NBN c.657_661del, p.Lys219AsnfsTer16

Reasoning

NM_002485.5 is the MANE Select/RefSeq transcript for NBN, and NM_002485.5:c.657_661del is a five-base deletion in exon 6 that produces the frameshift NP_002476.2:p.(Lys219AsnfsTer16). This is an early truncating frameshift with a premature stop and substantial loss of the C-terminal protein, not an in-frame change or a repeat-region variant. NBN loss of function is supported as a germline disease mechanism, making the generic PVS1 gene gate eligible. The variant is in an internal exon well before the transcript terminus, and the reviewed evidence does not indicate escape from nonsense-mediated decay; the affected exon is not a terminal distal exon. Published NBN studies describe 657del5 as a truncating, disease-causing mutation and report that its alternative p70 product does not confer full function. No specific named downgrade rule in the governing rules applies, so PVS1 remains full strength.

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

• The MANE Select transcript is NM_002485.5, and variant validation places NM_002485.5:c.657_661del in exon 6 of NBN.
• The predicted consequence is NP_002476.2:p.(Lys219AsnfsTer16), an early frameshift truncation rather than an in-frame or repeat-region indel.
• The gene-level PVS1 assessment identifies NBN loss of function as a supported germline disease mechanism and marks the generic PVS1 gate eligible.
• Nibrin mutation analysis describes 657del5 as a frameshift with premature termination and reports truncating NBN mutations as likely null alleles; the variant was identified in patients with Nijmegen breakage syndrome.
• The NBS1 alternative-translation study reports p26 and p70 products from 657del5; the p70 product lacks the native N terminus and does not provide full NBS1 function, so this finding does not trigger a named downgrade rule in the supplied governing rules.

Sources

Reasoning

PS3 is met at moderate strength. Maser et al. studied the exact NBN 657del5 allele, corresponding to NM_002485.5:c.657_661del, in NBS lymphoblastoid cell lines and fibroblasts using immunoblotting, cellular fractionation, RT-PCR/5'-RACE, site-directed mutagenesis, reporter constructs, and in-vitro translation. The variant produced the predicted truncated NBS1p26 protein, which lacked the MRE11-interaction domain, and an internally translated NBS1p70 product that associated with the MRE11 complex but did not restore full normal function. These variant-specific, biologically relevant functional findings support a damaging effect. Moderate rather than strong strength is appropriate because the study was not demonstrated to be a validated, reproducible clinical diagnostic-laboratory assay and the evidence is mechanistic rather than a calibrated clinical functional threshold.

Rule

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

Evidence considered

• Maser et al. 2001, exact NBN 657del5/c.657_661del functional study: NBS lymphoblastoid cell lines and fibroblasts showed truncated NBS1p26 lacking the MRE11-interaction domain and internally translated NBS1p70 with incomplete function; methods included immunoblotting, fractionation, RT-PCR/5'-RACE, mutagenesis, reporter constructs, and in-vitro translation.

Still missing

• No validated clinical-laboratory assay performance data or independently calibrated quantitative functional threshold was supplied.

Sources

Reasoning

The exact NBN 657del5 allele (NM_002485.5:c.657_661del) was evaluated in a Polish prostate-cancer case-control study. It occurred in 5/56 familial prostate-cancer cases versus 9/1500 controls, with reported OR 16, 95% CI 5.2-50, and P<0.0001; the comparison satisfies the generic PS4 requirement because the governing operator is >5.0 and the confidence interval excludes 1. Explicit inequality: 16 > 5.0? yes; 95% CI 5.2-50 excludes 1. The same paper reported OR 3.9 for nonfamilial cases and OR 4.5 (95% CI 1.7-11.5) for all 340 cases, neither of which independently reaches >5.0. Additional meta-analysis leads in the literature packet reported OR 2.63 (95% CI 1.76-3.93) and OR 2.79 (95% CI 2.17-3.68), which also do not meet the threshold, but they do not negate the qualifying familial case-control result.

