rs200449517

Variant names:

• chr2-151519656-A-G
• rs200449517
• NM_001164507.2:c.22590+2T>C

Variant summary

Our verdict is Uncertain significance. The variant received 0 ACMG points: 4P and 4B. BS1 PVS1_Strong

This summary comes from the ClinGen Evidence Repository: The NM_001164508.2:c.22590+2T>C variant in NEB occurs within the canonical splice donor site (+2) of intron 154. It is predicted to cause skipping of biologically-relevant-exon 154/182, resulting in an in-frame deletion (removes amino acids V7494-E7530). As per ClinGen Congenital Myopathies VCEP specifications, in-frame exon skipping of the NEB gene is an established disease mechanism in autosomal recessive nemaline myopathy (PVS1_Strong). The highest population minor allele frequency in gnomAD v4.1 is 0.0004030 (469/1163744 alleles) in European (non-Finnish) population, which is higher than the ClinGen Congenital Myopathies threshold ≥0.000237 for BS1 and therefore meets this criterion (BS1). Although this variant has been seen in the general population in a heterozygous state, with no homozygous observations, its frequency is not high enough to rule out a pathogenic role. This variant has been observed in individuals with congenital myopathies in a heterozygous state without a second variant. Therefore, no codes were applied (Internal data: GeneDx SCV000619698.7, CeGaT Center for Human Genetics Tuebingen SCV001152434.31, Fulgent Genetics SCV005650398.1). In summary, due to conflicting evidence, this variant is classified as a variant of uncertain significance for autosomal recessive nemaline myopathy based on the ACMG/AMP criteria applied, as specified by the ClinGen Congenital Myopathies: PVS1_Strong, BS1. (ClinGen Congenital Myopathies VCEP specifications version 1.0.0; 06/16/2025). LINK:https://erepo.genome.network/evrepo/ui/classification/CA1906606/MONDO:0018958/146

• Frequency:
  • Genomes: 𝑓 0.000092 (0 hom., cov: 32)
  • Exomes 𝑓: 0.00033 (0 hom.)
• Consequence: NEB NM_001164507.2 splice_donor, intron
• Scores: Splicing: ADA: 0.9960
• Clinical Significance: Uncertain significance reviewed by expert panel P:9 U:3
• Conservation: PhyloP100: 6.75
• Publications: 4 publications found

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]

RIF1 (HGNC:23207): (replication timing regulatory factor 1) This gene encodes a protein that shares homology with the yeast teleomere binding protein, Rap1 interacting factor 1. This protein localizes to aberrant telomeres may be involved in DNA repair. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Apr 2010]

ACMG classification

Our verdict: Uncertain_significance. The variant received 0 ACMG points.

• PVS1: For more information check the summary or visit ClinGen Evidence Repository.
• BS1: For more information check the summary or visit ClinGen Evidence Repository.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
NEB	NM_001164507.2	c.22590+2T>C		splice_donor_variant, intron_variant	Intron 154 of 181	ENST00000427231.7	NP_001157979.2
NEB	NM_001164508.2	c.22590+2T>C		splice_donor_variant, intron_variant	Intron 154 of 181	ENST00000397345.8	NP_001157980.2

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
NEB	ENST00000397345.8	c.22590+2T>C		splice_donor_variant, intron_variant	Intron 154 of 181	5	NM_001164508.2	ENSP00000380505.3
NEB	ENST00000427231.7	c.22590+2T>C		splice_donor_variant, intron_variant	Intron 154 of 181	5	NM_001164507.2	ENSP00000416578.2

Frequencies

GnomAD3 genomes AF: 0.0000920 AC: 14 AN: 152226 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.0000241

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: 0.000191

Gnomad OTH AF: 0.00

GnomAD2 exomes AF: 0.0000926 AC: 23 AN: 248252 AF XY: 0.0000742

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.000196

Gnomad OTH exome AF: 0.000166

GnomAD4 exome AF: 0.000328 AC: 474 AN: 1443192 Hom.: 0 Cov.: 29 AF XY: 0.000310 AC XY: 223 AN XY: 719150

