BLM c.2207_2212delinsTAGATTC, p.Tyr736LeufsTer5

Reasoning

NM_000057.4 is the MANE Select BLM transcript, and c.2207_2212delinsTAGATTC is an exonic frameshift in exon 10 producing NP_000048.1:p.(Tyr736LeufsTer5), with termination at codon 740 in a 1,418-amino-acid protein. The premature termination is far upstream of the final exon and is consistent with loss of the normal transcript/protein rather than a retained near-full-length product. Direct variant-specific functional evidence identifies the same blmAsh allele, reports approximately 80% reduction of steady-state BLM mRNA and premature termination at codon 740, and shows that homozygous cells lack detectable normal or full-length BLM protein. BLM loss of function is an established disease mechanism for Bloom syndrome: the cited functional study describes absent BLM as producing the cellular Bloom-syndrome phenotype, and the variant-specific study demonstrates rescue after introduction of normal BLM cDNA. Therefore, this is a null frameshift in a disease gene for which LOF is a known mechanism, and no named downgrade rule in the governing text applies.

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_000057.4 is the MANE Select BLM transcript; the variant is an exonic frameshift in exon 10 with protein consequence NP_000048.1:p.(Tyr736LeufsTer5), terminating at codon 740 in a 1,418-amino-acid protein.
• Ellis et al. (PMID:10521302) explicitly studied the matching BLM c.2207_2212delATCTGAinsTAGATTC blmAsh allele and reported approximately 80% reduction in steady-state BLM mRNA, premature translation termination at codon 740, absent detectable normal BLM protein in homozygous cells, and rescue of BLM expression and sister-chromatid-exchange phenotype after normal BLM cDNA transfection.
• Kohzaki et al. (PMID:26788541) identified the same founder frameshift as introducing a premature stop at codon 740; homozygous Bloom-syndrome fibroblasts did not express full-length BLM and showed the expected Bloom-syndrome cellular defects.
• The premature stop occurs well before the end of the coding sequence, and the cited variant-specific evidence supports transcript and full-length protein loss; no named downgrade rule from the governing PVS1 definition applies.

Sources

Reasoning

The exact BLM founder frameshift variant c.2207_2212delinsTAGATTC (blmAsh; p.(Y736Lfs*5)) was evaluated in Bloom-syndrome cell models. Homozygous blmAsh cells showed approximately 80% reduction of steady-state BLM mRNA, premature termination at codon 740, absent detectable normal BLM protein, and a high sister-chromatid-exchange phenotype. In cell-based complementation experiments, introduction of wild-type BLM cDNA restored detectable nuclear BLM expression and reduced the mean sister-chromatid-exchange count toward normal. Independent cellular characterization of homozygous blmAsh fibroblasts also documented hydroxyurea hypersensitivity, elevated sister-chromatid exchanges, quadriradial chromosomes, delayed DNA-damage response, and impaired DNA repair. These variant-specific, biologically relevant loss-of-function findings with wild-type rescue support PS3 at strong strength under the generic ACMG/AMP definition.

Rule

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

Evidence considered

• PMID:10521302, Materials and Methods and Results: the exact blmAsh variant was homozygous in Bloom-syndrome cell lines; the paper reports approximately 80% lower steady-state BLM mRNA, premature translation termination at codon 740, absent detectable normal BLM protein, and high sister-chromatid exchange.
• PMID:10521302, Results, Western Blot Analysis of Transfected Cells and SCE Analysis: stable transfection with normal BLM cDNA restored detectable nuclear BLM expression and reduced the mean sister-chromatid-exchange count toward normal.
• PMID:26788541, Introduction, Materials and Methods, and Table 2 footnote 6: the exact blmAsh frameshift is identified as NM_000057.3(BLM):c.2207_2212delATCTGAinsTAGATTC, p.Tyr736Leufs; homozygous Bloom-syndrome fibroblasts lacked full-length BLM and showed hydroxyurea hypersensitivity, elevated sister-chromatid exchanges, quadriradial chromosomes, delayed DNA-damage response, and impaired DNA repair.