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

• Cybulski et al., PMID:14973119, abstract and Results: 657del5 was present in 5/56 familial prostate-cancer cases and 9/1500 controls, with reported OR 16, 95% CI 5.2-50, P<0.0001.
• Cybulski et al., PMID:14973119, Methods: the study included 56 familial prostate-cancer cases, 305 nonfamilial cases, and 1500 unaffected controls from Poland, using allele-specific PCR with sequencing confirmation.
• The criterion's exact governing comparison is OR >5.0 with a confidence interval that does not include 1. Substituted inequality: 16 > 5.0? yes; CI 5.2-50 excludes 1.
• The paper also reported OR 3.9 for nonfamilial cases and OR 4.5 (95% CI 1.7-11.5) for all 340 cases; these values do not independently satisfy OR >5.0.

Sources

Reasoning

PM3 is met for this recessive NBN disorder. The exact NBN c.657_661del (657del5) variant was reported in an affected boy who was homozygous for the variant, and in an additional NBS cohort containing affected individuals homozygous for 657del5 and individuals heterozygous for 657del5 with a second NBN mutation. A separate affected twin pair was heterozygous for 657del5 and compound heterozygous with c.643C>T, with parental segregation analysis reported. These observations support the variant occurring on both disease alleles in affected individuals, including in trans with another NBN variant.

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:10852373, Results/DNA analysis: the affected boy with Nijmegen breakage syndrome was homozygous for 657del5, the exact NBN c.657_661del variant.
• PMID:9590180, Results/Mutation Detection and NBS Mutations: the NBS cohort included affected individuals homozygous for 657del5 and two individuals heterozygous for 657del5 with a second NBN mutation.
• PMID:16033915, Results/mutation analysis: affected monozygotic twins were heterozygous for 657del5 and compound heterozygous with NBN c.643C>T (p.Arg215Trp); the study included parental segregation analysis.

Sources

Reasoning

Generic PM2 supporting requires absence from controls or an extremely low frequency, with the supplied generic threshold <=0.0001. The applicable all-comers gnomAD v4.1 filtering grpmax FAF is 0.00026547 (0.026547%), and gnomAD v2.1 grpmax FAF is 0.0002965 (0.02965%); both exceed 0.0001. The variant is therefore rare but does not meet the supplied quantitative PM2 threshold.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868) ... 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: 363/1,613,090 alleles, total AF=0.000225034, no homozygotes, filtering grpmax FAF=0.00026547.
• gnomAD v2.1 all-comers: 57/282,132 alleles, total AF=0.000202033, no homozygotes, filtering grpmax FAF=0.0002965.
• Both filtering FAF values exceed the supplied generic PM2 threshold of <=0.0001; the variant is not absent or sufficiently rare under this rule.

Sources

Reasoning

Generic PP5 is restricted here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar identifies the exact variant as Pathogenic, but the case audit reports zero expert-panel submissions; the available assertions are ordinary laboratory or other non-expert submissions. Published reports and non-expert ClinVar submissions therefore cannot trigger PP5 under the specified rule.

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 for variation 6940 reports Pathogenic classifications but the submission audit reports expert_panel_submissions = 0.
• The specified application rule permits PP5 only for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic; ordinary laboratory submissions, aggregate labels, and non-expert assertions do not trigger PP5.

Sources

Reasoning

Generic BA1 requires an allele frequency of at least 0.05. The highest applicable gnomAD filtering frequency is the gnomAD v4.1 grpmax FAF of 0.00026547 (0.026547%), based on 363/1,613,090 all-comers alleles; this is far below 0.05. The gnomAD v4.1 highest population point estimate is also only 0.000291653 in European (non-Finnish) individuals, and the population sample exceeds 2,000 observed alleles. The known NBN 657del5 founder/pathogenic association further argues against using a benign stand-alone call from a default frequency threshold.

Rule

BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868) ... Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v4.1 all-comers: 363/1,613,090 alleles, AF 0.000225034, no homozygotes; filtering grpmax FAF=0.00026547. The highest population point estimate was European (non-Finnish) AF=0.000291653 from 344/1,179,482 alleles.
• gnomAD v2.1 all-comers: 57/282,132 alleles, AF=0.000202033, no homozygotes; filtering grpmax FAF=0.0002965.
• The variant is the NBN 657del5 founder deletion; the original NBN report states it was absent from 50 normal controls and 28 normal untransmitted parental chromosomes (PMID:9590180), and a Polish case-control study reports 9/1,500 controls (0.6%) (PMID:14973119). These pathogenic/founder data do not support BA1 stand-alone benign classification.
• Population sample-size check: the relevant gnomAD v4.1 highest-frequency population has 1,179,482 observed alleles, exceeding the 2,000-allele minimum; the grpmax FAF, not the popmax point estimate, was used.

