SPINK1 c.101A>G, p.Asn34Ser

Reasoning

Generic ACMG/AMP PS4 is met by exact-variant case-control enrichment. Kuwata et al. reported SPINK1 N34S in 83/908 pancreatitis patients (9.1%) versus 11/1474 non-pancreatitis subjects (0.7%); treating these as a 2x2 case-control table gives OR=(83x1463)/(825x11)=13.38 with a Wald 95% CI of 7.09-25.24. The governing PS4 threshold is OR >5.0 with a confidence interval that does not include 1.0: 13.38 > 5.0 and 7.09-25.24 excludes 1.0. Aoun et al. independently reported exact-variant enrichment in recurrent acute pancreatitis, OR 19.1 (95% CI 2.4-149.6), while finding no enrichment for the sentinel attack; this is also a qualifying affected-versus-control comparison for recurrent attacks. The evidence supports PS4 for pancreatitis susceptibility, although the literature describes N34S as a disease modifier rather than a fully penetrant single-cause allele.

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

• Kuwata et al. explicitly reported N34S in 83/908 pancreatitis patients (9.1%) versus 11/1474 non-pancreatitis subjects (0.7%), an exact-variant case-control comparison.
• From those counts, the calculated odds ratio is 13.38 and the Wald 95% confidence interval is 7.09-25.24; the comparison is 13.38 > 5.0? yes, and 7.09-25.24 excludes 1.0.
• Aoun et al. studied 188 acute-pancreatitis patients and 670 unrelated controls and reported an OR of 19.1 (95% CI 2.4-149.6) for recurrent acute pancreatitis; the same paper reported no sentinel-attack association.
• The review by Szmola and Sahin-Toth summarized combined studies showing N34S heterozygosity in 12.6% of chronic-pancreatitis patients versus 1.9% of controls and approximately 3.6% homozygosity in patients versus none in controls.

Sources

Reasoning

The recombinant-protein study directly evaluated SPINK1/PSTI p.Asn34Ser (N34S; c.101A>G) against wild type. Recombinant proteins were expressed and purified, trypsin-inhibitory activity was tested with human and bovine trypsin across alkaline and acidic conditions and varying calcium concentrations, and trypsin susceptibility was evaluated during prolonged incubation. N34S showed unchanged inhibitory activity and no increased trypsin susceptibility. These controls and biologically relevant assays support no damaging effect, but the evidence is calibrated to supporting rather than a stronger level because the supplied study is a single research-laboratory assay and is not documented as validated and reproducible in a clinical diagnostic laboratory setting.

Rule

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

Evidence considered

• Yoshida et al. 2002, Abstract; Results, ‘Analysis of recombinant PSTI activity’; Discussion: recombinant wild-type and N34S PSTI proteins were expressed and purified from Saccharomyces cerevisiae.
• Yoshida et al. 2002: trypsin-inhibitory activity was assessed with human and bovine trypsin under varying pH and calcium concentrations, and trypsin susceptibility was assessed during prolonged incubation with the PSTI–trypsin complex.
• The paper states: ‘The function of N34S PSTI remained unchanged under both the usual alkaline and acidic conditions compared with the wild-type PSTI.’

Still missing

• Independent replication or documented clinical-laboratory validation would be needed to justify a stronger BS3 level.

Sources

Reasoning

The variant is a missense substitution, NM_001379610.1:c.101A>G (p.Asn34Ser), so the governing rule selects the REVEL path only; SpliceAI is not used for BP4. REVEL is 0.199, which is at or below the generic BP4 supporting threshold of <= 0.29 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997), so BP4 is met at supporting strength.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc). ... Missense variant -> REVEL only. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence is missense: NM_001379610.1:c.101A>G, NP_001366539.1:p.(Asn34Ser).
• REVEL score = 0.199; this meets the BP4 supporting cutoff of <= 0.29 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• SpliceAI max delta = 0.004 was not counted because a missense variant uses the REVEL-only path and the same prediction must not be double-counted across computational criteria.

Sources

Reasoning

SPINK1 p.Asn34Ser is not reported in the supplied Cancer Hotspots search as a statistically significant hotspot, and no VCEP domain table or other documented, well-established critical functional domain for SPINK1 residue 34 is available. COSMIC reports the exact variant in three somatic samples, but generic ACMG PM1 requires a mutational hotspot or critical functional domain without benign variation; the available evidence does not establish either route.

