NTHL1 c.503T>C, p.Ile168Thr

Reasoning

The variant is a missense SNV, NM_002528.7:c.503T>C, p.(Ile168Thr), so the generic PP3/BP4 missense path applies and REVEL is the sole applicable predictor; SpliceAI is not used for this path. REVEL is 0.876, meeting the supplied generic ClinGen SVI moderate PP3 threshold of >=0.773 but not the strong threshold of >=0.932. BayesDel is not used because no generic ACMG-strength calibration is established. Therefore PP3 is met at moderate strength.

Rule

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

Evidence considered

• Variant consequence: missense_variant; NM_002528.7:c.503T>C produces NP_002519.2:p.(Ile168Thr), so the missense REVEL path applies.
• Local REVEL lookup assigns a score of 0.876 for this variant.
• The supplied generic ClinGen SVI REVEL calibration sets PP3 moderate at >=0.773 and PP3 strong at >=0.932; 0.876 therefore supports PP3 at moderate strength. Threshold source supplied in the governing prompt: Pejaver et al. 2022, PMID:36413997.
• SpliceAI max delta 0.014 is not counted because the governing generic path for a missense variant is REVEL only; BayesDel 0.543418 is not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The variant is a missense SNV, p.Ile168Thr. Multiple reviewed NTHL1 sources describe the associated syndrome as autosomal recessive and caused by biallelic inactivating variants, and report truncating p.Gln90* disease-associated alleles; the available gene-level evidence therefore supports a primarily truncating loss-of-function disease mechanism rather than a missense mechanism.

Rule

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

Evidence considered

• PMID:31285513 describes NTHL1 tumor syndrome biology and reports the truncating c.268C>T (p.Gln90*) allele.
• PMID:31243857 reports two unrelated homozygous c.268C>T (p.Gln90*) carriers and describes NTHL1-associated polyposis as biallelic.
• PMID:31068090 and PMID:31227763 describe NTHL1-associated disease as recessive and discuss truncating or inactivating alleles.

Sources

Reasoning

The variant is NP_002519.2:p.(Ile168Thr). The reviewed exact-variant literature and ClinVar-linked records do not identify a previously established pathogenic variant producing the same amino-acid change at residue 168. The reported NTHL1 missense comparator p.Ile176Thr is at a different residue and is described as a VUS, not an established pathogenic same-amino-acid change.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• The target variant is NP_002519.2:p.(Ile168Thr), while the reported missense comparator in the reviewed NTHL1 studies is c.527T>C (p.Ile176Thr), a different residue and not an established pathogenic variant.
• The reviewed exact-variant records and paper extracts contain no established pathogenic same-amino-acid p.Ile168Thr comparator.

Sources

Reasoning

The variant is p.Ile168Thr. vcep_materials.json has no NTHL1 domain-table entry because no VCEP framework is available, and the evidence reports that p.Ile168 is not in a statistically significant hotspot. No independently documented critical, well-established functional domain or hotspot covering residue 168 was identified in the supplied evidence; therefore the PM1 location requirement is not met.

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

• vcep_materials.json reports domain_tables as an empty object for NTHL1, so there is no VCEP-approved domain list to apply.
• The hotspot evidence states: "This variant does not lie in a statistically significant hotspot."

Sources

Reasoning

PM2 is not met. The supplied generic supporting threshold is <=0.0001, but the variant has gnomAD v4.1 all-comers AF=0.0018092911 (2909/1607812 alleles; 9 homozygotes), gnomAD v2.1 all-comers AF=0.0018025813 (500/277380 alleles; 1 homozygote), gnomAD v2.1 non-cancer exome AF=0.0019433719, and gnomAD v3.1 non-cancer genome AF=0.0015003041. These frequencies are not absent or extremely low under the supplied PM2 threshold.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers: AF=0.0018092911, 2909/1607812 alleles, 9 homozygotes.
• gnomAD v2.1 all-comers: AF=0.0018025813, 500/277380 alleles, 1 homozygote.
• gnomAD v2.1 non-cancer exomes: AF=0.0019433719, 453/233100 alleles, 1 homozygote.
• gnomAD v3.1 non-cancer genomes: AF=0.0015003041, 222/147970 alleles, 1 homozygote; v3.1 has no exome cohort.
• The generic PM2 supporting threshold supplied for this case is <=0.0001.

