MET c.3028+1G>T, p.?

Reasoning

PS3 is met at strong strength. Gray et al. directly studied MET c.3028+1G>T in affected individuals from multiple families and showed by RT-PCR and sequence verification that the variant excludes exon 14 from the mature transcript. The assay used a normal control and an established MET exon-14-skipping control context, and the result was observed in lymphoblastoid cell lines and lesional tissue. Exon 14 exclusion removes the 47-amino-acid juxtamembrane domain; complementary biochemical experiments using the resulting METΔ14 protein showed loss of CBL-mediated receptor destabilization, while the osteoblast assay with induced exon skipping showed reduced osteogenic differentiation and mineralization relative to a scrambled oligonucleotide control. These variant-specific splicing results, concordant biological mechanism, and disease-relevant cellular effects support a damaging functional effect. The study was not described as a validated clinical diagnostic-laboratory assay, but the evidence is sufficiently direct and biologically relevant for the standard PS3 strong code.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Gray et al. 2015, Results, ‘Expression of MET in Familial OFD’: c.3028+1G>T was detected in affected families and RT-PCR with exon-flanking primers showed exclusion of exon 14 from the mature MET transcript; sequencing verified the skipped product.
• Gray et al. 2015, Figure 2A and accompanying legend: affected-individual lymphoblastoid cell lines and OFD lesional tissue were compared with normal control material; the assay distinguished the 507-bp exon-14-inclusive product from the 367-bp exon-14-excluded product.
• Gray et al. 2015, Results, ‘METΔJMD and METp.Tyr1003Ser Receptors Are Resistant to CBL-Mediated Destabilization’: METΔ14 was not destabilized by CBL, unlike wild-type MET, while the unrelated ligase SIAH1 did not produce this differential effect.
• Gray et al. 2015, Results, ‘Diminished Osteoblastic and Osteoclastic Function Resulting from MET Exon Skipping’: induced exon skipping in MC3T3 cells reduced Alp expression, increased Rankl expression, and markedly repressed matrix mineralization compared with a scrambled oligoribonucleotide control.

Sources

Reasoning

PM2 is met at supporting strength. The variant is absent from gnomAD v2.1 and gnomAD v4.1, including the reported non-cancer subsets, which is below the supplied PM2 supporting threshold of <=0.0001. The available population evidence contains no observed allele, so no ancestry-specific frequency or homozygote is present to weaken this conclusion.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: variant absent; reported all-comers dataset has no observed allele or frequency.
• gnomAD v4.1: variant absent; reported all-comers dataset has no observed allele or frequency.
• The evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets; no non-cancer-specific observed allele or frequency is present.
• Generic calibration threshold supplied for this case: PM2 supporting <=0.0001, attributed in the governing materials to the ClinGen SVI recommendation (PMID:25741868).

Sources

Reasoning

PMID:26637977 reports the exact MET c.3028+1G>T variant in affected individuals from families 2, 3, and 4. In family 2, the variant segregated with osteofibrous dysplasia in six penetrant carriers, while family 3 included two penetrant carriers; the paper therefore provides segregation with disease in multiple affected family members across families. The report also experimentally linked the variant to MET exon 14 skipping.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• Gray et al., PMID:26637977, Results and Table 1: exact MET c.3028+1G>T was found in affected individuals from families 2, 3, and 4; family 2 included six penetrant carriers and family 3 included two penetrant carriers.
• Gray et al., PMID:26637977: familial segregation analysis was performed in individuals with bilateral osteofibrous dysplasia; RT-PCR demonstrated exclusion of MET exon 14 from the mature transcript.