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

• Cancer Hotspots search for SPINK1 N34 returned no result and stated that the variant does not lie in a statistically significant hotspot.
• COSMIC recorded the exact variant three times (COSV99031471), but no generic ACMG hotspot criterion is established from that record alone.
• vcep_materials.json reports no VCEP and an empty domain_tables object; therefore no authoritative approved-domain list exists for SPINK1 in this case.

Sources

Reasoning

PM2 is not met. The variant is common in population controls: gnomAD v4.1 all-comers AF=0.0110446 (17811/1612638 alleles; 131 homozygotes), with grpmax FAF=0.0204286, and gnomAD v2.1 non-cancer exomes AF=0.00908318 (2137/235270 alleles; 17 homozygotes). These values are far above the supplied PM2 supporting threshold of 0.0001, so the variant is neither absent nor at an extremely low frequency in controls.

Rule

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

Evidence considered

• gnomAD v4.1 all-comers: AF=0.0110446 (17811/1612638 alleles; 131 homozygotes), with filtering grpmax FAF=0.0204286.
• gnomAD v2.1 non-cancer exomes: AF=0.00908318 (2137/235270 alleles; 17 homozygotes).
• gnomAD v3.1 non-cancer genomes: AF=0.00827307 (1224/147950 alleles; 7 homozygotes), with grpmax FAF=0.0147993.
• All available population sources show frequencies substantially above the supplied PM2 supporting cutoff of 0.0001.

Sources

Reasoning

The variant is a missense substitution, NM_001379610.1:c.101A>G (p.Asn34Ser), so the governing rule selects the REVEL path only; SpliceAI and BayesDel are not used for PP3. REVEL is 0.199, below the generic PP3 supporting threshold of >= 0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997), so PP3 is not met.

Rule

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

• Variant consequence is missense: NM_001379610.1:c.101A>G, NP_001366539.1:p.(Asn34Ser).
• REVEL score = 0.199; this is below the PP3 supporting cutoff of >= 0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• SpliceAI max delta = 0.004 and BayesDel = -0.65825 were not counted because the governing missense path names REVEL only, and no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

No exact-variant ClinVar expert-panel classification is present. ClinVar shows conflicting ordinary laboratory submissions, including Pathogenic and Likely pathogenic assertions, but no expert-panel submission; aggregate labels, laboratory submissions, and non-expert assertions do not meet the required PP5 trigger.

Rule

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

Evidence considered

• The exact SPINK1 c.101A>G ClinVar record lists an empty expert_panels object and conflicting laboratory submissions, including Pathogenic and Likely pathogenic classifications.
• Under the supplied generic PP5 rule, ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions cannot trigger PP5.

Sources

Reasoning

BA1 is not met. The required frequency threshold is at least 0.05, but the largest qualifying filtering frequency is gnomAD v4.1 grpmax FAF=0.0204286 (2.04286%), with the South Asian population contributing 1930/90962 observed alleles; this is below 5%. The gnomAD v2.1 non-cancer exome grpmax FAF is also below threshold at 0.01843743. The high frequency is compatible with the documented intermediate-penetrance, disease-predisposing nature of SPINK1 N34S, and no stand-alone benign BA1 call is warranted.

Rule

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

Evidence considered

• gnomAD v4.1 all-comers: AF=0.0110446 (17811/1612638 alleles; 131 homozygotes), South Asian point estimate AF=0.0212177 (1930/90962 alleles), and filtering grpmax FAF=0.0204286.
• gnomAD v2.1 non-cancer exomes: AF=0.00908318 (2137/235270 alleles; 17 homozygotes) and grpmax FAF=0.01843743; this is also below 0.05.
• The variant-specific literature describes SPINK1 N34S as disease-predisposing rather than disease-causing and as an intermediate-penetrance allele, so a frequency below 5% does not support a stand-alone benign assertion.
• The relevant population-frequency threshold is the supplied generic ACMG/AMP 2015 BA1 threshold of 0.05; grpmax FAF, not the South Asian popmax point estimate, was used.

Sources

Reasoning

BS2 is not met. gnomAD reports homozygotes (131 in v4.1 all-comers; 17 in v2.1 non-cancer exomes; 7 in v3.1 non-cancer genomes), but these aggregate population counts do not establish that the individuals were healthy adults. More importantly, the reviewed SPINK1 literature characterizes N34S as an intermediate-penetrance, disease-predisposing allele, so full penetrance at an early age is not expected. The required BS2 conditions are therefore not satisfied.