Sources

Reasoning

The target change is p.Ile168Thr. No different missense substitution at residue Ile168 with an established pathogenic determination was identified in the supplied comparator collection, ClinVar-linked evidence, or extracted literature. The reported p.Ile176Thr variant is at residue 176 and therefore cannot satisfy the same-residue PM5 requirement.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• pm5_candidates.json records zero same-residue candidates and zero expert-panel or non-expert-majority pathogenic/likely pathogenic candidates.
• The reviewed NTHL1 papers report p.Ile176Thr, not a pathogenic different missense substitution at Ile168.

Sources

Reasoning

The supplied evidence characterizes NTHL1-associated disease as recessive and caused by biallelic inactivating variants, with reported truncating examples; it does not establish that missense variants are a common disease mechanism or that NTHL1 has a low rate of benign missense variation. The PP2 gene-level prerequisites are therefore not met.

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 literature describes NTHL1 tumor syndrome as resulting from biallelic inactivation and reports truncating p.Gln90* variants, rather than establishing a common missense disease mechanism.
• The reviewed sources provide no quantitative or curated demonstration of a low rate of benign NTHL1 missense variation.

Sources

Reasoning

BA1 is not met. Under the generic ACMG/AMP stand-alone threshold of >=0.05, the highest observed gnomAD v4.1 population allele frequency is 0.0086148649 in the Middle Eastern population, while the all-comers allele frequency is 0.0018092911 (2909/1607812); gnomAD v2.1 all-comers and non-cancer exomes are also below 0.05 at 0.0018025813 and 0.0019433719, respectively. The variant is therefore far below the 5% stand-alone benign threshold.

Rule

BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers: AF=0.0018092911, 2909/1607812 alleles, 9 homozygotes; highest population AF=0.0086148649 in the Middle Eastern population.
• gnomAD v2.1 all-comers: AF=0.0018025813, 500/277380 alleles; highest population AF=0.0054464112 in the Ashkenazi Jewish population.
• gnomAD v2.1 non-cancer exomes: AF=0.0019433719, 453/233100 alleles, 1 homozygote; this non-cancer subset is below the BA1 threshold as well.
• The generic ACMG/AMP 2015 stand-alone benign frequency threshold supplied for this case is >=0.05.

Sources

Reasoning

BS1 is not met under the supplied generic threshold of >=0.01. The highest observed gnomAD v4.1 population frequency is 0.0086148649 in the Middle Eastern population and the gnomAD v4.1 group maximum frequency is 0.00673071, both below 0.01; the gnomAD v2.1 all-comers and non-cancer exome group maxima are 0.00259472 and 0.00261814, respectively. Although NTHL1-related disease is recessive, these frequencies do not meet the supplied generic BS1 cutoff.

Rule

BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v4.1 all-comers: highest population AF=0.0086148649 in the Middle Eastern population; group maximum FAF=0.00673071; overall AF=0.0018092911.
• gnomAD v2.1 all-comers: group maximum FAF=0.00259472 and overall AF=0.0018025813.
• gnomAD v2.1 non-cancer exomes: group maximum FAF=0.00261814 and exome AF=0.0019433719.
• gnomAD v3.1 non-cancer genomes: AF=0.0015003041 and group maximum FAF=0.00360011; no exome cohort is available in v3.1.
• The generic BS1 calibration threshold supplied for this case is >=0.01.

Sources

Reasoning

The variant is a missense SNV, so BP4 must be evaluated through the missense REVEL path only; the low SpliceAI score cannot substitute for a non-benign REVEL result. REVEL is 0.876, which is above the supplied BP4 supporting cutoff of <=0.29 and is therefore gray-zone for BP4 rather than evidence of no impact. BayesDel is not used because no generic ACMG-strength calibration is established. Therefore BP4 is not met.

Rule

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

Evidence considered

• Variant consequence: missense_variant; NM_002528.7:c.503T>C produces NP_002519.2:p.(Ile168Thr), so the missense REVEL path applies.
• Local REVEL lookup assigns a score of 0.876 for this variant.
• The supplied generic ClinGen SVI REVEL calibration sets BP4 supporting at <=0.29; 0.876 does not meet BP4 and is an indeterminate/gray-zone REVEL result. Threshold source supplied in the governing prompt: Pejaver et al. 2022, PMID:36413997.
• SpliceAI max delta 0.014 is not counted because a missense variant's BP4 must use REVEL only; the clean splice prediction cannot establish no impact on the amino-acid substitution. BayesDel 0.543418 is not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

No case-specific evidence establishes that NM_002528.7:c.503T>C is de novo in an affected proband. The available material does not document the proband's phenotype, family history, testing of both biological parents, or confirmed maternity and paternity. Therefore the complete PS2 definition cannot be assessed.