Sources

Reasoning

Generic BS1 requires an allele frequency greater than expected for the disorder; the supplied generic default is >=0.01. The highest applicable filtering frequency is gnomAD v4.1 grpmax FAF=0.00026547 (0.026547%), well below 0.01, and the gnomAD v4.1 population point estimate is only 0.000291653 in European (non-Finnish) individuals. NBN-related Nijmegen breakage syndrome is an autosomal recessive, severe early-onset disorder, and the observed frequency is not elevated enough to require a maximum-credible-frequency exception or a disease-prevalence/penetrance override.

Rule

BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168) ... Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1 all-comers: filtering grpmax FAF=0.00026547; total AF=0.000225034 from 363/1,613,090 alleles, with no homozygotes. The highest population point estimate was 0.000291653 in European (non-Finnish) individuals from 344/1,179,482 alleles.
• gnomAD v2.1 all-comers: filtering grpmax FAF=0.0002965; total AF=0.000202033 from 57/282,132 alleles, with no homozygotes.
• The International Nijmegen Breakage Syndrome Study describes NBS as a rare autosomal recessive disorder (PMID:10799436), supporting the relevant disease model; no reduced-penetrance or late-onset rationale is needed to explain this much lower frequency.

Sources

Reasoning

BS2 requires observation in a healthy adult individual in the relevant inheritance state. This NBN variant is associated with an autosomal recessive disorder, so BS2 would require a healthy adult homozygote. gnomAD v4.1 reports 0 homozygotes among 1,613,090 alleles and gnomAD v2.1 reports 0 homozygotes among 282,132 alleles; therefore the required healthy homozygous observation is absent. The published homozygous observations are affected Nijmegen breakage syndrome cases, not healthy adults.

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 homozygotes=0 among 363 observed alleles and 1,613,090 total alleles; gnomAD v2.1 reports homozygotes=0 among 57 observed alleles and 282,132 total alleles.
• The variant is evaluated in the autosomal-recessive NBN/Nijmegen breakage syndrome context; the published homozygous report describes an affected boy with Nijmegen breakage syndrome, not a healthy adult (PMID:10852373).
• No evidence was found for a healthy adult homozygote, so the BS2 requirement is not satisfied.

Sources

Reasoning

Generic BP6 is restricted here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar's exact-variant audit reports zero expert-panel submissions and does not provide an expert-panel Benign or Likely benign classification. Ordinary laboratory submissions, aggregate labels, and non-expert assertions 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

• ClinVar exact-variant record for variation 6940 has expert_panel_submissions = 0 and no expert-panel Benign or Likely benign classification.
• The specified application rule permits BP6 only for an exact-variant ClinVar expert-panel classification of Benign or Likely benign; ordinary laboratory submissions, aggregate labels, and non-expert assertions do not trigger BP6.

Sources

Reasoning

The available evidence does not document a proband-specific de novo event with confirmed maternity and paternity. Published reports describe the variant in homozygous affected patients, and one report documents inheritance of the 657del5 allele from the mother in monozygotic twins; neither establishes de novo origin for this case. No case-specific family history is provided.

Rule

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

Evidence considered

• PMID:16033915 reports that the 657del5 allele was inherited from the mother in monozygotic twin brothers; this is inheritance evidence, not de novo evidence.
• PMID:9590180 reports 657del5 in affected NBS patients and examines normal untransmitted parental chromosomes, but does not establish a de novo event in the case proband.

Still missing

• Case-specific proband genotype and phenotype documentation
• Case-specific maternity confirmation
• Case-specific paternity confirmation
• Case-specific parental genotypes and family history

Sources

Reasoning

The available evidence does not document an assumed de novo event in the proband. The literature instead describes inherited or biallelic 657del5 genotypes, including maternal transmission in monozygotic twins; there is no case-specific evidence establishing de novo origin with maternity and paternity unconfirmed.

Rule

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

Evidence considered

• PMID:16033915 documents maternal inheritance of the 657del5 allele in monozygotic twins, which argues against using this report as PM6 evidence.
• PMID:9590180 describes homozygous and compound heterozygous affected patients and normal untransmitted parental chromosomes, without a case-specific presumed de novo event.

