VCV003024113.1 - ClinVar

NM_001927.4(DES):c.1056GGA[3] (p.Glu353_Asp354insGlu)

Germline

Classification

The aggregate germline classification for this variant, typically for a monogenic or Mendelian disorder as in the ACMG/AMP guidelines, or for response to a drug. This value is calculated by NCBI based on data from submitters. Read our rules for calculating the aggregate classification.

(1)

Stars represent the aggregate review status, or the level of review supporting the aggregate germline classification for this VCV record. This value is calculated by NCBI based on data from submitters. Read our rules for calculating the review status. The number of submissions which contribute to this review status is shown in parentheses.

Pathogenic

no assertion criteria provided

Somatic

No data submitted for somatic clinical impact

Somatic

No data submitted for oncogenicity

Variant Details

Identifiers

NM_001927.4(DES):c.1056GGA[3] (p.Glu353_Asp354insGlu)

Variation ID: 3024113 Accession: VCV003024113.1

Type and length

Microsatellite, 3 bp

Location

Cytogenetic: 2q35 2: 219421371-219421372 (GRCh38) [ NCBI UCSC ] 2: 220286093-220286094 (GRCh37) [ NCBI UCSC ]

Timeline in ClinVar

	First in ClinVar Help The date this variant first appeared in ClinVar with each type of classification.	Last submission Help The date of the most recent submission for each type of classification for this variant.	Last evaluated Help The most recent date that a submitter evaluated this variant for each type of classification.
Germline	Feb 29, 2024	Feb 28, 2024

HGVS

Nucleotide	Protein	Molecular consequence
NM_001927.4:c.1056GGA[3] MANE Select Help Transcripts from the Matched Annotation from the NCBI and EMBL-EBI (MANE) collaboration.	NP_001918.3:p.Glu353_Asp354insGlu	inframe insertion
NM_001927.4:c.1059_1061dupGGA MANE Select Help Transcripts from the Matched Annotation from the NCBI and EMBL-EBI (MANE) collaboration.		inframe insertion
NM_001382708.1:c.1053GGA[3]	NP_001369637.1:p.Glu352_Asp353insGlu	inframe insertion
NM_001382709.1:c.736-112GGA[3]		intron variant
NM_001382710.1:c.1024-37GGA[3]		intron variant
NM_001382711.1:c.1035GGA[3]	NP_001369640.1:p.Glu346_Asp347insGlu	inframe insertion
NM_001382712.1:c.1056GGA[3]	NP_001369641.1:p.Glu353_Asp354insGlu	inframe insertion
NM_001382713.1:c.786GGA[3]	NP_001369642.1:p.Glu263_Asp264insGlu	inframe insertion
NC_000002.12:g.219421372GGA[3]
NC_000002.11:g.220286094GGA[3]
NG_008043.1:g.7996GGA[3]
LRG_380:g.7996GGA[3]
LRG_380t1:c.1056_1058GGA[3]	LRG_380p1:p.Glu353_Asp354insGlu

... more HGVS ... less HGVS

Protein change

Other names

Canonical SPDI

NC_000002.12:219421371:GGAGGA:GGAGGAGGA

Functional consequence Help The effect of the variant on RNA or protein function, based on experimental evidence from submitters.

Global minor allele frequency (GMAF) Help The global minor allele frequency calculated by the 1000 Genomes Project. The minor allele at this location is indicated in parentheses and may be different from the allele represented by this VCV record.

Allele frequency Help The frequency of the allele represented by this VCV record.

Links

VarSome

Genes

Gene	OMIM	ClinGen Gene Dosage Sensitivity Curation		Variation Viewer Help Links to Variation Viewer, a genome browser to view variation data from NCBI databases.	Related variants
Gene	OMIM	HI score Help The haploinsufficiency score for the gene, curated by ClinGen’s Dosage Sensitivity Curation task team.	TS score Help The triplosensitivity score for the gene, curated by ClinGen’s Dosage Sensitivity Curation task team.		Within gene Help The number of variants in ClinVar that are contained within this gene, with a link to view the list of variants.	All Help The number of variants in ClinVar for this gene, including smaller variants within the gene and larger CNVs that overlap or fully contain the gene.
DES		-	-	GRCh38 GRCh37	1062	1108

Conditions - Germline

Condition Help The condition for this variant-condition (RCV) record in ClinVar.	Classification Help The aggregate germline classification for this variant-condition (RCV) record in ClinVar. The number of submissions that contribute to this aggregate classification is shown in parentheses. (# of submissions)	Review status Help The aggregate review status for this variant-condition (RCV) record in ClinVar. This value is calculated by NCBI based on data from submitters. Read our rules for calculating the review status.	Last evaluated Help The most recent date that a submitter evaluated this variant for the condition.	Variation/condition record Help The RCV accession number, with most recent version number, for the variant-condition record, with a link to the RCV web page.
Desmin-related myofibrillar myopathy	Pathogenic (1)	no assertion criteria provided	-	RCV003881698.2