African (AFR) AF: 0.0000606 AC: 2 AN: 32998

American (AMR) AF: 0.00 AC: 0 AN: 44658

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 26010

East Asian (EAS) AF: 0.00 AC: 0 AN: 39540

South Asian (SAS) AF: 0.00 AC: 0 AN: 85748

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53074

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5668

European-Non Finnish (NFE) AF: 0.000416 AC: 456 AN: 1095698

Other (OTH) AF: 0.000268 AC: 16 AN: 59798

GnomAD4 genome AF: 0.0000920 AC: 14 AN: 152226 Hom.: 0 Cov.: 32 AF XY: 0.0000403 AC XY: 3 AN XY: 74368

African (AFR) AF: 0.0000241 AC: 1 AN: 41458

American (AMR) AF: 0.00 AC: 0 AN: 15286

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 3470

East Asian (EAS) AF: 0.00 AC: 0 AN: 5202

South Asian (SAS) AF: 0.00 AC: 0 AN: 4832

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 10612

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 316

European-Non Finnish (NFE) AF: 0.000191 AC: 13 AN: 68046

Other (OTH) AF: 0.00 AC: 0 AN: 2092

Alfa AF: 0.000219 Hom.: 1

Bravo AF: 0.0000945

TwinsUK AF: 0.000270 AC: 1

ALSPAC AF: 0.000778 AC: 3

ESP6500AA AF: 0.00 AC: 0

ESP6500EA AF: 0.000122 AC: 1

ExAC AF: 0.000116 AC: 14

EpiCase AF: 0.000164

EpiControl AF: 0.000297

ClinVar

Significance: Uncertain significance

Submissions summary: Pathogenic:9 Uncertain:3

Revision: reviewed by expert panel

Submissions by phenotype

Nemaline myopathy 2 Pathogenic:4

Sep 16, 2020

Natera, Inc.

Significance: Likely pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Jan 08, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change affects a donor splice site in intron 155 of the NEB gene. It is expected to disrupt RNA splicing. Variants that disrupt the donor or acceptor splice site typically lead to a loss of protein function (PMID: 16199547), and loss-of-function variants in NEB are known to be pathogenic (PMID: 25205138). This variant is present in population databases (rs200449517, gnomAD 0.02%). This variant has not been reported in the literature in individuals affected with NEB-related conditions. ClinVar contains an entry for this variant (Variation ID: 451052). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. -

Apr 05, 2017

Counsyl

Significance: Likely pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

This submission and the accompanying classification are no longer maintained by the submitter. For more information on current observations and classification, please contact variantquestions@myriad.com. -

Jun 24, 2022

Victorian Clinical Genetics Services, Murdoch Childrens Research Institute

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as likely 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) 0115 - Variants in this gene are known to have variable expressivity (OMIM). (I) 0211 - Canonical splice site variant without proven consequence on splicing (no functional evidence available). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (28 heterozygotes, 0 homozygotes). (SP) 0505 - Abnormal splicing is predicted by in silico tools and affected nucleotide is highly conserved. (SP) 0705 - No comparable canonical splice variants have previous evidence for pathogenicity. (I) 0802 - This variant has moderate previous evidence of pathogenicity in unrelated individuals. This variant has been reported once as a VUS, but multiple times as likely pathogenic but with limited clinical information. One individual had progressive muscle weakness and foot drop with a variant of uncertain significance in trans (ClinVar, personal communication). It has also been observed in a cohort with retinopathy (PMID: 28132693). (SP) 0905 - No published segregation evidence has been identified for this variant. (I) 1007 - No published functional evidence has been identified for this variant. (I) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -

not provided Pathogenic:3

Oct 02, 2020

Revvity Omics, Revvity

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Feb 03, 2025

GeneDx

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Canonical splice site variant predicted to result in an in-frame loss of the adjacent exon in a gene for which loss of function is a known mechanism of disease; Deletions involving coding exons of this gene are a known mechanism of disease (HGMD); Has not been previously published as pathogenic or benign to our knowledge -

Sep 01, 2017

CeGaT Center for Human Genetics Tuebingen

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Nemaline myopathy Pathogenic:1 Uncertain:2