Rule

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

Evidence considered

• The reviewed case evidence contains no case-specific de novo report, parental genotype results, or confirmed maternity and paternity for NM_002528.7:c.503T>C.
• The reviewed literature extracts do not report the exact variant c.503T>C or p.(Ile168Thr) with de novo or parental-testing data.

Still missing

• Affected proband phenotype and relevant disease diagnosis
• Family history assessment
• Variant testing results for both biological parents
• Confirmed maternity and paternity

Reasoning

PS3 cannot be assessed because the reviewed literature does not provide a variant-specific in vitro or in vivo functional study for NTHL1 NM_002528.7:c.503T>C (NP_002519.2:p.(I168T)). PMID:31243857 explicitly reports no variant-specific functional assay for this variant, and PMID:31285513 reports other NTHL1 variants and cohort observations but not c.503T>C. Computational prediction and clinical classification statements do not establish a validated, reproducible functional assay supportive of a damaging effect.

Rule

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

Evidence considered

• Terradas et al. 2019 (PMID:31243857), Section 6, reports NTHL1 variants in polyposis cohorts but does not mention c.503T>C/p.I168T and describes no variant-specific functional assay for this variant.
• Lorca et al. 2019 (PMID:31285513), Discussion/Table 2 and cohort results, reports other NTHL1 variants and tissue-expression observations but does not report c.503T>C/p.I168T or a functional assay for it.
• Belhadj et al. 2019 (PMID:31227763), Results and Discussion, does not mention c.503T>C/p.I168T and provides no functional assay result for this variant.
• Ricci et al. 2019 (PMID:31068090), Results/Table 1, does not mention c.503T>C/p.I168T and provides no variant-specific functional assay result.

Still missing

• A validated, reproducible in vitro or in vivo assay directly testing NTHL1 c.503T>C (p.I168T) and demonstrating a damaging effect is not available in the reviewed evidence.

Sources

Reasoning

No exact-variant case-control study was identified for NM_002528.7:c.503T>C (NP_002519.2:p.(Ile168Thr)). The reviewed literature reports other NTHL1 variants or gene-level cohorts, but provides neither a prevalence comparison for this variant in affected individuals versus controls nor an exact-variant relative risk/odds ratio and confidence interval. Therefore the PS4 threshold cannot be evaluated.

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

• Belhadj et al. 2019 (PMID:31227763) searched for c.503T>C/p.(I168T) but reported no exact-variant observation; its cited NTHL1 findings concern other variants and a biallelic patient.
• Terradas et al. 2019 (PMID:31243857) reported NTHL1 prevalence among polyposis cohorts, including 1.9% (5/263) biallelic mutations, but no exact-variant case-control estimate for c.503T>C.
• Lorca et al. 2019 (PMID:31285513) searched for c.503T>C/p.(I168T) but reported other NTHL1 variants and no exact-variant prevalence comparison, relative risk, odds ratio, or confidence interval.

Still missing

• An exact-variant case-control prevalence comparison with relative risk or odds ratio and its confidence interval is unavailable.

Sources

Reasoning

NTHL1-associated disease is recessive, but the case evidence does not document this exact variant in an affected proband with a second pathogenic NTHL1 variant, nor does it establish phase by parental or offspring testing. The reviewed literature reports other NTHL1 variants and biallelic cases, while the exact NM_002528.7:c.503T>C (p.Ile168Thr) variant is not reported in the extracted papers. gnomAD reports homozygotes, but population observations do not establish an affected-proband genotype, a pathogenic allele in trans, or phase. Therefore PM3 cannot be assessed.

Rule

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

Evidence considered

• ClinVar variation 587357 identifies NM_002528.7:c.503T>C (p.Ile168Thr) but provides conflicting submissions and no documented affected-proband phase or trans relationship for this criterion.
• gnomAD v4.1 reports 9 homozygotes for the variant (AF 0.00180929); this is population evidence without affected-proband status, a pathogenic partner allele, or phase information.
• Belhadj et al. 2019 (PMID:31227763) reports a different biallelic NTHL1 case with c.268C>T and c.550-1G>A in trans; the exact c.503T>C variant is not reported.
• Terradas et al. 2019 (PMID:31243857) reports different homozygous NTHL1 variants and a different heterozygous c.527T>C (p.I176T) carrier; the exact c.503T>C variant is not reported.
• The reviewed case evidence contains no parental or offspring testing result establishing phase for c.503T>C.

