KLLN c.136T>G, p.(Phe46Val)

Reasoning

PM2 is met at supporting strength. The supplied generic SVI threshold is AF <=0.0001. Because no VCEP governs KLLN, the default all-comers sources apply: gnomAD v2.1 exomes report AF 1.29808e-05 (2/154074 alleles), and gnomAD v4.1 reports AF 8.38204e-06 (13/1550936 alleles). The highest observed subgroup frequencies, 8.78194e-05 in gnomAD v2.1 and 5.94983e-05 in gnomAD v4.1, also remain below 0.0001.

Rule

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• Generic ACMG/AMP PM2 definition and supplied SVI supporting threshold: AF <=0.0001; the threshold is identified in the case data as the ClinGen SVI 2020 recommendation to downgrade PM2 to supporting (PMID:25741868).
• gnomAD v2.1 exomes report AF 1.29808e-05 (2/154074 alleles; zero homozygotes), with highest subgroup AF 8.78194e-05 in South Asian individuals.
• gnomAD v4.1 reports AF 8.38204e-06 (13/1550936 alleles; zero homozygotes), with highest subgroup AF 5.94983e-05 in South Asian individuals; all values are <=0.0001.

Sources

Reasoning

The variant is a missense substitution, p.(Phe46Val), so the governing generic rule selects the REVEL path only; the SpliceAI result is not used for BP4. REVEL is 0.124, which is at or below the generic BP4 supporting threshold of <= 0.29 supplied in this case, supporting BP4. BayesDel is not usable because no generic ACMG-strength calibration is established.

Rule

BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) ... Missense variant -> REVEL only.

Evidence considered

• NM_001126049.2:c.136T>G is an exonic missense variant producing NP_001119521.1:p.(Phe46Val), so only REVEL is applicable for this criterion.
• The local REVEL lookup reports 0.124 for the variant (source_registry key: revel).
• The supplied generic calibration assigns BP4 supporting at REVEL <= 0.29; this threshold is from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• The available SpliceAI max delta of 0.002 was not used because splice prediction is not the governing path for a missense variant; the BayesDel score was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The variant is a missense substitution, p.(Phe46Val), so the governing generic rule selects the REVEL path only; SpliceAI is not considered for PP3. REVEL is 0.124, below the generic PP3 supporting threshold of >= 0.644 supplied in this case and therefore does not support PP3. BayesDel is not usable because no generic ACMG-strength calibration is established.

Rule

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... Missense variant -> REVEL only.

Evidence considered

• NM_001126049.2:c.136T>G is an exonic missense variant producing NP_001119521.1:p.(Phe46Val), so only REVEL is applicable for this criterion.
• The local REVEL lookup reports 0.124 for the variant (source_registry key: revel).
• The supplied generic calibration assigns PP3 supporting at REVEL >= 0.644; this threshold is from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• The available SpliceAI max delta of 0.002 was not used because splice prediction is not the governing path for a missense variant; the BayesDel score was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

BA1 is not met. The governing generic ACMG/AMP stand-alone threshold supplied for this case is allele frequency >=0.05. Using the default all-comers population sources because no VCEP governs KLLN, gnomAD v2.1 reports overall AF 1.29808e-05 and highest subgroup AF 8.78194e-05 in South Asian individuals; gnomAD v4.1 reports overall AF 8.38204e-06 and highest subgroup AF 5.94983e-05. All observed frequencies are far below 0.05.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• Generic ACMG/AMP 2015 BA1 stand-alone threshold supplied for this case: allele frequency >=0.05 (PMID:25741868).
• gnomAD v2.1 exomes show AF 1.29808e-05 (2/154074 alleles; zero homozygotes), with highest subgroup AF 8.78194e-05 in South Asian individuals (2/22774 alleles).
• gnomAD v4.1 combined data show AF 8.38204e-06 (13/1550936 alleles; zero homozygotes), with highest subgroup AF 5.94983e-05 in South Asian individuals (5/84036 alleles).

