rs1276026682

Variant names:

• chr2-151679718-T-TA
• rs1276026682
• NM_001164507.2:c.3255+2dupT

Variant summary

Our verdict is Uncertain significance. The variant received 5 ACMG points: 5P and 0B. PVS1_Moderate PM2 PP5

The NM_001164507.2(NEB):​c.3255+2dupT variant causes a splice donor, intron change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000274 in 1,457,936 control chromosomes in the GnomAD database, with no homozygous occurrence. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: 𝑓 0.0000068 (0 hom., cov: 30)
  • Exomes 𝑓: 0.0000023 (0 hom.)
• Consequence: NEB NM_001164507.2 splice_donor, intron
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:2 U:4
• Conservation: PhyloP100: 9.29
• Publications: 0 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]

NEB Gene-Disease associations (from GenCC):

• nemaline myopathy 2

  Inheritance: AR, AD Classification: DEFINITIVE, STRONG, LIMITED Submitted by: ClinGen, Labcorp Genetics (formerly Invitae), Myriad Women’s Health, G2P, Ambry Genetics
• childhood-onset nemaline myopathy

  Inheritance: AD Classification: SUPPORTIVE Submitted by: Orphanet
• intermediate nemaline myopathy

  Inheritance: AD Classification: SUPPORTIVE Submitted by: Orphanet
• typical nemaline myopathy

  Inheritance: AD Classification: SUPPORTIVE Submitted by: Orphanet
• lethal multiple pterygium syndrome

  Inheritance: AR Classification: SUPPORTIVE Submitted by: Orphanet
• severe congenital nemaline myopathy

  Inheritance: AR Classification: SUPPORTIVE Submitted by: Orphanet

ACMG classification

Our verdict: Uncertain_significance. The variant received 5 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.0042223786 fraction of the gene. Cryptic splice site detected, with MaxEntScore 3.8, offset of 44, new splice context is: tggGTgggt. Cryptic site results in frameshift change. If cryptic site found is not functional and variant results in exon loss, it results in inframe change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 2-151679718-T-TA is Pathogenic according to our data. Variant chr2-151679718-T-TA is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 551957.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
NEB	NM_001164507.2	c.3255+2dupT		splice_donor_variant, intron_variant	Intron 32 of 181	ENST00000427231.7	NP_001157979.2
NEB	NM_001164508.2	c.3255+2dupT		splice_donor_variant, intron_variant	Intron 32 of 181	ENST00000397345.8	NP_001157980.2

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
NEB	ENST00000397345.8	c.3255+2_3255+3insT		splice_donor_variant, intron_variant	Intron 32 of 181	5	NM_001164508.2	ENSP00000380505.3
NEB	ENST00000427231.7	c.3255+2_3255+3insT		splice_donor_variant, intron_variant	Intron 32 of 181	5	NM_001164507.2	ENSP00000416578.2
NEB	ENST00000409198.5	c.3255+2_3255+3insT		splice_donor_variant, intron_variant	Intron 32 of 149	5		ENSP00000386259.1

Frequencies

GnomAD3 genomes AF: 0.00000682 AC: 1 AN: 146694 Hom.: 0 Cov.: 30

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: 0.0000151

Gnomad OTH AF: 0.00

GnomAD2 exomes AF: 0.00000403 AC: 1 AN: 247968 AF XY: 0.00000743

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: 0.00000893

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000229 AC: 3 AN: 1311242 Hom.: 0 Cov.: 31 AF XY: 0.00000307 AC XY: 2 AN XY: 650506

African (AFR) AF: 0.00 AC: 0 AN: 29080

American (AMR) AF: 0.00 AC: 0 AN: 40174

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 20848

East Asian (EAS) AF: 0.00 AC: 0 AN: 28308

South Asian (SAS) AF: 0.00 AC: 0 AN: 84756

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 39152

Middle Eastern (MID) AF: 0.000198 AC: 1 AN: 5038

European-Non Finnish (NFE) AF: 0.00000197 AC: 2 AN: 1013360

Other (OTH) AF: 0.00 AC: 0 AN: 50526

GnomAD4 genome AF: 0.00000682 AC: 1 AN: 146694 Hom.: 0 Cov.: 30 AF XY: 0.00 AC XY: 0 AN XY: 71566

African (AFR) AF: 0.00 AC: 0 AN: 40434

American (AMR) AF: 0.00 AC: 0 AN: 14834

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 3402

East Asian (EAS) AF: 0.00 AC: 0 AN: 4552

South Asian (SAS) AF: 0.00 AC: 0 AN: 4232

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 9616

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 310

European-Non Finnish (NFE) AF: 0.0000151 AC: 1 AN: 66392

Other (OTH) AF: 0.00 AC: 0 AN: 2044

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:2 Uncertain:4

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Nemaline myopathy 2 Uncertain:2

Aug 26, 2021

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change falls in intron 32 of the NEB gene. It does not directly change the encoded amino acid sequence of the NEB protein. It affects a nucleotide within the consensus splice site of the intron. This variant is not present in population databases (ExAC no frequency). This variant has been observed in individuals with nemaline myopathy (PMID: 25205138). ClinVar contains an entry for this variant (Variation ID: 551957). 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. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -

May 19, 2017

Counsyl

Significance: Uncertain significance

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. -

not provided Pathogenic:1

Mar 14, 2024

GeneDx

Significance: 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); Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 25205138) -

Arthrogryposis multiplex congenita 6 Pathogenic:1

Feb 06, 2024

Baylor Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

not specified Uncertain:1

Feb 29, 2024

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

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: NEB c.3255+2dupT alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Four predict the variant abolishes the canonical 5' splicing donor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 4e-06 in 247968 control chromosomes. c.3255+2dupT has been reported in the literature at a homozygous state in at-least one individual affected with Nemaline Myopathy (example, Lehtokari_2014). These data indicate that the variant may be associated with disease. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publication have been ascertained in the context of this evaluation (PMID: 25205138). ClinVar contains an entry for this variant (Variation ID: 551957). Based on the evidence outlined above, the variant was classified as VUS-possibly pathogenic. -

Nemaline myopathy Uncertain:1

Mar 20, 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.3255+2dup variant in NEB has been identified in two individuals with nemaline myopathy (PMID: 25205138), and has been identified in 0.0003% (3/1079752) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs991716529). Although this variant has been seen in the general population in a heterozygous state, its frequency is low enough to be consistent with a recessive carrier frequency. This variant has also been reported in ClinVar (Variation ID: 551957) and has been interpreted as a variant of uncertain significance by Counsyl and Invitae. Of the 2 affected individuals, one of those was a homozygote, which increases the likelihood that the c.3255+2dup variant is pathogenic (PMID: 25205138). This variant is located in the 3’ splice region. Computational tools do suggest an impact to splicing. However, this information is not predictive enough to determine pathogenicity. Four additional likely pathogenic/pathogenic variants, predicted to induce the same splicing effect as this variant, have been reported in ClinVar as being associated with nemaline myopathy, supporting that the c.3255+2dup variant may be pathogenic (Variation ID: 449502, 552035, 553174, 556478). In summary, while there is some suspicion for a pathogenic role, the clinical significance of this variant is uncertain. ACMG/AMP Criteria applied: PS1_moderate, PP3, PM2_supporting, PM3_supporting (Richards 2015). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
PhyloP100		9.3
Mutation Taster		=0/100	disease causing

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.40		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.40		Position offset: 3

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

Other links and lift over

dbSNP: rs1276026682; hg19: chr2-152536232;