Whole exome sequencing highlights rare variants in CTCF, DNMT1, DNMT3A, EZH2 and SUV39H1 as associated with FSHD

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Abstract

Introduction: Despite the progress made in the study of Facioscapulohumeral Dystrophy (FSHD), the wide heterogeneity of disease complicates its diagnosis and the genotype-phenotype correlation among patients and within families. In this context, the present work employed Whole Exome Sequencing (WES) to investigate known and unknown genetic contributors that may be involved in FSHD and may represent potential disease modifiers, even in presence of a D4Z4 Reduced Allele (DRA).

Methods: A cohort of 126 patients with clinical signs of FSHD were included in the study, which were characterized by D4Z4 sizing, methylation analysis and WES. Specific protocols were employed for D4Z4 sizing and methylation analysis, whereas the Illumina ^® Next-Seq 550 system was utilized for WES. The study included both patients with a DRA compatible with FSHD diagnosis and patients with longer D4Z4 alleles. In case of patients harboring relevant variants from WES, the molecular analysis was extended to the family members.

Results: The WES data analysis highlighted 20 relevant variants, among which 14 were located in known genetic modifiers ( SMCHD1, DNMT3B and LRIF1) and 6 in candidate genes ( CTCF, DNMT1, DNMT3A, EZH2 and SUV39H1). Most of them were found together with a permissive short (4–7 RU) or borderline/long DRA (8–20 RU), supporting the possibility that different genes can contribute to disease heterogeneity in presence of a FSHD permissive background. The segregation and methylation analysis among family members, together with clinical findings, provided a more comprehensive picture of patients.

Discussion: Our results support FSHD pathomechanism being complex with a multigenic contribution by several known ( SMCHD1, DNMT3B, LRIF1) and possibly other candidate genes ( CTCF, DNMT1, DNMT3A, EZH2, SUV39H1) to disease penetrance and expressivity. Our results further emphasize the importance of extending the analysis of molecular findings within the proband’s family, with the purpose of providing a broader framework for understanding single cases and allowing finer genotype-phenotype correlations in FSHD-affected families.

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Most cited references 58

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Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology

Sue Richards, Nazneen Aziz, Sherri Bale … (2015)

The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants. 1 In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next generation sequencing. By adopting and leveraging next generation sequencing, clinical laboratories are now performing an ever increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes and epigenetic assays for genetic disorders. By virtue of increased complexity, this paradigm shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context, the ACMG convened a workgroup in 2013 comprised of representatives from the ACMG, the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP and CAP stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories including genotyping, single genes, panels, exomes and genomes. This report recommends the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants identified in Mendelian disorders. Moreover, this recommendation describes a process for classification of variants into these five categories based on criteria using typical types of variant evidence (e.g. population data, computational data, functional data, segregation data, etc.). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a CLIA-approved laboratory with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or equivalent.

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REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants.

Nilah M. Ioannidis, Joseph H. Rothstein, Vikas Rao Pejaver … (2016)

The vast majority of coding variants are rare, and assessment of the contribution of rare variants to complex traits is hampered by low statistical power and limited functional data. Improved methods for predicting the pathogenicity of rare coding variants are needed to facilitate the discovery of disease variants from exome sequencing studies. We developed REVEL (rare exome variant ensemble learner), an ensemble method for predicting the pathogenicity of missense variants on the basis of individual tools: MutPred, FATHMM, VEST, PolyPhen, SIFT, PROVEAN, MutationAssessor, MutationTaster, LRT, GERP, SiPhy, phyloP, and phastCons. REVEL was trained with recently discovered pathogenic and rare neutral missense variants, excluding those previously used to train its constituent tools. When applied to two independent test sets, REVEL had the best overall performance (p < 10(-12)) as compared to any individual tool and seven ensemble methods: MetaSVM, MetaLR, KGGSeq, Condel, CADD, DANN, and Eigen. Importantly, REVEL also had the best performance for distinguishing pathogenic from rare neutral variants with allele frequencies <0.5%. The area under the receiver operating characteristic curve (AUC) for REVEL was 0.046-0.182 higher in an independent test set of 935 recent SwissVar disease variants and 123,935 putatively neutral exome sequencing variants and 0.027-0.143 higher in an independent test set of 1,953 pathogenic and 2,406 benign variants recently reported in ClinVar than the AUCs for other ensemble methods. We provide pre-computed REVEL scores for all possible human missense variants to facilitate the identification of pathogenic variants in the sea of rare variants discovered as sequencing studies expand in scale.