Sources

Reasoning

Scope: this is a canonical intronic splice-donor variant, so PP3 uses the SpliceAI path only; REVEL is not applicable and BayesDel has no generic ACMG-strength calibration. SpliceAI max delta is 0.943 (DS_DL), exceeding the generic PP3 moderate threshold of >=0.5 from the SpliceAI high-precision cutoff (Jaganathan et al. 2019, PMID:30661751). The splice prediction is used for PP3 and is not also counted for BP4 or BP7.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• Variant NM_000245.4:c.3028+1G>T is an intronic canonical donor (+1) variant; therefore the applicable computational path is SpliceAI only.
• SpliceAI Lookup reports DS_AG 0.000, DS_AL 0.388, DS_DG 0.506, and DS_DL 0.943; max delta = 0.943.
• Under the supplied generic calibration, SpliceAI PP3 moderate requires >=0.5; the threshold is the SpliceAI high-precision cutoff from Jaganathan et al. 2019 (PMID:30661751).
• BayesDel score 0.312606 was not used because no generic ACMG-strength calibration is established, and REVEL was not applicable to this splice variant.

Sources

Reasoning

PVS1 is not met. NM_000245.4:c.3028+1G>T is a canonical donor-site variant in the MANE Select transcript and therefore has the qualifying splice-site location, but variant-specific RNA and functional evidence show exon 14 skipping rather than an established null transcript: RT-PCR demonstrated exclusion of the 141-nucleotide exon 14, preserving the reading frame and deleting 47 amino acids from the cytoplasmic juxtamembrane domain. The reported consequence is MET receptor stabilization caused by loss of CBL-mediated destabilization, with prolonged ligand-dependent signaling, not demonstrated loss of MET function or nonsense-mediated decay. Consequently, the observed in-frame exon-skipping product does not satisfy the required null-variant/known-LOF-mechanism combination for PVS1. The affected exon is not a repeat-region or protein-length-extension event, and no evidence establishes NMD for this transcript product.

Rule

PVS1: Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.

Evidence considered

• NM_000245.4:c.3028+1G>T is at the invariant canonical +1 donor position of exon 14 in the MANE Select transcript.
• Gray et al. (PMID:26637977) report RT-PCR evidence that the variant excludes MET exon 14 from mature transcript; the 141-nucleotide exon is skipped in frame, deleting 47 amino acids from the juxtamembrane domain.
• The same study reports loss of CBL-mediated receptor destabilization, MET stabilization, and prolonged ligand-dependent signaling, which is a demonstrated altered-signaling consequence rather than a demonstrated null loss-of-function product or NMD-mediated transcript loss.

Sources

Reasoning

The governing PP4 rule requires a patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. PMID:26637977 documents the exact variant in patients with osteofibrous dysplasia (OFD), including characteristic periosteal cortical lesions of the tibia and fibula, but the paper states that OFD is typically sporadic and describes both germline and somatic MET mechanisms. The supplied evidence therefore does not establish a phenotype with a single genetic etiology for applying PP4.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• PMID:26637977 describes OFD as a congenital disorder with radiolucent lesions affecting cortical bone under the periosteum, predominantly the tibia and fibula.
• The same paper states that OFD is typically sporadic and reports germline MET mutations in familial or bilateral disease as well as a somatic MET mutation in unilateral disease; this does not support a single genetic etiology.

Sources

Reasoning

PP5 requires a reputable source to recently report the variant as pathogenic when the underlying evidence is unavailable for independent evaluation. The exact-variant ClinVar record has no expert-panel submission; it contains an OMIM risk-factor submission without assertion criteria and a single-laboratory uncertain-significance submission. The available primary paper is independently reviewable and does not constitute an eligible exact-variant ClinVar expert-panel pathogenic or likely pathogenic classification.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 225227 is classified Uncertain significance with criteria provided by a single submitter; the submission audit reports zero expert-panel submissions.
• The exact-variant OMIM submission is labeled risk factor with no assertion criteria, not Pathogenic or Likely pathogenic.
• The exact-variant Labcorp submission is Uncertain significance, not Pathogenic or Likely pathogenic.

