rs201553266

Positions:

• chr2-151551738-C-T

Variant summary

Our verdict is Pathogenic. Variant got 10 ACMG points: 10P and 0B. PM2 PP5_Very_Strong

The NM_004543.5(NEB):​c.14841G>A​(p.Ser4947Ser) variant causes a splice region, synonymous change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.0000136 in 1,612,444 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★).

• Frequency:
  • Genomes: 𝑓 0.0000066 (0 hom., cov: 32)
  • Exomes 𝑓: 0.000014 (0 hom.)
• Consequence: NEB NM_004543.5 splice_region, synonymous
• Scores: Splicing: ADA: 0.9999
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:12 U:2
• Conservation: PhyloP100: 0.745

Genes affected

NEB (HGNC:7720): (nebulin) This gene encodes nebulin, a giant protein component of the cytoskeletal matrix that coexists with the thick and thin filaments within the sarcomeres of skeletal muscle. In most vertebrates, nebulin accounts for 3 to 4% of the total myofibrillar protein. The encoded protein contains approximately 30-amino acid long modules that can be classified into 7 types and other repeated modules. Protein isoform sizes vary from 600 to 800 kD due to alternative splicing that is tissue-, species-,and developmental stage-specific. Of the 183 exons in the nebulin gene, at least 43 are alternatively spliced, although exons 143 and 144 are not found in the same transcript. Of the several thousand transcript variants predicted for nebulin, the RefSeq Project has decided to create three representative RefSeq records. Mutations in this gene are associated with recessive nemaline myopathy. [provided by RefSeq, Sep 2009]

ACMG classification

Verdict is Pathogenic. Variant got 10 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 2-151551738-C-T is Pathogenic according to our data. Variant chr2-151551738-C-T is described in ClinVar as [Likely_pathogenic]. Clinvar id is 235402.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr2-151551738-C-T is described in Lovd as [Pathogenic]. Variant chr2-151551738-C-T is described in Lovd as [Likely_pathogenic]. Variant chr2-151551738-C-T is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
NEB	NM_001164507.2	c.19944G>A	p.Ser6648Ser	synonymous_variant	129/182	ENST00000427231.7	NP_001157979.2
NEB	NM_001164508.2	c.19944G>A	p.Ser6648Ser	synonymous_variant	129/182	ENST00000397345.8	NP_001157980.2

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
NEB	ENST00000397345.8	c.19944G>A	p.Ser6648Ser	synonymous_variant	129/182	5	NM_001164508.2	ENSP00000380505.3
NEB	ENST00000427231.7	c.19944G>A	p.Ser6648Ser	synonymous_variant	129/182	5	NM_001164507.2	ENSP00000416578.2

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152136 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.00

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.0000147

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000282 AC: 7 AN: 248206 Hom.: 0 AF XY: 0.0000297 AC XY: 4 AN XY: 134644

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.0000582

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.000112

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000178

Gnomad OTH exome AF: 0.000166

GnomAD4 exome AF: 0.0000144 AC: 21 AN: 1460308 Hom.: 0 Cov.: 30 AF XY: 0.0000138 AC XY: 10 AN XY: 726526

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.0000896

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.0000505

Gnomad4 SAS exome AF: 0.0000116

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.0000108

Gnomad4 OTH exome AF: 0.0000332

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152136 Hom.: 0 Cov.: 32 AF XY: 0.00 AC XY: 0 AN XY: 74306

Gnomad4 AFR AF: 0.00

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.0000147

Gnomad4 OTH AF: 0.00

Bravo AF: 0.0000189

EpiCase AF: 0.00

EpiControl AF: 0.0000593

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:12 Uncertain:2

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Nemaline myopathy 2 Pathogenic:3 Uncertain:1

Pathogenic, criteria provided, single submitter	clinical testing	Victorian Clinical Genetics Services, Murdoch Childrens Research Institute	Oct 09, 2024	Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with arthrogryposis multiplex congenita 6 (MIM#619334) and nemaline myopathy 2 (MIM#256030). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0210 - Splice site variant proven to affect splicing of the transcript with known effect on protein sequence. RT-PCR studies on blood from an individual homozygous for this variant have shown it causes loss of the canonical donor splice site, and gain of a cryptic splice site in the intron. This causes the retention of 120 intronic nucleotides creating a premature stop codon that is expected to lead to nonsense-mediated decay (PMID: 26841830). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (7 heterozygotes, 0 homozygotes). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been classified as pathogenic or likely pathogenic by multiple clinical laboratories in ClinVar, and has been observed as homozygous or compound heterozygous in individuals with congenital myopathy in the literature (PMIDs: 26841830, 35081925). (SP) 1205 - This variant has been shown to be maternally inherited (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -
Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Nov 14, 2023	This sequence change affects codon 6648 of the NEB mRNA. It is a 'silent' change, meaning that it does not change the encoded amino acid sequence of the NEB protein. This variant also falls at the last nucleotide of exon 129, which is part of the consensus splice site for this exon. This variant is present in population databases (rs201553266, gnomAD 0.01%). This variant has been observed in individuals with nemaline myopathy (PMID: 25205148; Invitae). ClinVar contains an entry for this variant (Variation ID: 235402). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. For these reasons, this variant has been classified as Pathogenic. -
Likely pathogenic, no assertion criteria provided	clinical testing	Counsyl	Jul 30, 2018	- -
Uncertain significance, flagged submission	research	Muscle and Diseases Team, Institut de Génétique et Biologie Moléculaire et Cellulaire	Mar 01, 2024	PM3+PM2+PP3 -