Sources

Reasoning

PM2 is met at supporting strength. The variant is reported absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer cohort views, which is consistent with an allele frequency at or below the generic supporting threshold of <=0.0001. The result is supported across the available datasets, but the ACMG/AMP indel caveat warrants moderate rather than high confidence in a purely absence-based call.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers result: absent; the associated non-cancer view is also reported absent.
• gnomAD v4.1 all-comers result: absent; the associated non-cancer view is also reported absent.
• No grpmax FAF or allele count is available because the variant was not observed.
• The case variant is a deletion-insertion indel, and ACMG/AMP cautions that population data for indels may be poorly called by next-generation sequencing.

Sources

Reasoning

BLM-related Bloom syndrome is recessive. The exact variant, BLM c.2207_2212delinsTAGATTC (blmAsh), was reported as homozygous in individuals with Bloom syndrome in two independent full-text reports. Homozygosity establishes that two copies of this pathogenic variant are present on opposite homologues, satisfying the required trans relationship for a recessive disorder; no separate pathogenic allele is needed because the second allele is the same variant. PMID:10521302 reports an affected individual homozygous for blmAsh and describes the associated BLM deficiency and Bloom-syndrome cellular phenotype. PMID:26788541 reports a Bloom-syndrome fibroblast line homozygous for the same variant and lacking full-length BLM.

Rule

PM3: For recessive disorders, detected in trans with a pathogenic variant ... Note: This requires testing of parents (or offspring) to determine phase.

Evidence considered

• PMID:10521302, Materials and Methods/Results: an individual with Bloom syndrome was homozygous for BLM c.2207-2212delATCTGAinsTAGATTC (blmAsh); the report describes approximately 80% reduced BLM mRNA, premature termination at codon 740, absent detectable normal BLM protein, and correction of the cellular phenotype after wild-type BLM complementation.
• PMID:26788541, Introduction/Materials and Methods: GM08505 Bloom-syndrome fibroblasts were homozygous for NM_000057.3(BLM):c.2207_2212delATCTGAinsTAGATTC (p.Tyr736Leufs), lacked full-length BLM, and showed Bloom-syndrome cellular defects.

Sources

Reasoning

Lowery et al. report the exact BLM c.2207_2212delinsTAGATTC (p.Tyr736Leufs*5) in a patient with an exocrine pancreatic neoplasm who also carried the BRCA1 founder variant c.68_69delAG (p.Glu23Valfs*17). The concurrent BRCA1 pathogenic germline alteration is an alternate molecular basis for the patient's cancer, so BP5 is met under the generic definition.

Rule

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

Evidence considered

• Lowery et al. 2018, Table 4, row 'BRCA1 + BLM': BRCA1 c.68_69delAG (p.Glu23Valfs*17) plus BLM c.2207_2212delATCTGAinsTAGATTC (p.Tyr736Leufs*5), age 51-55, female, stage I/II, family history yes, Ashkenazi Jewish.
• Lowery et al. 2018, Table 3 lists the BLM variant as a recessive founder alteration and the BRCA1 alteration as a pathogenic founder alteration in the same patient cohort; this provides the alternate molecular basis required by BP5.

Sources

Reasoning

The exact BLM c.2207_2212delinsTAGATTC variant is reported in one pancreatic-neoplasm patient, but no case-control comparison for this exact variant was found. De Voer et al. performed a qualifying case-control study of aggregated deleterious BLM mutations: 3/185 early-onset CRC cases versus 1/532 controls, with OR=8.67 and 95% CI=0.69-455.36; this CI includes 1. The paper's aggregate result cannot be transferred to the exact variant, and the reported Ashkenazi BLMAsh comparison was only described as more than twice as frequent without exact variant-specific counts. Therefore the ACMG PS4 requirement is not met.