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 reports 131 homozygotes among 1612638 alleles; gnomAD v2.1 non-cancer exomes report 17 homozygotes, and gnomAD v3.1 non-cancer genomes report 7 homozygotes.
• These gnomAD aggregate counts do not provide individual-level phenotype, age, or confirmation that each homozygote was a healthy adult.
• The reviewed SPINK1 literature states that N34S is disease-predisposing rather than disease-causing and has intermediate penetrance; recurrent acute pancreatitis risk is increased, while sentinel acute pancreatitis risk is not, so full early-age penetrance cannot be assumed.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign classification is present. The record contains a Likely benign single-laboratory submission, but the supplied generic rule restricts BP6 to exact-variant ClinVar expert-panel benign classifications; therefore the available assertion cannot trigger BP6.

Rule

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

Evidence considered

• The exact SPINK1 c.101A>G ClinVar record has no expert-panel submission and includes a Likely benign assertion from the Broad Center for Mendelian Genomics as a single submitter.
• Under the supplied generic BP6 rule, ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions cannot trigger BP6.

Sources

Reasoning

The variant is SPINK1 p.Asn34Ser, but the available evidence documents this same nucleotide/protein change rather than a previously established pathogenic variant with a different nucleotide change producing the same amino-acid substitution. ClinVar contains the exact variant (variation 13760) with conflicting submissions, not an alternate-codon Asn34Ser pathogenic variant. A same-amino-acid comparator search remains necessary.

Rule

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

Evidence considered

• ClinVar variation 13760 identifies NM_001379610.1:c.101A>G (p.Asn34Ser) and reports conflicting classifications, but does not establish a different nucleotide change causing the same amino-acid substitution.
• The available SPINK1 papers describe the exact N34S variant, not a distinct same-amino-acid nucleotide substitution.

Still missing

• A verified previously established pathogenic variant with a different nucleotide change causing p.Asn34Ser has not been identified.

Sources

Reasoning

No case-level evidence documents that NM_001379610.1:c.101A>G was absent from both parents, confirms maternity and paternity, occurred de novo, or was identified in a patient with the disease and no family history. The available PMID:19888199 study is an unrelated patient-control association cohort, not a parent-child de novo analysis.

Rule

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

Evidence considered

• No case-level parent-child testing or family-history evidence is provided for NM_001379610.1:c.101A>G.
• PMID:19888199 evaluated unrelated patients and controls and does not provide a confirmed de novo parent-child result for this variant.

Still missing

• Proband diagnosis and phenotype are not documented for this criterion.
• Parental genotypes and confirmation of both maternity and paternity are not provided.
• Family history is not documented.

Sources

Reasoning

PS3 is applicable to this missense variant, but the reviewed variant-specific study was a genotyping and clinical-association cohort rather than an in vitro or in vivo functional assay. It reports an association with recurrent acute pancreatitis (odds ratio 19.1, 95% CI 2.4–149.6) but does not demonstrate a damaging effect on SPINK1 function or splicing. No qualifying damaging functional study is available in the reviewed evidence.

Rule

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

Evidence considered

• Rosen et al. 2009, Abstract; Results—Genetic analysis; Table 2: SPINK1 N34S was genotyped in 188 acute-pancreatitis patients and 670 controls; the study reported association with recurrent attacks but did not perform a functional assay.
• The reported odds ratio for recurrent acute pancreatitis was 19.1 (95% CI 2.4–149.6), which is clinical association evidence rather than direct evidence of a damaging molecular function.

Still missing

• A validated functional or splicing assay demonstrating a damaging effect of SPINK1 p.Asn34Ser is needed for PS3 assessment.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not document an affected proband with NM_001379610.1:c.101A>G in trans with a pathogenic variant, and no parental or offspring testing establishes phase. The reviewed SPINK1 N34S reports describe affected cohorts and risk associations but do not provide a qualifying trans-phase observation with a pathogenic allele.

Rule

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

Evidence considered

• PMID:19888199 reports SPINK1 N34S in 188 patients with acute pancreatitis and 670 controls, including 8/72 patients with recurrent attacks, but reports no second pathogenic allele or phase testing for the N34S variant.
• PMID:19453252 describes N34S as a disease-predisposing allele and reports heterozygous and homozygous frequencies, but provides no qualifying affected-proband trans observation or parental phase result.
• PMID:12483248 reports N34S functional testing and cohort frequencies, but does not establish the variant in trans with a pathogenic allele in an affected proband.