Submissions - Germline

Classification Help The submitted germline classification for each SCV record. (Last evaluated)	Review status Help Stars represent the review status, or the level of review supporting the submitted (SCV) record. This value is calculated by NCBI based on data from the submitter. Read our rules for calculating the review status. This column also includes a link to the submitter’s assertion criteria if provided, and the collection method. (Assertion criteria)	Condition Help The condition for the classification, provided by the submitter for this submitted (SCV) record. This column also includes the affected status and allele origin of individuals observed with this variant.	Submitter Help The submitting organization for this submitted (SCV) record. This column also includes the SCV accession and version number, the date this SCV first appeared in ClinVar, and the date that this SCV was last updated in ClinVar.	More information Help This column includes more information supporting the classification, including citations, the comment on classification, and detailed evidence provided as observations of the variant by the submitter.
Pathogenic (-)	no assertion criteria provided Method: clinical testing, in vitro	Desmin-related myofibrillar myopathy Affected status: yes, not applicable Allele origin: inherited, not applicable	Laboratorio de Investigaciones Aplicadas a Neurociencias, Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia Accession: SCV004565348.1 First in ClinVar: Feb 28, 2024 Last updated: Feb 28, 2024	Comment: The c.1059_1061dupGGA mutation in DES involves the duplication of Glu at position 353 of DES. According to guidelines of the American College of Medical Genetics … (more) The c.1059_1061dupGGA mutation in DES involves the duplication of Glu at position 353 of DES. According to guidelines of the American College of Medical Genetics and Genomics, in-frame insertions are considered moderate indicative of pathogenicity (Richards et al 2015). Additionally, this mutation is located in exon 6 of the protein, which is part of the highly conserved coil 2B domain (known to govern DES polypeptide assembly into and subsequent higher-order structures; Bar et al 2004; Goebel et al 2011; Walter et al 2007) and where many pathogenic mutations were observed in patients. Moreover, disruptive DES filament assembly was in vitro observed in the presence of the mutation (doi.org/10.21203/rs.3.rs-3805874/v1). (less) Observation 1: Number of individuals with the variant: 3 Clinical Features: Myofibrillar myopathy (present) Age: 30-39 years Sex: male Method: The index patient and his 2 siblings signed an informed consent to perform the clinical exome and to share their data anonymously. Genomic DNA from peripheral blood mononuclear cells (PBMCs) derived from the index patient and his siblings was extracted using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer's protocol. The exon regions were enriched using an exome sequencing capture kit [SureSelect V6-Post (Agilent Technologies)] and these regions were sequenced on an Illumina Novaseq sequencer using 150-bp paired-end-reads with a 97,2% average coverage. After sequencing, the raw reads from each sample were sorted by index sequences. The adapter sequences were trimmed using Trim galore (bioinformatics.babraham.ac.uk). To remove low-quality bases (QD<2.0) GATK software was used. Clean reads were aligned to the human reference genome (hg19) using the Burrows-Wheeler Aligner (BWA) software package . Following alignment, PCR duplicates were removed using the Picard tools Mark Duplicates package (http://picard.sourceforge.net/). Realignment around known indel sites and base quality score recalibration (BQSR) were performed using GATK software. GATK Haplotype Caller software was used to call raw variants. The indels and single nucleotide polymorphisms (SNPs) were annotated using SnpEff. Public versions of the databases dbNSFP, 1000 Genome Project, Exome Sequencing Project, ClinVar were used. Prediction of functional effects was evaluated using SnpEff. From annotated VCF, a bioinformatic panel of 80 genes known to cause muscular myopathy or dystrophy was analyzed and the panel mean depth was 65x as calculated. Variants with low allele frequency (<1%) or absent in population databases, localized in coding regions or splicing sites were prioritized. All candidate variants were manually inspected for sequencing and variant calling quality using the Integrative Genomics Viewer[38]. Pathogenic and likely pathogenic variants were confirmed by conventional Sanger sequencing using Macrogen sequencing service. Lastly, variants were correlated with patient phenotypes and the results of clinical investigations. Observation 2: Method: To evaluate whether the index patient mutation in DES generates protein aggregates in cells, two plasmids were synthesized by SynBio technology for DES expression: Plasmid DES_WT, that expresses the patient wild-type DES sequence (containing the SNPs of the patient´s wild-type DES allele), and the plasmid DES_dupGGA, that expresses the patient mutant DES sequence (containing the dupGGA mutation and the SNPs of the patient mutant DES allele). Briefly, the constructs contain piggybac transposon, puromycin resistance and a doxycycline inducible promoter for DES-P2A-mCherry expression. 250,000 HEK cells were transfected with 1.2 µg of each DES plasmid + 0.4 ug of a plasmid containing the transposase, using 5 µl X-tremeGENE Transfection Reagent (6365787001, Roche). After 24 h, 2 µg/ml puromycin (#ant-pr 1, InvivoGene) was added for 48 h to select the transfected cells, and again 1 week after that another pulse of 48 h was given to select the cells that incorporated the plasmid in the genome. DES expression was induced with 200 ng/ml doxycycline (Sigma-Aldrich) for 72 h. At that time, HEK cells of each experimental group and a non-transfected negative control were fixed with 2% PFA for immunofluorescence staining (explained below). Moreover, EnzChek® Caspase-3 Assay Kit #2 (Molecular Probes) was used to evaluate apoptosis in HEK cells that overexpress wild-type and dupGGA DES sequences after 72 h of doxycycline, following the manufacturer's instructions. Control cells with and without doxycycline treatment were used as controls for this experiment. Result: To assess the functional properties of the mutation in DES and determine if it induces aggregate formation, we overexpressed this variant and the WT isoform in HEK cells. DES_dupGGA or DES_WT were co-expressed with mCherry, separated after protein translation by an autocleavage peptide. For this experiment, we used a pool of cells with random integration of different quantities of the exogenous vector. This resulted in cells with varying levels of DES/mCherry expression within each cell line, allowing us to compare the behavior of DES_dupGGA and DES_WT in HEK cells based on their expression levels after doxycycline induction. In terms of DES staining, control HEK cells displayed an extended filamentous network, which was also observed in cells of the other two cell lines where mCherry expression was absent or very low. Since mCherry expression correlates directly with DES expression in our system, we can infer that these cells also had low expression of exogenous DES, and their cytoskeleton remained unaltered. HEK cells overexpressing DES_dupGGA did not form the normal filamentous network and instead developed small aggregates around the cell membrane. Interestingly, the endogenous filamentous network observed in control HEK cells disappeared completely in these cells. In contrast, overexpressing DES_WT did not result in aggregate formation. However, the normal extended filamentous network was not formed, and denser DES filaments were observed, likely due to an artifact of the technique resulting from the high amounts of DES in the cells. The presence of small aggregates or small DES structures in HEK cells overexpressing DES_dupGGA was also confirmed by Western blot in native conditions, where lower molecular weight bands in PAGE were observed compared to HEK cells overexpressing DES_WT. Control SDS-PAGE confirmed the same result for both experimental groups. Similar to DES, VIM is another intermediate filament that can experience abnormal assembly in cells expressing certain mutations. In this study, we examined the effects of DES_dupGGA mutation on VIM. When we analyzed VIM expression in the three cell lines, we observed an extended filamentous network in HEK cells and HEK cells overexpressing DES_WT. These aggregates specifically occurred in mCherry-expressing cells and were not found in mCherry-negative cells, suggesting that DES_dupGGA affected VIM polymerization. To evaluate the possibility of cytotoxic effects of DESdupGGA mutation, we evaluated the activation of Caspase 3 in control HEK cells and HEK cells that overexpress DES_dupGGA and DES_WT after 72 h of doxycycline induction. We did not observe any differences among the groups. This indicates that overexpression of DES_dupGGA or DES_WT did not induce cell apoptosis under the conditions tested.