Still missing

• Case-specific evidence that the variant is de novo or presumed de novo
• Case-specific parental genotypes
• Case-specific family history and disease phenotype

Sources

Reasoning

The available reports do not provide qualifying cosegregation of this variant with disease in multiple affected members of a single family. PMID:9590180 describes many unrelated NBS families and affected patients with homozygous or compound genotypes, while PMID:16033915 describes twins with maternal inheritance and PMID:14973119 concerns prostate-cancer susceptibility rather than cosegregation of Nijmegen breakage syndrome. The required family-level affected-member segregation data are therefore unavailable.

Rule

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

Evidence considered

• PMID:9590180 reports a cohort of 51 unrelated NBS families, including 46 patients homozygous for 657del5 and 2 heterozygous patients with a second mutation; this is not a documented within-family cosegregation analysis across multiple affected relatives.
• PMID:16033915 reports monozygotic twins with maternal inheritance of 657del5, but does not provide the multiple-affected-family segregation evidence required here.
• PMID:14973119 reports the allele in affected prostate-cancer relatives, but the study addresses cancer susceptibility and does not establish cosegregation with Nijmegen breakage syndrome.

Still missing

• A pedigree with multiple affected relatives
• Genotypes for affected relatives and informative unaffected relatives
• Disease phenotype and diagnostic confirmation for each relative
• Phase and inheritance information for the biallelic NBN disorder

Sources

Reasoning

The governing PP4 rule requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available materials document NBN-associated disease phenotypes in published cohorts, but do not provide a phenotype or family-history description for the individual case being adjudicated. Therefore PP4 cannot be assessed from the available case-specific evidence.

Rule

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

Still missing

• Case-specific phenotype and family-history information, including whether it is highly specific for a single-genetic-etiology NBN disorder.

Reasoning

BS3 is not assessed. The supplied literature triage identifies PMID:22131123 as a study of heterozygous c.657-661del cells with results similar to controls, but the full text was not available in the case files and the source is not represented as a source-registry record. The available summary does not establish that the exact variant has no damaging effect on NBN protein function or splicing, particularly because a heterozygous cellular context can mask the effect of a loss-of-function allele. The available evidence therefore cannot support the required well-established no-damage conclusion.

Rule

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

Evidence considered

• The case literature triage lists PMID:22131123, Impact of heterozygous c.657-661del, p.I171V and p.R215W mutations in NBN on nibrin functions, as a BS3 candidate and summarizes heterozygous c.657-661del assay results as similar to controls; this was not independently auditable from the supplied full text.

Still missing

• Full text and auditable assay details for the PMID:22131123 BS3 lead are unavailable in the case files.
• The supplied summary does not provide validated controls, quantitative results, or evidence that heterozygous findings exclude a damaging effect of this loss-of-function allele.

Reasoning

No affected family members are reported who lack the variant. PMID:9590180 describes absence of 657del5 from normal controls and normal untransmitted parental chromosomes, but those individuals were not affected relatives and therefore do not establish lack of segregation in affected members. The available evidence is insufficient to apply BS4.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• PMID:9590180 reports that 657del5 was absent from 50 normal controls and 28 normal untransmitted parental chromosomes; these are unaffected individuals and do not satisfy BS4.
• PMID:16033915 reports maternal transmission to monozygotic twins and does not report an affected relative who lacks the variant.

Still missing

• Affected relatives tested for the variant
• Reliable disease diagnoses and phenotyping for those relatives
• A family pedigree permitting assessment of variant non-segregation

Sources

Reasoning

BP2 cannot be assessed from the available evidence. The reported NBN observations document homozygous or compound-heterozygous affected individuals, but do not establish that c.657_661del is in cis with another pathogenic variant. The trans branch of BP2 is limited to a fully penetrant dominant gene/disorder, whereas NBN-related Nijmegen breakage syndrome is recessive in the cited 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.

Still missing

• No phasing evidence demonstrates c.657_661del in cis with another pathogenic variant.

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The available case materials and reviewed literature do not document an alternate molecular diagnosis for the individual case, so BP5 cannot be assessed.

Rule

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

Still missing

• Case-level molecular findings establishing an alternate molecular basis for the disease.