Sources

Reasoning

BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, so no allele frequency reaches the generic stand-alone threshold of >=0.05. Because the variant is absent, no grpmax FAF or population allele count is available to support a BA1 call.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: variant absent; no allele frequency, grpmax FAF, or observed-allele count reported.
• gnomAD v4.1: variant absent; no allele frequency, grpmax FAF, or observed-allele count reported.
• Generic calibration threshold supplied for this case: BA1 >=0.05, attributed in the governing materials to ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, so the observed frequency is not greater than expected for the disorder and does not reach the generic default threshold of >=0.01. No grpmax FAF or population with at least 2,000 observed alleles is available because the variant was not observed.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: variant absent; no filtering allele frequency, grpmax FAF, or observed-allele count reported.
• gnomAD v4.1: variant absent; no filtering allele frequency, grpmax FAF, or observed-allele count reported.
• Generic BS1 default supplied for this case: >=0.01; the supplied guidance requires use of grpmax FAF rather than a popmax point estimate and consideration of disease prevalence, penetrance, and allelic heterogeneity before applying BS1.

Sources

Reasoning

BS3 is not met. The available functional study does not show that the variant has no damaging effect on protein function or splicing; instead, it directly shows that MET c.3028+1G>T causes exon 14 exclusion and links the resulting METΔ14 protein to impaired receptor destabilization and disease-relevant osteogenic effects. No benign functional or normal-splicing result was identified for this variant.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• Gray et al. 2015 directly reports aberrant splicing for c.3028+1G>T, with exclusion of MET exon 14 from the mature transcript.
• Gray et al. 2015 reports that METΔ14 was resistant to CBL-mediated receptor destabilization and that induced exon skipping impaired osteogenic differentiation and matrix mineralization.

Sources

Reasoning

The available variant-specific evidence does not show lack of segregation in affected family members. Instead, it reports segregation with osteofibrous dysplasia in multiple affected carriers. The four non-penetrant carriers reported in family 2 were unaffected carriers and therefore do not constitute affected members in whom the variant fails to segregate.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• Gray et al., PMID:26637977, Results and Table 1: family 2 included six penetrant and four non-penetrant carriers; families 2 and 3 showed segregation among affected individuals rather than lack of segregation.

Sources

Reasoning

Scope: this is an intronic canonical splice-donor variant, so BP4 uses the SpliceAI path only; REVEL is not applicable. SpliceAI max delta is 0.943, which indicates predicted splice impact and is above the generic BP4 supporting threshold of <=0.1 from Jaganathan et al. 2019 (PMID:30661751); therefore BP4 is not met. The SpliceAI result is not double-counted with PP3.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• Variant NM_000245.4:c.3028+1G>T is an intronic canonical donor (+1) variant; therefore the applicable computational path is SpliceAI only.
• SpliceAI Lookup reports max delta = 0.943 (DS_DL), above the generic BP4 supporting threshold of <=0.1.
• The BP4 threshold is the SpliceAI recommended cutoff from Jaganathan et al. 2019 (PMID:30661751); the result does not support no impact on splicing.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The available exact-variant report describes MET c.3028+1G>T in affected OFD cases and familial segregation, while the evidence contains no alternate pathogenic molecular diagnosis for those cases. No BP5 evidence is documented.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• PMID:26637977 reports the exact variant in affected OFD individuals and attributes the disease mechanism to MET alterations; it does not report an alternate molecular diagnosis in those cases.

Sources

Reasoning

BP6 requires a reputable source to recently report the variant as benign when the underlying evidence is unavailable for independent evaluation. The exact-variant ClinVar record has no expert-panel submission and no Pathogenic/Likely pathogenic or Benign/Likely benign expert-panel classification; its reported classifications are risk factor and uncertain significance. Therefore BP6 is not met.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 225227 has zero expert-panel submissions; the recorded submissions are OMIM risk factor and Labcorp Uncertain significance.
• Neither submission is an exact-variant expert-panel Benign or Likely benign assertion.

Sources

Reasoning

No case evidence documents a de novo occurrence with both maternity and paternity confirmed, a disease-consistent phenotype, and no family history. The available report describes familial osteofibrous dysplasia and segregation, not a de novo event.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• The available variant-specific report describes affected individuals in families with familial osteofibrous dysplasia and does not report a de novo proband with confirmed maternity and paternity.

Still missing

• Documented maternity and paternity confirmation, parental variant testing, and absence of family history for a de novo proband are unavailable.

