Variant classification summary

NM_005188.3:c.1096-1G>T

CBL · NP_005179.2:p.? · NM_005188.3

GRCh37: chr11:119148875 G>T · GRCh38: chr11:119278165 G>T

Gene: CBL Transcript: NM_005188.3

Final call

Pathogenic

PVS1 very strong PS2 strong PS3 supporting PM1 moderate PM2 supporting PP5 supporting

Variant details

Gene

CBL

Transcript

NM_005188.3

Protein

NP_005179.2:p.?

gnomAD AF

6.287433809040576e-07 (v4.1)

ClinVar

Pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_005188.3:c.1096-1G>T is a canonical splice acceptor variant in CBL, disrupting the AG dinucleotide at the intron 7 / exon 8 boundary.

2

CBL loss-of-function is an established disease mechanism for a RASopathy phenotype with predisposition to juvenile myelomonocytic leukemia (CBL mutation-associated syndrome).

3

The variant has been observed as a de novo occurrence in two unrelated individuals with features consistent with CBL-related RASopathy, with confirmed paternity in both cases.

4

A minigene assay demonstrated that c.1096-1G>T results in complete skipping of exon 8, which encodes the linker region and RING finger domain critical for CBL E3 ubiquitin ligase function.

5

The variant affects the RING finger domain / linker region (exons 8-9), a well-established mutational hotspot where approximately 50% of germline CBL mutations cluster and no benign variation is observed.

6

The variant is extremely rare in population databases: absent from gnomAD v2.1 and gnomAD-Canada, and observed in a single heterozygous carrier in gnomAD v4.1 (1/1,590,474 alleles; AF = 0.00006%).

7

This variant is classified as Pathogenic in ClinVar (Variation ID: 180815) by five independent clinical laboratories.

8

Applying generic ACMG/AMP 2015 combination rules: PVS1 (very_strong) + PS2 (strong) + PM1 (moderate) + PS3 (supporting) + PM2 (supporting) + PP5 (supporting) is sufficient for a Pathogenic classification.