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Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2

Richard Lemmers, Rabi Tawil, Lisa Petek … (2012)

Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded DUX4 gene in skeletal muscle. The more common form, autosomal dominant FSHD1, is caused by a contraction of the D4Z4 array, whereas the genetic determinants and inheritance of D4Z4 array contraction-independent FSHD2 are unclear. Here we show that mutations in SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein levels and segregate with genome-wide D4Z4 CpG hypomethylation in human kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4 expression. Reducing SMCHD1 levels in skeletal muscle results in contraction-independent DUX4 expression. Our study identifies SMCHD1 as an epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic determinant of FSHD2 and possibly other human diseases subject to epigenetic regulation.

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Author and article information

Contributors

Claudia Strafella: URI : https://loop.frontiersin.org/people/575017/overview

Valerio Caputo: URI : https://loop.frontiersin.org/people/721524/overview

Sara Bortolani: URI : https://loop.frontiersin.org/people/973552/overview

Domenica Megalizzi: URI : https://loop.frontiersin.org/people/1647262/overview

Giulia Trastulli: URI : https://loop.frontiersin.org/people/2404516/overview

Mauro Monforte: URI : https://loop.frontiersin.org/people/976578/overview

Luca Colantoni: URI : https://loop.frontiersin.org/people/606724/overview

Carlo Caltagirone: URI : https://loop.frontiersin.org/people/113537/overview

Giorgio Tasca: URI : https://loop.frontiersin.org/people/584648/overview

Raffaella Cascella: URI : https://loop.frontiersin.org/people/2213428/overview

Emiliano Giardina: URI : https://loop.frontiersin.org/people/611135/overview

Journal

Journal ID (nlm-ta): Front Genet

Journal ID (iso-abbrev): Front Genet

Journal ID (publisher-id): Front. Genet.

Title: Frontiers in Genetics

Publisher: Frontiers Media S.A.

ISSN (Electronic): 1664-8021

Publication date (Electronic): 22 August 2023

Publication date Collection: 2023

Volume: 14

Electronic Location Identifier: 1235589

Affiliations

[1] ¹ Genomic Medicine Laboratory UILDM , IRCCS Santa Lucia Foundation , Rome, Italy

[2] ² Unità Operativa Complessa di Neurologia , Fondazione Policlinico Universitario A. Gemelli IRCCS , Rome, Italy

[3] ³ Department of Clinical and Behavioral Neurology , IRCCS Fondazione Santa Lucia , Rome, Italy

[4] ⁴ Istituto di Neurologia , Università Cattolica del Sacro Cuore , Rome, Italy

[5] ⁵ John Walton Muscular Dystrophy Research Centre , Newcastle University and Newcastle Hospitals NHS Foundation Trusts , Newcastle UponTyne, United Kingdom

[6] ⁶ Department of Biomedical Sciences , Catholic University Our Lady of Good Counsel , Tirana, Albania

[7] ⁷ Medical Genetics Laboratory , Department of Biomedicine and Prevention , Tor Vergata University , Rome, Italy

Author notes

Edited by: Jordi Pérez-Tur, Spanish National Research Council (CSIC), Spain

Reviewed by: Frederique Magdinier, Institut National de la Santé et de la Recherche Médicale (INSERM), France

Edoardo Malfatti, Hôpitaux Universitaires Henri Mondor, France

*Correspondence: Emiliano Giardina, emiliano.giardina@ 123456uniroma2.it

Article

Publisher ID: 1235589

DOI: 10.3389/fgene.2023.1235589

PMC ID: 10477786

PubMed ID: 37674478

SO-VID: d89915e8-a6b4-4fda-b091-09dde33349d6

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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

History

Date received : 06 June 2023

Date accepted : 09 August 2023

Funding

This research was supported by FSHD Society Research Grant #Winter 2021-0992658837 (COD:FSHD) to CS and by Ministry of Health (Ricerca Corrente) to CC.

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section-at-acceptance Genetics of Common and Rare Diseases

ScienceOpen disciplines: Genetics

Keywords: fshd,exome,d4z4,genetics,muscular dystrophy

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ScienceOpen disciplines: Genetics

Keywords: fshd, exome, d4z4, genetics, muscular dystrophy

Whole exome sequencing highlights rare variants in CTCF, DNMT1, DNMT3A, EZH2 and SUV39H1 as associated with FSHD

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Most cited references 58

Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology

REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants.

Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2

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