Sources

Reasoning

BS1 is not met. The generic SVI default supplied for this case is AF >=0.01 for strong evidence. The highest observed frequency is 8.78194e-05 in gnomAD v2.1 South Asian individuals; gnomAD v2.1 overall AF is 1.29808e-05 and gnomAD v4.1 overall AF is 8.38204e-06. None reaches 0.01.

Rule

BS1: Allele frequency is greater than expected for disorder.

Evidence considered

• Generic BS1 definition applies, with the supplied generic SVI strong threshold AF >=0.01; the threshold source named in the case data is Whiffin et al. 2017 (PMID:28518168).
• gnomAD v2.1 exomes show overall AF 1.29808e-05 and highest subgroup AF 8.78194e-05 in South Asian individuals.
• gnomAD v4.1 combined data show overall AF 8.38204e-06 and highest subgroup AF 5.94983e-05 in South Asian individuals; all reported frequencies are below 0.01.

Sources

Reasoning

The variant is NM_001126049.2:c.136T>G, NP_001119521.1:p.(Phe46Val). The supplied evidence does not verify a previously established pathogenic alternate nucleotide change producing the same amino-acid change at residue 46. ClinVar returned no result for the exact variant, but that does not establish whether another nucleotide change causing p.Phe46Val has been classified as pathogenic. SpliceAI predicts no significant splice impact, with maximum delta score 0.002, so the protein-level caveat is not the limiting issue. A verified same-amino-acid pathogenic comparator is required.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• The case variant is NM_001126049.2:c.136T>G, NP_001119521.1:p.(Phe46Val).
• ClinVar returned no result for the exact queried variant; this does not exclude a different nucleotide change producing p.Phe46Val.
• SpliceAI maximum delta score is 0.002, indicating no significant predicted splice impact.

Still missing

• No verified same-amino-acid pathogenic comparator variant for p.Phe46Val.
• No targeted evidence establishing pathogenicity for another nucleotide change in codon 46.

Sources

Reasoning

No proband-level de novo observation is documented, and there is no information confirming both maternity and paternity, excluding a family history, or establishing that the patient has the relevant disease. The criterion therefore cannot be assessed from the available evidence.

Rule

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

Still missing

• Documented proband phenotype and disease diagnosis
• Parental testing results for both maternity and paternity
• Family history assessment

Reasoning

No variant-specific functional study for NM_001126049.2:c.136T>G (NP_001119521.1:p.Phe46Val) was identified in the supplied evidence. The available materials contain no validated, reproducible in vitro or in vivo assay result testing this variant's effect on KLLN function or product, so the evidence is insufficient to assess PS3.

Rule

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

Evidence considered

• The supplied case materials contain no variant-specific functional assay result for NM_001126049.2:c.136T>G or NP_001119521.1:p.Phe46Val.
• No validated, reproducible in vitro or in vivo study establishing a damaging effect on KLLN function or product was available for this variant.

Still missing

• A variant-specific functional assay with appropriate positive and negative controls and demonstrated biological relevance is needed to assess PS3.

Reasoning

No case-control study or variant-specific affected-case/control counts, odds ratio or relative risk, and confidence interval were provided. Population frequency in gnomAD is not a case-control enrichment analysis and cannot establish PS4. The governing ACMG/AMP note requires OR or RR >5.0 with a confidence interval that excludes 1.0; no such value is available.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... 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

• No variant-specific case-control enrichment result, odds ratio, relative risk, or confidence interval is present.
• gnomAD v2.1 and v4.1 provide population frequencies only, not affected-case versus control enrichment; therefore they cannot satisfy PS4.

Still missing

• Variant-specific affected-case and control counts or a case-control OR/RR with its confidence interval are needed.

Reasoning

The variant affects KLLN residue F46. vcep_materials.json has no KLLN entry in domain_tables, so no authoritative approved critical-domain list is available for this gene. The supplied hotspot result reports no statistically significant hotspot for KLLN F46, but the available evidence does not establish whether residue 46 lies in a critical, well-established functional domain without benign variation. Domain-specific evidence is required.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• The variant is NP_001119521.1:p.(Phe46Val), affecting residue 46.
• vcep_materials.json contains an empty domain_tables object, with no KLLN domain entry.
• The KLLN F46 hotspot query returned no statistically significant hotspot row.