Final determination: Generic ACMG/AMP 2015 fallback rules support a Pathogenic classification based on the observed combination of very strong, strong, moderate, and supporting pathogenic criteria.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	Met	This variant affects the canonical splice acceptor site of CBL intron 7 (c.1096-1G>T), disrupting the AG dinucleotide at position -1. Germline loss-of-function variants in CBL are an established disease mechanism for CBL mutation-associated syndrome (a RASopathy with predisposition to JMML). SpliceAI predicts acceptor loss with a max delta score of 0.99. Under ClinGen SVI PVS1 recommendations (PMC6185798), canonical ±1,2 splice site variants in genes with an established LoF mechanism are assigned PVS1 at full strength.	pvs1_gene_context pvs1_variant_assessment pvs1_generic_framework spliceai PMID:25952305
PS1	N/A	PS1 applies to a missense variant at the same amino acid position as a known pathogenic missense change. This is a canonical splice site variant and does not produce a defined amino acid change.
PS2	Met	NM_005188.3:c.1096-1G>T has been observed as a de novo occurrence in two unrelated individuals with features consistent with CBL-related RASopathy, with confirmed paternity in both cases. Martinelli et al. (2015) reported this variant as de novo in a sporadic case (patient 827-10) with parental DNA confirmation. Seaby et al. (2017) reported the same variant as de novo in a child with atypical hemolytic uremic syndrome and RASopathy features.	PMID:25952305 PMID:28589114 clinvar
PS3	Met	A minigene assay performed by Martinelli et al. (2015) demonstrated that c.1096-1G>T results in aberrant splicing with complete skipping of exon 8, which encodes essential regions of the linker region and RING finger domain. This well-established in vitro functional assay confirms a damaging effect on mRNA processing and protein function. Applied at supporting level because this evidence confirms the null effect already captured by PVS1 rather than providing independent functional evidence of a distinct mechanism.	PMID:25952305
PS4	Not assessed	Insufficient data to calculate a statistically significant difference in variant prevalence between affected individuals and the general population. While the variant has been reported in multiple affected individuals through ClinVar submissions and published cases, formal case-control comparison is not available.	clinvar
PS5	N/A	PS5 (also known as PM5) applies to a novel missense change at an amino acid residue where a different pathogenic missense change has been previously observed. This is a canonical splice site variant, not a missense change, and does not produce a defined amino acid substitution.
PM1	Met	The c.1096-1G>T variant affects splicing of exon 8, which encodes the linker region and RING finger domain of CBL. Exons 8 and 9 are well-established mutational hotspots: approximately 50% of germline CBL mutations affect Tyr371 within this region, and splice site mutations affecting introns 7 and 8 account for approximately 10% of CBL defects in myeloid malignancies. The affected region is a critical functional domain with a high density of pathogenic variants and no benign variation.	PMID:25952305
PM2	Met	NM_005188.3:c.1096-1G>T is absent from gnomAD v2.1 and gnomAD-Canada v1.0. In gnomAD v4.1, it is observed in a single heterozygous carrier (1/1,590,474 alleles; AF = 6.29e-07, ~0.00006%), well below the PM2 threshold of 0.1%. The variant is absent from all subpopulations except European (non-Finnish) where it is present in 1/1,159,346 alleles.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	PM5 requires a different pathogenic missense change at the same amino acid residue. This is a canonical splice site variant affecting intron 7; it does not produce a defined amino acid change and no classic same-residue comparator can be established. The pm5_candidates pipeline confirmed no eligible comparators.	pm5_candidates
PM6	N/A	PM6 applies when a variant is assumed de novo but paternity/maternity has not been confirmed. PS2 has already been applied with confirmed paternity in both reported de novo cases; PM6 is not applied when the higher-level PS2 criterion is met.
PP1	Not assessed	No segregation data are available for this variant. The two reported de novo cases are sporadic and no multi-generational families with this variant have been described.
PP2	N/A	PP2 applies to missense variants in genes with a low rate of benign missense variation where missense variants are a common disease mechanism. This is a canonical splice site variant, not a missense change.
PP3	N/A	Multiple in silico tools support a damaging effect (SpliceAI max delta 0.99, acceptor loss). However, under ClinGen SVI PVS1 recommendations, PP3 should not be applied for the same splice prediction evidence already captured by PVS1 for canonical splice variants. The splice effect is already accounted for under PVS1.	spliceai pvs1_variant_assessment
PP4	Not assessed	CBL-related RASopathy has overlapping features with Noonan syndrome and other RASopathies; the phenotype is not sufficiently specific to a single genetic etiology to apply PP4. Clinical features of reported cases include prenatal pleural effusions/hydrops, feeding difficulties, failure to thrive, short stature, hypotonia, psychomotor retardation, and mild pulmonary valve stenosis.	PMID:25952305 PMID:28589114
PP5	Met	NM_005188.3:c.1096-1G>T is classified as Pathogenic in ClinVar (Variation ID: 180815) by five clinical laboratories (GeneDx, Laboratory for Molecular Medicine, Ambry Genetics, Invitae/Labcorp, and King Faisal Specialist Hospital). While the review status is 'criteria provided, single submitter' and no expert panel review is available, multiple independent clinical laboratories have reached the same conclusion based on overlapping evidence.	clinvar
BA1	N/A	BA1 requires an allele frequency >1% in population databases. The variant has an allele frequency of 6.29e-07 (0.00006%) in gnomAD v4.1, far below the 1% threshold.	gnomad_v4
BS1	N/A	BS1 requires an allele frequency >0.3% in population databases. The variant has an allele frequency of 6.29e-07 (0.00006%) in gnomAD v4.1, far below the 0.3% threshold.	gnomad_v4
BS2	Not met	The variant is observed in a single heterozygous carrier in gnomAD v4.1 (1/1,590,474 alleles). For a fully penetrant dominant disorder, a single observation in a population database does not provide sufficient evidence for a benign interpretation. The carrier may have subclinical features or represent incomplete penetrance, which has been described in CBL-related disorders.	gnomad_v4
BS3	Not met	Well-established in vitro functional studies (minigene assay, Martinelli 2015) demonstrate that c.1096-1G>T causes aberrant splicing with complete skipping of exon 8, supporting a damaging rather than benign effect. BS3 requires functional studies showing no damaging effect.	PMID:25952305
BS4	Not assessed	No segregation data are available to assess for lack of cosegregation with disease in affected families. The reported cases are sporadic de novo occurrences; no multi-generational families have been described.
BP1	N/A	BP1 applies to missense variants in genes where primarily truncating variants cause disease. This is a canonical splice site variant, not a missense change.
BP2	N/A	BP2 applies to a variant observed in trans with a pathogenic variant for a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern. No such observations have been reported for this variant.
BP3	N/A	Skipped: trivially not applicable. BP3 applies to in-frame deletions/insertions in repetitive regions without known function; this variant is a canonical splice site substitution.
BP4	Not met	Multiple in silico tools predict a damaging effect on splicing. SpliceAI predicts acceptor loss with a max delta score of 0.99. BP4 requires multiple lines of computational evidence suggesting no impact; the available evidence supports the opposite conclusion.	spliceai
BP5	N/A	BP5 applies when a variant is found in a case with an alternate molecular basis for disease. No such observation has been reported for this variant.
BP6	Not met	ClinVar classifies this variant as Pathogenic (Variation ID: 180815), not Benign or Likely Benign. BP6 requires classification as benign by a reputable source.	clinvar
BP7	N/A	BP7 applies to synonymous variants for which splicing prediction algorithms predict no impact. This variant is a canonical splice site substitution at the intron 7 acceptor site (c.1096-1G>T), not a synonymous variant.

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