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

• de Voer et al. 2015, Results, targeted resequencing: 3/185 CRC cases (1.6%) versus 1/532 controls (0.2%) for aggregated deleterious BLM mutations, OR=8.67, 95% CI=0.69-455.36; the CI includes 1.
• The same paper states that prior Ashkenazi studies of BLMAsh found the allele more than twice as frequent in CRC cases versus matched controls, but this report does not provide exact-variant counts and the paper's own cohort did not assay BLMAsh.
• Lowery et al. 2018 reports the exact BLM variant in one of 615 patients with exocrine pancreatic neoplasms, without unaffected controls or an exact-variant prevalence comparison.
• The required PS4 threshold is an RR or OR >5.0 with a confidence interval that does not include 1.0; the available aggregate result is 8.67 > 5.0, but 0.69-455.36 includes 1.0 and is not exact-variant evidence.

Still missing

• An exact-variant case-control study with affected and unaffected or population-control counts and a confidence interval is not available.

Sources

Reasoning

The available clinical report concerns an unselected cohort of patients with exocrine pancreatic neoplasms. Pancreatic neoplasia, family history, Ashkenazi Jewish ancestry, and a concurrent BRCA1 founder variant are not a phenotype or family-history pattern highly specific for a disease with a single genetic etiology; the reported BLM-related condition is recessive and the phenotype is not diagnostic of Bloom syndrome.

Rule

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

Evidence considered

• Lowery et al. 2018 studied 615 unselected patients with exocrine pancreatic neoplasms; the exact BLM variant occurred in one 51-55-year-old Ashkenazi Jewish woman with stage I/II disease and family history of cancer.
• The same patient also carried the BRCA1 founder variant c.68_69delAG, and the paper describes multiple genes and multiple pathogenic germline alterations in this pancreatic-neoplasm cohort rather than a single etiologic phenotype.

Sources

Reasoning

ClinVar lists the exact variant as Pathogenic/Likely pathogenic with a two-star review status and multiple laboratory submissions, but the record contains no expert-panel classification. Ordinary laboratory submissions and an aggregate ClinVar label do not satisfy the required reputable-source basis for PP5.

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 5454 reports Pathogenic/Likely pathogenic, review status 'criteria provided, multiple submitters, no conflicts,' review stars=2, and expert_panels={}.
• The exact variant has 23 Pathogenic and 1 Likely pathogenic clinical-laboratory submissions plus one additional lowercase pathogenic submission; these are not an expert-panel assertion.

Still missing

• No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification was identified.

Sources

Reasoning

BA1 is not met. The variant is reported absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer cohort views, so no filtering allele frequency reaches the generic stand-alone threshold of >=0.05. No popmax point estimate is available to override this conclusion, and the variant is an indel for which population calling can be imperfect.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers result: absent; the associated non-cancer view is also reported absent.
• gnomAD v4.1 all-comers result: absent; the associated non-cancer view is also reported absent.
• The case variant is a deletion-insertion indel; ACMG/AMP cautions that population data for indels may be poorly called by next-generation sequencing.

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer cohort views, rather than having a filtering allele frequency greater than expected for the disorder. Thus the generic 0.01 default is not reached; no ancestry-specific elevation, homozygote observation, or maximum-credible-frequency calculation is needed to support BS1.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers result: absent; the associated non-cancer view is also reported absent.
• gnomAD v4.1 all-comers result: absent; the associated non-cancer view is also reported absent.
• No grpmax filtering allele frequency or ancestry-specific frequency is available because the variant was not observed.

Sources

Reasoning

The reviewed variant-specific functional studies do not show a lack of damaging effect. Instead, homozygous blmAsh cells demonstrated reduced BLM mRNA, premature termination, absent detectable normal BLM protein, abnormal sister-chromatid exchange, and impaired DNA-repair-associated cellular phenotypes, with correction toward normal after wild-type BLM complementation. Therefore the available functional evidence is discordant with the BS3 definition and does not support a benign functional interpretation.

Rule

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

Evidence considered

• PMID:10521302, Results: homozygous blmAsh cells had approximately 80% reduced BLM mRNA, premature termination, absent detectable normal BLM protein, and elevated sister-chromatid exchange; wild-type BLM complementation reduced the abnormal SCE phenotype toward normal.
• PMID:26788541, Materials and Methods and cellular phenotype results: homozygous blmAsh fibroblasts lacked full-length BLM and showed cellular defects including hydroxyurea hypersensitivity, elevated sister-chromatid exchanges, quadriradial chromosomes, delayed DNA-damage response, and impaired DNA repair.