Still missing

• No verified critical functional-domain annotation covering KLLN residue 46.
• No evidence establishing absence of benign variation within a qualifying domain.

Reasoning

PM3 cannot be assessed because the available evidence does not establish a recessive KLLN disorder, does not document an affected proband carrying NM_001126049.2:c.136T>G with a pathogenic variant, and provides no parental or offspring testing to establish trans phase.

Rule

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

Evidence considered

• No affected-proband, pathogenic-in-trans partner, or family phase observation is documented for NM_001126049.2:c.136T>G.
• No parental or offspring testing is documented to determine phase.

Still missing

• Establish whether the relevant KLLN disorder is recessive.
• Document an affected proband carrying NM_001126049.2:c.136T>G and a pathogenic variant in the same gene.
• Obtain parental or offspring testing demonstrating that the variants are in trans.

Reasoning

The variant is p.Phe46Val. The supplied evidence does not verify a different missense substitution at KLLN residue 46 that has been determined to be pathogenic. The exact variant is absent from ClinVar, and the PM5 candidate record contains zero same-residue candidates; these results do not prove that no pathogenic alternate missense variant exists elsewhere. A verified pathogenic alternate missense change at F46 is required.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• The case variant is NP_001119521.1:p.(Phe46Val), at residue 46.
• The PM5 candidate record reports zero same-residue candidates and does not establish a pathogenic comparator.
• ClinVar returned no result for the exact queried variant.

Still missing

• No verified pathogenic alternate missense variant at KLLN residue 46.
• No targeted same-residue literature or database evidence establishing a pathogenic comparator.

Sources

Reasoning

No proband-level de novo observation is documented. The available evidence does not establish that the variant was assumed de novo or provide the parental testing context needed to distinguish PM6 from PS2.

Rule

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

Still missing

• Evidence that the variant is de novo in the proband
• Parental testing information showing whether maternity and paternity were confirmed
• Documented disease phenotype

Reasoning

No affected family members, variant-positive relatives, or meioses are documented. Consequently, there is no segregation series with which to assess co-segregation, and no stronger PP1 level can be assigned.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... May be used as stronger evidence with increasing segregation data.

Still missing

• Pedigree with affected family members
• Genotypes of affected and unaffected relatives
• Phenotype definitions and segregation across informative meioses

Reasoning

The supplied gene-context evidence supports KLLN loss of function as a germline disease mechanism, but it does not establish both required PP2 components: a low rate of benign missense variation across KLLN and missense variants as a common disease mechanism. No gene-wide benign missense-rate metric or reliable disease-variant mechanism distribution showing missense as common was supplied. Therefore PP2 cannot be adjudicated under the generic rule.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• The supplied KLLN gene-context assessment supports loss of function as a germline disease mechanism.
• The supplied evidence does not provide a gene-wide benign missense-rate metric or a disease-variant mechanism distribution showing missense as common.

Still missing

• No verified gene-wide evidence that KLLN has a low rate of benign missense variation.
• No verified evidence that missense variants are a common KLLN disease mechanism.

Sources

Reasoning

The case materials do not provide the patient's phenotype or family history, nor do they establish that the phenotype is highly specific for a disease with a single genetic etiology. Gene-level disease literature alone cannot establish PP4 for this patient.

Rule

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

Evidence considered

• No patient-level phenotype or family-history evidence is available for assessing PP4.
• The available KLLN literature is gene- and disease-context information, not patient-specific phenotype evidence for this variant.

Still missing

• Patient phenotype and family history, with documentation of specificity for a single genetic etiology, are needed.

Reasoning

The exact variant is absent from the retrieved ClinVar record and no expert-panel Pathogenic or Likely pathogenic classification from a reputable source is available. Ordinary or aggregate assertions would not qualify, and none are present. Therefore PP5 cannot be assessed as met.