Jun 16, 2025

ClinGen Congenital Myopathies Variant Curation Expert Panel, ClinGen

Significance: Uncertain significance

Review Status: reviewed by expert panel

Collection Method: curation

The NM_001164508.2:c.22590+2T>C variant in NEB occurs within the canonical splice donor site (+2) of intron 154. It is predicted to cause skipping of biologically-relevant-exon 154/182, resulting in an in-frame deletion (removes amino acids V7494-E7530). As per ClinGen Congenital Myopathies VCEP specifications, in-frame exon skipping of the NEB gene is an established disease mechanism in autosomal recessive nemaline myopathy (PVS1_Strong). The highest population minor allele frequency in gnomAD v4.1 is 0.0004030 (469/1163744 alleles) in European (non-Finnish) population, which is higher than the ClinGen Congenital Myopathies threshold ≥0.000237 for BS1 and therefore meets this criterion (BS1). Although this variant has been seen in the general population in a heterozygous state, with no homozygous observations, its frequency is not high enough to rule out a pathogenic role. This variant has been observed in individuals with congenital myopathies in a heterozygous state without a second variant. Therefore, no codes were applied (Internal data: GeneDx SCV000619698.7, CeGaT Center for Human Genetics Tuebingen SCV001152434.31, Fulgent Genetics SCV005650398.1). In summary, due to conflicting evidence, this variant is classified as a variant of uncertain significance for autosomal recessive nemaline myopathy based on the ACMG/AMP criteria applied, as specified by the ClinGen Congenital Myopathies: PVS1_Strong, BS1. (ClinGen Congenital Myopathies VCEP specifications version 1.0.0; 06/16/2025). -

Apr 10, 2025

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

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: NEB c.22695+2T>C is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing and loss of NEB function. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes a 5 splicing donor site. One predict the variant no significant impact on splicing. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 9.3e-05 in 248252 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in NEB causing Nemaline Myopathy 2 (9.3e-05 vs 0.0035), allowing no conclusion about variant significance. c.22695+2T>C has been reported in the literature in an individual affected with simplex Retinitis Pigmentosa without any familial history of Nemaline Myopathy and who did not have a reported second pathogenic variant (Arno_2017). This report does not provide unequivocal conclusions about association of the variant with Nemaline Myopathy 2. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publication has been ascertained in the context of this evaluation (PMID: 28132693). ClinVar contains an entry for this variant (Variation ID: 451052). Based on the evidence outlined above, the variant was classified as likely pathogenic. -

Mar 03, 2025

Broad Center for Mendelian Genomics, Broad Institute of MIT and Harvard

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: curation

The c.22590+2T>C variant in NEB has not been previously reported in the literature in individuals with nemaline myopathy, but has been identified in 0.04% (469/1163744) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs200449517). Although this variant has been seen in the general population in a heterozygous state, its frequency is not high enough to rule out a pathogenic role. This variant has also been reported in ClinVar (Variation ID: 451052) and has been interpreted as likely pathogenic by multiple submitters, and as a variant of uncertain significance by Illumina Laboratory Services. This variant is located in the 5' splice region. Computational tools predict a splicing impact, though this information is not predictive enough to determine pathogenicity. This variant is adjacent to an in-frame exon and is more likely to escape nonsense mediated decay (NMD) and result in a truncated protein. In-frame exon skipping of the NEB gene is an established disease mechanism in autosomal recessive nemaline myopathy. In summary, the clinical significance of the c.22590+2T>C variant is uncertain. ACMG/AMP Criteria applied: PVS1_strong (Richards 2015). -

Nemaline myopathy 2;C5543431:Arthrogryposis multiplex congenita 6 Pathogenic:1

Jun 12, 2024

Fulgent Genetics, Fulgent Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Arthrogryposis multiplex congenita 6 Uncertain:1

Aug 09, 2023

Baylor Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.41	D
BayesDel_noAF	Pathogenic	0.38
CADD	Pathogenic	30
DANN	Uncertain	0.99
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.0
FATHMM_MKL	Pathogenic	0.99	D
PhyloP100		6.8
GERP RS		6.2
Mutation Taster		=3/97	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.72
SpliceAI score (max)		0.97		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.97		Position offset: 2

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

Other links and lift over

dbSNP: rs200449517; hg19: chr2-152376170; COSMIC: COSV50836851;