Reasoning

The governing PS4 rule requires a significant case-control prevalence comparison, with RR or OR >5.0 and a confidence interval that excludes 1.0. PMID:26637977 reports this exact variant in affected individuals from families 2, 3, and 4 and reports non-penetrant carriers within familial pedigrees, but it does not provide an unrelated unaffected control cohort or a case-control count table from which an OR and 95% CI can be computed. The two literature_pass PS4 candidate papers (PMID:16203897 and PMID:9234973) have no abstract or full-text record available in the case materials, so their possible case-control data could not be verified. PS4 therefore cannot be adjudicated without reviewing those papers for affected-case and control counts.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• PMID:26637977 reports NM_000245.4:c.3028+1G>T in affected individuals from families 2, 3, and 4; Table 1 lists six penetrant and four non-penetrant carriers in family 2 and two penetrant carriers in family 3.
• The non-penetrant familial carriers are not an unrelated case-control cohort, and the paper supplies no case/control frequency, odds ratio, relative risk, or confidence interval for this exact variant.
• literature_pass.json identifies PMID:16203897 and PMID:9234973 as PS4 candidates, but neither has an abstract or full-text file available in this case for verification.

Still missing

• Obtain and review PMID:16203897 for affected-case and unaffected or population-control counts.
• Obtain and review PMID:9234973 for affected-case and unaffected or population-control counts.
• If counts are found, compute OR and 95% CI and test OR >5.0 with CI excluding 1.0.

Sources

Reasoning

PM3 cannot be assessed from the available evidence. PMID:26637977 reports the variant in affected individuals and familial segregation, but does not document a second pathogenic variant, testing of parents or offspring, or phase showing this variant in trans. The inheritance mode for the relevant MET disorder is also not established in the case evidence, so the recessive-disorder applicability of PM3 cannot be confirmed.

Rule

For recessive disorders, detected in trans with a pathogenic variant ... This requires testing of parents (or offspring) to determine phase.

Evidence considered

• Gray et al. (PMID:26637977) reports MET c.3028+1G>T in affected individuals from families 2, 3, and 4 and describes familial segregation, but the extracted report contains no second pathogenic allele or parental/offspring phase result.
• The case evidence does not establish that the relevant MET-associated disorder is recessive.

Still missing

• Determine the inheritance mode of the relevant MET-associated disorder.
• Identify a pathogenic or likely pathogenic variant on the other allele, if present.
• Obtain parental or offspring testing establishing trans phase.

Sources

Reasoning

No case evidence documents an assumed de novo occurrence without confirmed maternity and paternity. The available report describes familial osteofibrous dysplasia and segregation rather than a de novo event.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• The available variant-specific report describes affected individuals in families with familial osteofibrous dysplasia and does not report an assumed de novo occurrence.

Still missing

• A proband-level de novo observation and parental testing information are unavailable.

Reasoning

BS2 cannot be assessed from the available evidence. The population records document absence from gnomAD but do not report an identified healthy adult individual carrying this variant in the homozygous, heterozygous, or hemizygous state, and they provide no age, phenotype, inheritance, or penetrance information for such an individual.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent; these records do not identify a healthy adult carrier or provide individual-level zygosity, age, phenotype, or penetrance data.
• The case evidence does not provide a verified healthy-adult observation suitable for BS2.

Still missing

• No verified healthy adult individual with documented zygosity and phenotype was identified.
• No evidence establishes full penetrance at an early age for the relevant disorder.
• No inheritance-specific healthy-carrier observation is available for this variant.

Sources

Reasoning

BP2 cannot be assessed from the available evidence. PMID:26637977 reports affected carriers and segregation of MET c.3028+1G>T, but does not document a pathogenic variant in cis or in trans. The case evidence also does not establish a fully penetrant dominant MET disorder, so neither BP2 pathway can be confirmed.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• Gray et al. (PMID:26637977) reports MET c.3028+1G>T in affected individuals and familial segregation, but the extracted report does not identify a pathogenic variant in cis or trans.
• The case evidence does not establish a fully penetrant dominant MET disorder.

Still missing

• Determine whether the relevant MET-associated disorder is fully penetrant and dominant.
• Obtain allele-level testing establishing cis or trans phase with a pathogenic variant.

Sources