia Larizza

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article number23
JournalClinical Epigenetics
Volume8
Issue number1
DOIs
Publication statusPublished - Mar 1 2016

Fingerprint

Methylation
Mosaicism
Software Design
Growth Disorders
Cheek
Multiplex Polymerase Chain Reaction
Southern Blotting
Epigenomics
Single Nucleotide Polymorphism
Phenotype
Gene Expression

Keywords

  • Beckwith–Wiedemann syndrome
  • Borderline cases
  • Molecular diagnosis
  • Mosaic (epi)genetic alterations
  • MS-MLPA
  • Multi-method approach
  • Pyrosequencing
  • Silver–Russell syndrome
  • SNP array
  • Southern blot

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Developmental Biology
  • Genetics(clinical)

Cite this

A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver–Russell and Beckwith–Wiedemann syndromes. / Russo, Silvia; Calzari, Luciano; Mussa, Alessandro; Mainini, Ester; Cassina, Matteo; Di Candia, Stefania; Clementi, M.; Guzzetti, Sara; Tabano, Silvia; Miozzo, Monica Rosa; Sirchia, Silvia Maria; Finelli, Palma; Prontera, P.; Maitz, Silvia; Sorge, G.; Calcagno, Annalisa; Maghnie, Mohamad; Divizia, Maria Teresa; Melis, Daniela; Manfredini, Emanuela; Ferrero, G. B.; Pecile, Vanna; Larizza, Lidia.

In: Clinical Epigenetics, Vol. 8, No. 1, 23, 01.03.2016.

Research output: Contribution to journalArticle

Russo, S, Calzari, L, Mussa, A, Mainini, E, Cassina, M, Di Candia, S, Clementi, M, Guzzetti, S, Tabano, S, Miozzo, MR, Sirchia, SM, Finelli, P, Prontera, P, Maitz, S, Sorge, G, Calcagno, A, Maghnie, M, Divizia, MT, Melis, D, Manfredini, E, Ferrero, GB, Pecile, V & Larizza, L 2016, 'A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver–Russell and Beckwith–Wiedemann syndromes', Clinical Epigenetics, vol. 8, no. 1, 23. https://doi.org/10.1186/s13148-016-0183-8
Russo, Silvia ; Calzari, Luciano ; Mussa, Alessandro ; Mainini, Ester ; Cassina, Matteo ; Di Candia, Stefania ; Clementi, M. ; Guzzetti, Sara ; Tabano, Silvia ; Miozzo, Monica Rosa ; Sirchia, Silvia Maria ; Finelli, Palma ; Prontera, P. ; Maitz, Silvia ; Sorge, G. ; Calcagno, Annalisa ; Maghnie, Mohamad ; Divizia, Maria Teresa ; Melis, Daniela ; Manfredini, Emanuela ; Ferrero, G. B. ; Pecile, Vanna ; Larizza, Lidia. / A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver–Russell and Beckwith–Wiedemann syndromes. In: Clinical Epigenetics. 2016 ; Vol. 8, No. 1.
@article{61c6a68cc69f4352819fd426da0112bc,
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.",
keywords = "Beckwith–Wiedemann syndrome, Borderline cases, Molecular diagnosis, Mosaic (epi)genetic alterations, MS-MLPA, Multi-method approach, Pyrosequencing, Silver–Russell syndrome, SNP array, Southern blot",
author = "Silvia Russo and Luciano Calzari and Alessandro Mussa and Ester Mainini and Matteo Cassina and {Di Candia}, Stefania and M. Clementi and Sara Guzzetti and Silvia Tabano and Miozzo, {Monica Rosa} and Sirchia, {Silvia Maria} and Palma Finelli and P. Prontera and Silvia Maitz and G. Sorge and Annalisa Calcagno and Mohamad Maghnie and Divizia, {Maria Teresa} and Daniela Melis and Emanuela Manfredini and Ferrero, {G. B.} and Vanna Pecile and Lidia Larizza",
year = "2016",
month = "3",
day = "1",
doi = "10.1186/s13148-016-0183-8",
language = "English",
volume = "8",
journal = "Clinical Epigenetics",
issn = "1868-7075",
publisher = "Springer Verlag",
number = "1",

}

TY - JOUR

T1 - A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver–Russell and Beckwith–Wiedemann syndromes

AU - Russo, Silvia

AU - Calzari, Luciano

AU - Mussa, Alessandro

AU - Mainini, Ester

AU - Cassina, Matteo

AU - Di Candia, Stefania

AU - Clementi, M.

AU - Guzzetti, Sara

AU - Tabano, Silvia

AU - Miozzo, Monica Rosa

AU - Sirchia, Silvia Maria

AU - Finelli, Palma

AU - Prontera, P.

AU - Maitz, Silvia

AU - Sorge, G.

AU - Calcagno, Annalisa

AU - Maghnie, Mohamad

AU - Divizia, Maria Teresa

AU - Melis, Daniela

AU - Manfredini, Emanuela

AU - Ferrero, G. B.

AU - Pecile, Vanna

AU - Larizza, Lidia

PY - 2016/3/1

Y1 - 2016/3/1

N2 - 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.

AB - 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.

KW - Beckwith–Wiedemann syndrome

KW - Borderline cases

KW - Molecular diagnosis

KW - Mosaic (epi)genetic alterations

KW - MS-MLPA

KW - Multi-method approach

KW - Pyrosequencing

KW - Silver–Russell syndrome

KW - SNP array

KW - Southern blot

UR - http://www.scopus.com/inward/record.url?scp=84963626026&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84963626026&partnerID=8YFLogxK

U2 - 10.1186/s13148-016-0183-8

DO - 10.1186/s13148-016-0183-8

M3 - Article

VL - 8

JO - Clinical Epigenetics

JF - Clinical Epigenetics

SN - 1868-7075

IS - 1

M1 - 23

ER -