Still missing

• Affected-proband data for NM_002528.7:c.503T>C are not available.
• A second pathogenic NTHL1 variant and its trans phase are not documented.
• Parental or offspring testing establishing phase is not documented.

Sources

Reasoning

No case-specific evidence establishes an assumed de novo occurrence of NM_002528.7:c.503T>C in an affected patient. The available material does not document a disease phenotype, family history, or parental testing showing absence of the variant; consequently, PM6 cannot be assessed.

Rule

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

Evidence considered

• The reviewed case evidence contains no case-specific assumed-de-novo report or parental genotype results for NM_002528.7:c.503T>C.
• The reviewed literature extracts do not report the exact variant c.503T>C or p.(Ile168Thr) with de novo data.

Still missing

• Affected patient phenotype and diagnosis
• Family history assessment
• Parental genotype results or other evidence supporting de novo status

Reasoning

No case-specific family data show NM_002528.7:c.503T>C co-segregating with disease in affected relatives. The available material provides no affected relatives, informative meioses, genotype-phenotype observations, or segregation analysis for the exact variant, so PP1 cannot be assessed or upgraded.

Rule

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

Evidence considered

• The reviewed case evidence contains no pedigree, affected-relative genotypes, informative meioses, or exact-variant segregation results for NM_002528.7:c.503T>C.
• The reviewed literature extracts discuss other NTHL1 variants but do not report c.503T>C or p.(Ile168Thr) with segregation data.

Still missing

• Affected relatives and their diagnoses
• Exact-variant genotypes for affected and unaffected relatives
• Informative meioses and segregation analysis
• Disease phenotype consistency across relatives

Reasoning

The case materials do not provide a patient phenotype or family history for this individual that can be judged highly specific for an NTHL1-related disease with a single genetic etiology. The reviewed papers do not report this exact variant and cannot substitute for the patient's phenotype.

Rule

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

Evidence considered

• The case evidence contains no patient-level phenotype or family-history description for NM_002528.7:c.503T>C.
• Belhadj et al. 2019 (PMID:31227763) and Lorca et al. 2019 (PMID:31285513) describe NTHL1-associated cohorts and other variants, not the phenotype of this case or the exact variant.

Still missing

• Patient-level phenotype, family history, and diagnostic context are unavailable.

Sources

Reasoning

ClinVar contains no exact-variant expert-panel submission for NM_002528.7:c.503T>C: expert-panel submissions = 0. The available exact-variant classifications are ordinary laboratory or single-submitter assertions, including Likely benign and Uncertain significance, and therefore do not satisfy the required PP5 trigger. No reputable source reporting this exact variant as pathogenic was identified.

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 record NM_002528.7(NTHL1):c.503T>C (p.Ile168Thr) reports 17 submissions, 0 expert-panel submissions, and classifications of Likely benign or Uncertain significance among the usable submissions.
• The exact-variant ClinVar submissions are ordinary laboratory or single-submitter assertions; none is an expert-panel Pathogenic or Likely pathogenic classification.

Still missing

• No exact-variant ClinVar expert-panel pathogenic or likely pathogenic classification is available.

Sources

Reasoning

BS2 cannot be established from the available population data. The variant is observed in 9 homozygotes in gnomAD v4.1, including 1 Middle Eastern homozygote, and in 1 homozygote each in gnomAD v2.1 all-comers, gnomAD v2.1 non-cancer exomes, and gnomAD v3.1 non-cancer genomes. However, the records do not establish that these were healthy adults assessed to an age appropriate for the expected phenotype, and the generic BS2 definition requires that context for a recessive disorder.

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 v4.1 all-comers reports 9 homozygotes overall, including 1 in the Middle Eastern population and 8 in the European non-Finnish population.
• gnomAD v2.1 all-comers reports 1 homozygote; the non-cancer exome subset also reports 1 homozygote.
• gnomAD v3.1 non-cancer genomes reports 1 homozygote.
• Population database homozygote counts do not, by themselves, document that each individual was a healthy adult with full penetrance expected at an early age.

Still missing

• Healthy-adult status and age-appropriate clinical assessment are not documented for the observed homozygotes.