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 exact-variant search reported no result for NM_001126049.2:c.136T>G; no expert-panel classification or review status is available.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification would be required to evaluate PP5.

Sources

Reasoning

BS2 is not assessed. gnomAD reports zero homozygotes, but the available aggregate population records do not document an individual healthy adult, age, phenotype, inheritance state, or the full-penetrance early-onset condition required by the BS2 definition. Aggregate heterozygote observations therefore cannot establish BS2.

Rule

BS2: 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 reports 0 homozygotes among 2 variant alleles; gnomAD v4.1 reports 0 homozygotes among 13 variant alleles.
• These aggregate population records do not identify healthy adult carriers or provide individual-level age, phenotype, inheritance, or penetrance information.

Still missing

• No documented healthy adult individual-level observation with sufficient age, phenotype, and inheritance information to apply BS2.
• No documented full-penetrance early-onset disease context sufficient to apply the generic BS2 definition.

Sources

Reasoning

No variant-specific functional study for NM_001126049.2:c.136T>G (NP_001119521.1:p.Phe46Val) was identified in the supplied evidence. The available materials contain no validated, reproducible in vitro or in vivo assay result demonstrating that this variant has no damaging effect on KLLN protein function or splicing, so the evidence is insufficient to assess BS3.

Rule

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

Evidence considered

• The supplied case materials contain no variant-specific functional assay result for NM_001126049.2:c.136T>G or NP_001119521.1:p.Phe46Val.
• No validated, reproducible in vitro or in vivo study demonstrating no damaging effect on KLLN protein function or splicing was available for this variant.

Still missing

• A variant-specific functional assay with appropriate controls and demonstrated biological relevance is needed to assess absence of a damaging effect for BS3.

Reasoning

No affected relatives or family genotypes are documented, so there is no affected-member non-segregation observation. The BS4 phenocopy caveat cannot be evaluated without family phenotype and genetic data.

Rule

Lack of segregation in affected members of a family.

Still missing

• Affected-relative genotypes
• Family pedigree and phenotype verification
• Assessment for phenocopies or multiple pathogenic variants

Reasoning

The supplied gene-context evidence supports KLLN loss of function as a germline disease mechanism, but it does not establish that primarily truncating variants are the known disease-causing variant class. No sufficiently complete pathogenic-variant spectrum or curated comparison of truncating versus missense disease mechanisms was supplied. The missense consequence of p.Phe46Val alone is insufficient to apply BP1.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• The supplied KLLN gene-context assessment supports loss of function as a germline disease mechanism.
• The supplied evidence does not establish that primarily truncating variants are known to cause disease.

Still missing

• No verified evidence that disease-causing KLLN variants are primarily truncating.
• No complete curated comparison of truncating and missense disease mechanisms.

Sources

Reasoning

BP2 cannot be assessed because no affected-proband or family observation documents NM_001126049.2:c.136T>G in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern.

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

• No affected-proband or family observation is documented showing NM_001126049.2:c.136T>G with a pathogenic variant.
• No cis or trans phase result is documented.

Still missing

• Document the relevant inheritance model and, if dominant, whether the disorder is fully penetrant.
• Document an affected or informative individual carrying NM_001126049.2:c.136T>G with a pathogenic variant.
• Obtain phase testing showing cis or trans configuration.

Reasoning

No evidence shows that this variant was found in a case with an alternate molecular basis for disease. The available records do not document a competing molecular diagnosis in the relevant case.

Rule

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

Evidence considered

• No alternate molecular diagnosis or competing pathogenic variant is documented in the available case evidence.

Still missing

• A documented alternate molecular diagnosis in the same case is needed.

Reasoning

The exact variant is absent from the retrieved ClinVar record and no expert-panel Benign or Likely benign classification from a reputable source is available. Ordinary or aggregate assertions would not qualify, and none are present. Therefore BP6 cannot be assessed as 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 exact-variant search reported no result for NM_001126049.2:c.136T>G; no expert-panel classification or review status is available.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign classification would be required to evaluate BP6.

Sources