Submissions for variant NM_001164508.2(NEB):c.21417+3A>G

gnomAD frequency: 0.00004  dbSNP: rs148950085

Total submissions: 8

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
GeneDx	RCV000493255	SCV000582836	pathogenic	not provided	2024-08-01	criteria provided, single submitter	clinical testing	Observed multiple times with a second NEB variant in unrelated patients in the published literature with myopathy, but it is not known whether the variants occurred on the same (in cis) or on different (in trans) chromosomes (PMID: 33742414; Lee et al., (2020) Abstracts/Neuromuscular Disorders 30 S46-S150); In silico analysis supports a deleterious effect on splicing; RNA studies demonstrate a damaging effect: skipping of exon 143 which is in-frame (PMID: 32222963); Deletions involving coding exons of this gene are a known mechanism of disease (HGMD; other references); This variant is associated with the following publications: (PMID: 30990797, 31696431, 33742414, 27535533, 35821219, 38585796, 32222963, 37525074)
Labcorp Genetics (formerly Invitae), Labcorp	RCV001086213	SCV001020043	pathogenic	Nemaline myopathy 2	2024-01-31	criteria provided, single submitter	clinical testing	This sequence change falls in intron 144 of the NEB gene. It does not directly change the encoded amino acid sequence of the NEB protein. RNA analysis indicates that this variant induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs148950085, gnomAD 0.2%). This variant has been observed in individual(s) with nemaline myopathy (PMID: 31696431, 32222963). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. This variant is also known as c.21417+3A>G. ClinVar contains an entry for this variant (Variation ID: 430110). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in skipping of exon 144, but is expected to preserve the integrity of the reading-frame (PMID: 32222963). For these reasons, this variant has been classified as Pathogenic.
Revvity Omics, Revvity	RCV000493255	SCV003812156	uncertain significance	not provided	2019-12-10	criteria provided, single submitter	clinical testing
Broad Center for Mendelian Genomics, Broad Institute of MIT and Harvard	RCV001086213	SCV003922351	pathogenic	Nemaline myopathy 2	2023-05-02	criteria provided, single submitter	curation	The heterozygous c.21522+3A>G variant in NEB was identified by our study, in the compound heterozygous state with a likely pathogenic variant (NC_000002.12:g.151692086G>T), in two affected siblings with nemaline myopathy 2. Familial exome analysis confirmed that this variant was in trans with a likely pathogenic variant (NC_000002.12:g.151692086G>T). The c.21522+3A>G variant in NEB has been reported in 26 affected individuals (PMID: 32222963, PMID: 29246625, PMID: 33742414, PMID: 31696431, PMID: 30990797), but has been identified in 0.2% (28/12460) of East Asian chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs148950085). 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: 430110) and has conflicting interpretations of pathogenicity. Of the 26 affected individuals previously reported, one was a homozygote (PMID: 29246625) and six were compound heterozygotes who carried pathogenic or likely pathogenic variants in trans (PMID: 31696431, PMID: 32222963, ClinVar Variation ID: 632921, ClinVar Variation ID: 575054), which increases the likelihood that the c.21522+3A>G variant is pathogenic. RT-PCR analysis performed on affected tissue shows exon skipping of exon 143 (PMID: 32222963). This variant is located in the 5' splice region. Computational tools do suggest an impact to splicing. However, this information is not predictive enough to determine pathogenicity. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive nemaline myopathy 2. ACMG/AMP Criteria applied: PS3_Moderate, PM3_Very Strong (Richards 2015).
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV003230521	SCV003929204	uncertain significance	not specified	2023-04-06	criteria provided, single submitter	clinical testing	Variant summary: NEB c.21522+3A>G alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. 4/4 computational tools predict no significant impact on normal splicing. However, RT-PCR analysis of patient samples showed the variant to result in skipping of exon 143. It is unclear what impact this exon skipping has on protein function (Wang_2020). The variant allele was found at a frequency of 0.00019 in 157544 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in NEB causing Nemaline Myopathy 2 (0.00019 vs 0.0035), allowing no conclusion about variant significance. However the variant is found at a higher frequency in East Asian populations. c.21522+3A>G has been reported in the literature in individuals affected with Nemaline Myopathy 2, especially in populations of East Asian descent (examples: Lee_2017, Wen_2020, Wang_2020, Yin_2022) with many examples of heterozygotes carrying no second variant or a second variant with ClinVar classifications ranging from pathogenic to likely benign. At least 5 patients have been reported with pathogenic variants as second reported allele. These data provide conflicting evidence about the variant's association with disease. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation, classifying the variant as uncertain significance (n=1), likely pathogenic (n=1), or likely benign (n=1). Based on the evidence outlined above, the variant was classified as VUS - possibly pathogenic.
Baylor Genetics	RCV003470617	SCV004200047	pathogenic	Arthrogryposis multiplex congenita 6	2024-03-28	criteria provided, single submitter	clinical testing
Neuberg Centre For Genomic Medicine, NCGM	RCV001086213	SCV005042554	likely pathogenic	Nemaline myopathy 2		criteria provided, single submitter	clinical testing	The splice region, intron variant c.21522+3A>G in NEB gene has been reported previously in compound heterozygous state in individuals with nemaline myopathy Yin X, et al., 2022, Wen Q, et al., 2020. The variant is reported with 0.02% allele frequency in gnomAD Exomes and is novel not in any individuals in 1000 Genomes. This variant has been reported to the ClinVar database as Likely Benign/Uncertain Significance/Likely Pathogenic. However, functional studies are required to confirm its pathogenicity. The variant affects the position three nucleotides downstream of exon 144. The spliceAI tool predicts this splice site variant to be likely damaging. This variant is predicted to cause loss of normal protein function through protein truncation. Loss of function variants have been previously reported to be disease causing Lehtokari VL, et al., 2014. For these reasons, this variant has been classified as Likely Pathogenic.
PreventionGenetics, part of Exact Sciences	RCV004737563	SCV005360116	pathogenic	NEB-related disorder	2024-06-07	no assertion criteria provided	clinical testing	The NEB c.21522+3A>G variant is predicted to interfere with splicing. This variant has been reported with a second NEB variant in many individuals with nemaline myopathy and has been described as a possible founder variant in East Asian populations (Wen et al. 2019. PubMed ID: 31696431; Yin et al. 2021. PubMed ID: 33742414; reported as c.21417+3A>G (NM_001164508) in Wang et al. 2020. PubMed ID: 32222963). This variant is reported in 0.22% of alleles in individuals of East Asian descent in gnomAD. RT-PCR studies suggest this variant impacts mRNA splicing, resulting in skipping of exon 144 of NM_001271208 (Wang et al. 2020. PubMed ID: 32222963). An additional study using RNA-seq analysis on muscle tissue confirms the c.21522+3A>G variant results in impaired splicing (Silverstein et al. 2024. PubMed ID: 38585796, peer review in progress). Another nucleotide change at the same position (c.21522+3A>C) has also been reported in an individual with nemaline myopathy and indicated to interfere with normal splicing (Cummings et al. 2017. PubMed ID: 28424332). This variant is interpreted as pathogenic.