not provided Pathogenic:2 Uncertain:1

Pathogenic, criteria provided, single submitter	clinical testing	Center for Pediatric Genomic Medicine, Children's Mercy Hospital and Clinics	Aug 13, 2014	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	May 15, 2021	Cryptic splice site that introduces a premature stop codon in gene for which loss-of-function is a known mechanism of disease (Oliveira et al., 2016); Not observed at significant frequency in large population cohorts (Lek et al., 2016); This variant is associated with the following publications: (PMID: 9536098, 17576681, 32721234, 32222963, 26841830, 26403434, 25205138) -
Uncertain significance, flagged submission	clinical testing	Eurofins Ntd Llc (ga)	Feb 24, 2017	- -

Arthrogryposis multiplex congenita 6 Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	DASA	Feb 14, 2022	This sequence change has been observed in affected individual(s) and ClinVar contains an entry for this variant (ClinVar ID: 235402; PMID: 9536098; 17576681; 32721234; 32222963; 26841830; 26403434; 25205138) - PS4. The variant is present at low allele frequencies population databases (rs201553266 - gnomAD 0.00006573%; ABraOM no frequency - http://abraom.ib.usp.br/) - PM2_supporting. The p.(Ser6648=) was detected in trans with a pathogenic variant (PMID: 9536098; 17576681; 32721234) - PM3_strong. Multiple lines of computational evidence support a deleterious effect on the gene or gene product - PP3. In summary, the currently available evidence indicates that the variant is pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Mar 09, 2024	- -

Nemaline myopathy 2;C5543431:Arthrogryposis multiplex congenita 6 Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Fulgent Genetics, Fulgent Genetics	Jun 06, 2024	- -
Pathogenic, criteria provided, single submitter	clinical testing	Juno Genomics, Hangzhou Juno Genomics, Inc	-	PS3+PM2_Supporting+PM3_Strong+PP4 -

NEB-related disorder Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

PreventionGenetics, part of Exact Sciences

May 26, 2023

The NEB c.19944G>A variant is not predicted to result in an amino acid change (p.=). This variant is predicted to abolish the adjacent splice donor site (Alamut Visual Plus v1.6.1). This variant has been reported along with a second putative disease-causing variant in multiple individuals with nemaline myopathy; in most cases parental testing confirmed the variants were on opposite alleles (Lehtokari et al 2014. PubMed ID: 25205138; Wang Q et al 2020. PubMed ID: 32222963; Zhang Y et al 2022. PubMed ID: 35081925). This variant is reported in 0.011% of alleles in individuals of East Asian descent in gnomAD (http://gnomad.broadinstitute.org/variant/2-152408252-C-T). This variant is interpreted as pathogenic. -

Inborn genetic diseases Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jun 12, 2015

- -

Nemaline myopathy Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Jun 12, 2018

Variant summary: NEB c.19944G>A (p.Ser6648Ser) alters a non-conserved nucleotide that is the last nucleotide of an exon and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes a 5' splicing donor site, while two predict the variant weakens a 5' donor site. These predictions are supported by functional evidence reported by Oliveira_2016 using whole blood from a homozygous patient, which showed the preferential use of a cryptic splice site, leading to a predicted truncation of the protein. Truncations downstream of this variant have been classified as pathogenic by our laboratory (e.g., p.Arg7026X and p.Leu8137fsX18). The variant allele was found at a frequency of 2.9e-05 in 245250 control chromosomes, which does not exceed the maximal expected allele frequency for a pathogenic variant in NEB. c.19944G>A has been reported in the literature in several individuals affected with Nemaline Myopathy 2, including both homozygous and compound heterozygous patients, indicating that the variant is likely to be associated with disease. Four clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 with conflicting assessments, including pathogenic (1x), likely pathogenic (2x), and VUS (1x). Based on the evidence outlined above, the variant was classified as pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.34
CADD	Benign	21
DANN	Benign	0.53

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.98
SpliceAI score (max)		0.77		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.77		Position offset: 0

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs201553266; hg19: chr2-152408252;