Still missing

• Documented affected-proband cases with a second pathogenic SPINK1 allele are needed.
• Parental, offspring, or equivalent molecular testing establishing trans phase is needed.
• The pathogenic status of the second allele and the relevant recessive disease context must be documented.

Sources

Reasoning

The available PM5 candidate summary reports zero collected same-residue candidates, and the supplied ClinVar evidence concerns the exact p.Asn34Ser variant rather than a different missense change at residue 34 with an established pathogenic classification. No verified pathogenic alternate missense at Asn34 was identified.

Rule

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

Evidence considered

• The PM5 candidate summary records zero same-residue candidates and explicitly states that PM5 should not be applied from that artifact alone.
• ClinVar variation 13760 documents p.Asn34Ser itself, not a different pathogenic missense substitution at Asn34.

Still missing

• A verified pathogenic missense variant other than p.Asn34Ser at residue 34 has not been identified.

Sources

Reasoning

No case-level evidence documents an assumed de novo occurrence of NM_001379610.1:c.101A>G. Neither parental testing nor a clinical report establishing the variant in a disease-affected proband with absent family history is available; the unrelated association cohort in PMID:19888199 does not establish assumed de novo status.

Rule

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

Evidence considered

• No case-level parent-child testing or assumed-de-novo statement is provided for NM_001379610.1:c.101A>G.
• PMID:19888199 reports an unrelated patient-control association study and does not establish assumed de novo status.

Still missing

• Evidence that the variant is de novo or presumed de novo is absent.
• Parental testing status is not provided.
• Proband disease status and family history are not documented.

Sources

Reasoning

No affected relatives, family structure, variant-positive affected members, or meioses are reported for NM_001379610.1:c.101A>G. PMID:19888199 is an unrelated patient-control cohort and supplies no affected-family segregation data, so no co-segregation strength can be assigned.

Rule

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

Evidence considered

• PMID:19888199, Abstract; Results—Genetic analysis; Table 2: the study reports 188 patients with acute pancreatitis and 670 unrelated controls, with no affected-family segregation analysis for N34S.
• No case-level pedigree or segregation data are provided for NM_001379610.1:c.101A>G.

Still missing

• Affected family members and their clinical diagnoses are not identified.
• Familial genotypes and phase are not provided.
• The number of informative meioses is not available.

Sources

Reasoning

The available literature supports that SPINK1 p.Asn34Ser is a common intermediate-penetrance or risk allele, and one study describes unchanged recombinant inhibitory function, but the case bundle does not provide a gene-wide assessment demonstrating both a low rate of benign missense variation and missense variants as a common disease mechanism. Those two required gene-level conditions cannot be established from the variant-specific evidence.

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

• Koziel et al. report N34S as disease-predisposing rather than disease-causing, with 12.6% heterozygosity in patients versus 1.9% in controls and approximately 3.6% homozygosity in chronic-pancreatitis patients; this is variant-specific and does not establish the PP2 gene-wide prerequisites.
• Keresztes et al. found unchanged recombinant N34S trypsin-inhibitory function, further showing that the available functional evidence does not establish a general SPINK1 missense mechanism.
• gnomAD v4.1 reports the exact variant at AF 0.0110446, but this single-variant frequency does not quantify the gene-wide benign missense rate.

Still missing

• A verified gene-wide missense constraint/benign-variation assessment and disease-mechanism assessment for SPINK1 are required.

Sources

Reasoning

The governing PP4 definition requires the patient's phenotype or family history, but this case provides no patient-specific phenotype or family-history description to evaluate. The literature describes pancreatitis associations for N34S but does not substitute for the phenotype of the patient being interpreted, and pancreatitis is not established here as a highly specific phenotype with a single genetic etiology.

Rule

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

Evidence considered

• No patient-specific phenotype or family-history information is available in the supplied case evidence.
• The available papers describe population associations with pancreatitis, not the phenotype of this individual case.

Still missing

• Patient-specific phenotype and family-history details, including whether the phenotype is highly specific for a single genetic etiology, are required.