Sources

Reasoning

BS3 cannot be assessed because the reviewed literature does not provide a variant-specific in vitro or in vivo functional study showing no damaging effect on NTHL1 protein function or splicing for NM_002528.7:c.503T>C (NP_002519.2:p.(I168T)). The absence of a reported assay is not evidence of a normal functional effect, and the available clinical, computational, and cohort observations do not satisfy BS3.

Rule

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

Evidence considered

• Terradas et al. 2019 (PMID:31243857), Section 6, does not mention c.503T>C/p.I168T and reports no variant-specific functional assay that could support a normal functional effect.
• Lorca et al. 2019 (PMID:31285513), Discussion/Table 2 and cohort results, does not report c.503T>C/p.I168T or a normal-function/normal-splicing assay for this variant.
• Belhadj et al. 2019 (PMID:31227763), Results and Discussion, does not mention c.503T>C/p.I168T and provides no assay demonstrating absence of a damaging effect.
• Ricci et al. 2019 (PMID:31068090), Results/Table 1, does not mention c.503T>C/p.I168T and provides no variant-specific assay demonstrating preserved protein function or splicing.

Still missing

• A validated, reproducible in vitro or in vivo assay directly testing NTHL1 c.503T>C (p.I168T) and showing no damaging effect on protein function or splicing is not available in the reviewed evidence.

Sources

Reasoning

No case-specific evidence documents affected family members who lack NM_002528.7:c.503T>C. The available material contains no pedigree, phenotype-confirmed affected relatives, relative genotypes, or information sufficient to exclude phenocopies or additional pathogenic variants; therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The reviewed case evidence contains no affected-relative genotype results showing absence of NM_002528.7:c.503T>C.
• The reviewed literature extracts do not report the exact variant c.503T>C or p.(Ile168Thr) with non-segregation evidence.

Still missing

• Phenotype-confirmed affected relatives
• Exact-variant testing results in affected relatives
• Pedigree and family structure
• Assessment for phenocopies and additional pathogenic variants

Reasoning

The case evidence does not document the exact variant observed in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern. The reviewed papers do not report NM_002528.7:c.503T>C (p.Ile168Thr), and the available ClinVar and population records do not establish cis/trans phase with a pathogenic allele. Therefore BP2 cannot be assessed.

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

• ClinVar variation 587357 contains conflicting classifications for NM_002528.7:c.503T>C (p.Ile168Thr), but no submission-level cis/trans phase evidence with a pathogenic variant is documented in the case evidence.
• gnomAD v4.1 reports population allele counts and homozygotes, but does not provide the phase or clinical inheritance information required for BP2.
• The reviewed NTHL1 papers report other variants and genotypes, not the exact c.503T>C variant; none supplies a qualifying cis or trans observation for BP2.

Still missing

• No affected-person or family genotype establishes c.503T>C in trans with a pathogenic variant.
• No cis-phase observation with a pathogenic variant is documented.
• For a dominant-disorder trans path, fully penetrant dominant inheritance is not established for the relevant disorder.

Sources

Reasoning

No case-level evidence was identified showing NM_002528.7:c.503T>C in a patient with an alternate molecular basis for disease. Reports of other NTHL1 variants and general recessive inheritance observations do not establish an alternate molecular diagnosis for this case.

Rule

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

Evidence considered

• No case summary or reviewed publication documents an alternate pathogenic molecular diagnosis in a patient carrying the exact c.503T>C variant.
• Ricci et al. 2019 (PMID:31068090), Belhadj et al. 2019 (PMID:31227763), and Lorca et al. 2019 (PMID:31285513) discuss other NTHL1 variants or cohort-level recessive inheritance and do not establish BP5 for this exact variant.

Still missing

• Case-level evidence of a confirmed alternate molecular basis for the patient's disease is unavailable.

Sources

Reasoning

ClinVar contains no exact-variant expert-panel submission for NM_002528.7:c.503T>C: expert-panel submissions = 0. The available Likely benign assertions are ordinary laboratory or single-submitter classifications, not an exact-variant expert-panel benign or likely benign classification. Therefore the required BP6 trigger is absent.

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 record NM_002528.7(NTHL1):c.503T>C (p.Ile168Thr) reports 17 submissions and 0 expert-panel submissions.
• The available Likely benign classifications are ordinary laboratory or single-submitter assertions and do not meet the required exact-variant expert-panel condition for BP6.

Still missing

• No exact-variant ClinVar expert-panel benign or likely benign classification is available.

Sources