Sources

Reasoning

ClinVar contains no expert-panel classification of the exact variant as Benign or Likely benign. Its available submissions are predominantly Pathogenic or Likely pathogenic, with no qualifying benign expert-panel source, so 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 5454 reports Pathogenic/Likely pathogenic with review status 'criteria provided, multiple submitters, no conflicts'; expert_panels={} and no Benign or Likely benign expert-panel assertion is present.
• The ClinVar record's ordinary laboratory submissions and aggregate label cannot trigger BP6.

Still missing

• No exact-variant ClinVar expert-panel Benign or Likely benign classification was identified.

Sources

Reasoning

No proband phenotype or family-history information is documented, and no parental testing is provided; therefore, de novo status with confirmation of both maternity and paternity cannot be established.

Rule

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

Evidence considered

• No proband phenotype, pedigree, family-history assessment, or parental segregation results are available for this criterion.

Still missing

• Documented disease-consistent proband phenotype
• Documented family history or absence of family history
• Parental testing confirming maternity and paternity
• Evidence that the variant is absent in both parents

Reasoning

No proband phenotype or family-history information is documented, and no parental testing or other evidence establishing an assumed de novo event is provided; PM6 cannot be assessed.

Rule

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

Evidence considered

• No parental genotypes, de novo observation, pedigree, or proband phenotype relevant to PM6 are available.

Still missing

• Documented disease-consistent proband phenotype
• Documented family history or absence of family history
• Parental genotypes or other evidence supporting de novo occurrence

Reasoning

No affected relatives, informative meioses, variant-positive relatives, or variant-negative relatives with phenotype data are documented; co-segregation cannot be evaluated.

Rule

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

Evidence considered

• No family segregation data or informative meioses are available for the specified variant.

Still missing

• Pedigree with affected and unaffected relatives
• Genotypes for informative family members
• Phenotype status for tested relatives
• Number of informative meioses

Reasoning

BS2 cannot be assessed from the available evidence. The case materials do not document this variant in a healthy adult individual as a homozygote, heterozygote, or hemizygote, and they do not provide an age-qualified individual-level observation that can be evaluated against the criterion's full-penetrance and early-age requirements.

Rule

BS2

Evidence considered

• The available case evidence contains no individual-level healthy-adult observation for this variant.

Still missing

• A reliable report of this variant in a healthy adult, with genotype and age/phenotype context, is required to assess BS2.

Reasoning

No affected family members with documented negative testing for the variant are reported, so lack of segregation cannot be evaluated; phenocopy and multiple-variant explanations also cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-relative genotypes, family phenotype data, or segregation analysis are available for BS4.

Still missing

• Affected relatives with reliable phenotype ascertainment
• Variant testing results for affected relatives
• Assessment for phenocopies
• Assessment for additional pathogenic variants in the family

Reasoning

No evidence was identified that the exact BLM variant is in cis with another pathogenic variant. The available reports describe the variant as homozygous or as a single reported germline alteration, but do not provide a cis-linked pathogenic variant. Because Bloom syndrome due to BLM is recessive, the dominant-disorder trans branch of BP2 is not applicable; the cis branch remains assessable but lacks phase-resolved evidence.

Rule

BP2: 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:10521302: the exact variant is reported in a homozygous Bloom-syndrome cell line; no additional pathogenic variant in cis is reported.
• PMID:26788541: the exact variant is reported in a homozygous Bloom-syndrome fibroblast line; no cis-linked pathogenic variant is reported.
• PMID:29506128, Table 3: the exact BLM variant is listed as a recessive founder variant in one patient, but the report provides no cis/trans phase information for another pathogenic variant.

Still missing

• No parental, offspring, or read-level phasing data establish a pathogenic variant in cis with this BLM variant.

Sources