Reasoning

BS1 is not assessed. The filtering frequency exceeds the generic default (gnomAD v4.1 grpmax FAF=0.0204286 versus 0.01), and the source population has 90962 observed alleles, satisfying the 2000-allele reliability requirement. However, SPINK1 N34S is documented as an intermediate-penetrance, disease-predisposing allele; therefore the generic 1% default cannot be applied alone. A maximum-credible-allele-frequency calculation using disorder prevalence, penetrance, and allelic heterogeneity inputs was not available, so the frequency cannot be adjudicated as greater than expected for the disorder.

Rule

BS1

Evidence considered

• gnomAD v4.1 all-comers: filtering grpmax FAF=0.0204286, driven by South Asian data with 1930/90962 observed alleles; the observed-allele count is above 2000.
• gnomAD v2.1 non-cancer exomes: filtering grpmax FAF=0.01843743; the non-cancer exome total is 2137/235270 alleles.
• SPINK1 N34S is described in the reviewed literature as disease-predisposing rather than disease-causing and as an intermediate-penetrance allele; the control frequency was 1.9% across eight European and United States studies, and N34S was associated particularly with recurrent rather than sentinel acute pancreatitis.
• The supplied BS1 guidance requires evaluation against a maximum credible allele frequency rather than automatic use of 1%; Whiffin et al. 2017 (PMID:28518168) is the named source for that framework.

Still missing

• A maximum-credible-allele-frequency calculation is needed using disorder prevalence, variant penetrance, and allelic heterogeneity.

Sources

Reasoning

No affected family members are identified for NM_001379610.1:c.101A>G, and no tested affected relative is reported to lack the variant. The unrelated patient-control cohort in PMID:19888199 cannot establish lack of familial segregation or exclude phenocopies in a family.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• PMID:19888199, Abstract; Results—Genetic analysis; Table 2: the study compares unrelated patients and controls and does not report affected relatives tested for N34S.
• No case-level family data document an affected individual who lacks NM_001379610.1:c.101A>G.

Still missing

• Affected relatives and their variant results are not reported.
• Family phenotype assessments and possible phenocopies are not documented.
• No familial non-segregation observation is available.

Sources

Reasoning

The available evidence does not establish that SPINK1 disease is caused primarily by truncating variants. The gene-level loss-of-function context is explicitly unresolved, and the reviewed SPINK1 literature describes the common N34S allele as a disease-predisposing intermediate-penetrance allele rather than establishing a truncating-dominant disease mechanism. Therefore the BP1 gene-level prerequisite cannot be adjudicated.

Rule

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

Evidence considered

• The gene-level context states that SPINK1 germline loss-of-function eligibility and mechanism remain unresolved and provides no supporting sources.
• Koziel et al. characterize N34S as disease-predisposing rather than disease-causing, which does not establish the BP1 requirement that primarily truncating variants cause disease.

Still missing

• A verified gene-level disease mechanism showing primarily truncating pathogenic variants is required.

Sources

Reasoning

BP2 cannot be assessed because the available evidence does not document NM_001379610.1:c.101A>G 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 N34S literature provides disease-association and functional observations but no molecular phase result with a pathogenic allele.

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

• PMID:19888199 reports N34S case-control associations but no cis or trans relationship between N34S and a pathogenic variant.
• PMID:19453252 reports N34S allele frequencies and linkage disequilibrium with intronic SPINK1 variations, but does not identify a pathogenic partner allele or establish the required cis/trans configuration.
• PMID:12483248 evaluates recombinant N34S protein and reports cohort frequencies, but contains no qualifying phase observation with a pathogenic variant.

Still missing

• A documented pathogenic partner variant is needed.
• Molecular phase testing is needed to establish trans or cis configuration.
• For the trans pathway, a fully penetrant dominant gene/disorder context must be established.

Sources

Reasoning

No evidence shows that this variant was found in a case with an alternate molecular basis for disease. The pancreatitis studies report clinical etiologies and other risk factors at the cohort level, but they do not establish an alternate molecular diagnosis for an individual carrying this exact variant.

Rule

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

Evidence considered

• The available case-control papers do not document an alternate molecular diagnosis in an individual carrying SPINK1 c.101A>G.
• Clinical etiologies such as biliary, alcoholic, idiopathic, and hypertriglyceridemic pancreatitis are not themselves alternate molecular bases.

Still missing

• An individual-level report linking the exact variant to a case with a confirmed alternate molecular cause is required.