Comment:

The c.1059_1061dupGGA mutation in DES involves the duplication of Glu at position 353 of DES. According to guidelines of the American College of Medical Genetics and Genomics, in-frame insertions are considered moderate indicative of pathogenicity (Richards et al 2015). Additionally, this mutation is located in exon 6 of the protein, which is part of the highly conserved coil 2B domain (known to govern DES polypeptide assembly into and subsequent higher-order structures; Bar et al 2004; Goebel et al 2011; Walter et al 2007) and where many pathogenic mutations were observed in patients. Moreover, disruptive DES filament assembly was in vitro observed in the presence of the mutation (doi.org/10.21203/rs.3.rs-3805874/v1).

Germline Functional Evidence

There is no functional evidence in ClinVar for this variation. If you have generated functional data for this variation, please consider submitting that data to ClinVar.

Comment:

Citations for germline classification of this variant

There are no citations for germline classification of this variant in ClinVar. If you know of citations for this variation, please consider submitting that information to ClinVar.

Text-mined citations for this variant ...

Record last updated Jun 23, 2024

ClinVar Genomic variation as it relates to human health

NM_001927.4(DES):c.1056GGA[3] (p.Glu353_Asp354insGlu)

Variant Details

Genes

Conditions - Germline

Submissions - Germline

Germline Functional Evidence

Citations for germline classification of this variant

Text-mined citations for this variant ...