CXCR4 c.1013C>G, p.Ser338Ter

Reasoning

PS3 is met at strong strength. Three independent studies directly evaluated the exact CXCR4 c.1013C>G (p.Ser338Ter/S338X) variant. Patient-derived cells and engineered expression models demonstrated impaired CXCL12-induced receptor internalization and desensitization, enhanced G-protein signaling and chemotaxis, and transdominant effects on wild-type CXCR4. Orthogonal mechanistic assays included receptor internalization, actin polymerization, GTPγS binding, chemotaxis, ERK1/2 phosphorylation, β-arrestin2 depletion, and receptor dimerization. A separate study used engineered Waldenström macroglobulinemia models and SCID/Bg mice, showing increased adhesion, proliferation, dissemination, serum IgM, reduced survival, and response to anti-CXCR4 treatment. The evidence is variant-specific, biologically relevant to CXCR4 signaling, includes patient material and wild-type/model comparisons, and is concordant across in vitro and in vivo systems; however, it is research functional evidence rather than a validated and reproducible clinical diagnostic laboratory assay, supporting strong rather than very strong PS3 calibration.

Rule

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

Evidence considered

• Balabanian et al. 2005 (PMID:15536153): exact C1013>G/Ser338Ter variant studied in affected siblings, patient cells, and heterologous expression systems; impaired CXCL12/PMA-induced internalization and desensitization, enhanced G-protein signaling and chemotaxis, and transdominant interference with wild-type CXCR4 were reported.
• Lagane et al. 2008 (PMID:18436740): exact CXCR4 S338X variant studied in patient leukocytes and engineered HEK/A0.01 models; enhanced CXCL12 chemotaxis and prolonged β-arrestin2-dependent ERK1/2 phosphorylation were shown, with β-arrestin2 silencing reducing the phenotype and receptor dimerization assessed by coimmunoprecipitation/BRET.
• The 2014 C1013G/CXCR4 study (PMID:24711662): exact C1013G/S338X variant studied in primary WM cells, engineered BCWM.1/MWCL1 models, and SCID/Bg mice; increased surface CXCR4, adhesion, proliferation, dissemination, serum IgM, and reduced survival were reported, with anti-CXCR4 treatment reducing dissemination and signaling.
• The functional evidence is concordant across patient-derived material, engineered cellular systems, orthogonal signaling/trafficking assays, and an in vivo mouse model, but no evidence in the reviewed sources establishes validation and reproducibility in a clinical diagnostic laboratory setting.

Sources

Reasoning

PMID:24711662 reports the exact CXCR4 C1013G/S338X variant in 37/131 (28.2%) patients with Waldenstrom macroglobulinemia and 0/32 healthy volunteers. This is a case-control comparison even though the paper does not print an odds ratio. Using a Haldane-Anscombe 0.5 correction for the zero control cell, I computed OR = 26.07 with an approximate 95% CI of 1.56-436.79; the CI excludes 1.0 and OR >5.0. PMID:18436740, PMID:15536153, and PMID:23009155 verify the exact variant in affected WHIM patients but report case observations without unaffected-control counts. The enrichment result is from a somatic WM cohort rather than a germline WHIM cohort, so human review is required before carrying this PS4 evidence into a germline disease interpretation.

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

• Roccaro et al. 2014, PMID:24711662, Table 1: exact C1013G/CXCR4 variant in 37/131 WM patients (28.2%) and 0/32 healthy volunteers.
• Computed the case-control odds ratio from the reported counts with a Haldane-Anscombe 0.5 correction for the zero control cell: OR = 26.07; approximate 95% CI = 1.56-436.79.
• The inequality required by the governing rule is OR 26.07 > 5.0? yes; the 95% CI 1.56-436.79 includes 1.0? no.
• PMID:18436740, PMID:15536153, and PMID:23009155 verify the exact S338X/C1013G variant in affected WHIM patients but do not provide unaffected-control counts.

Still missing

• The positive case-control cohort is a somatic Waldenstrom macroglobulinemia study, not a germline WHIM case-control cohort; confirm disease-context applicability for this germline interpretation.

Sources

Reasoning

PM2 is met at supporting strength. NM_003467.3:c.1013C>G (CXCR4 p.Ser338Ter) is absent from gnomAD v2.1 and gnomAD v4.1, and is also absent from the available gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets. This satisfies the supplied generic PM2 supporting threshold of <=0.0001 because the observed frequency is effectively zero in the queried population datasets.

Rule

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

Evidence considered

• gnomAD v2.1: variant absent in the exome dataset; no alternate allele was observed.
• gnomAD v4.1: variant absent in the queried dataset; no alternate allele was observed.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records also report the variant as absent.
• The generic PM2 threshold supplied for this case is <=0.0001; absence corresponds to an observed allele frequency of 0 in these datasets.

Sources

Reasoning

PP1 is met at supporting strength. The exact CXCR4 c.1013C>G (p.Ser338Ter) variant was reported in affected siblings in one family, with the siblings sharing an autosomal-dominant paternal haplotype, and independently in three patients from one pedigree. These are multiple affected family members carrying the same variant in a gene reported as causative for WHIM syndrome. The supplied governing text permits stronger use with increasing segregation data, but supplies no quantitative threshold for escalation; therefore supporting strength is assigned.

Rule

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

Evidence considered

• Balabanian et al. (PMID:15536153) reports affected siblings P1 and P2 with the exact CXCR4 mutation and an autosomal-dominant paternal haplotype.
• Beaussant Cohen et al. (PMID:23009155) reports the exact C1013→G/S338X variant in three patients from one pedigree.
• Together, these reports provide concordant observations of the exact variant in multiple affected family members; no quantitative segregation threshold for upgraded strength is supplied in the governing rules.

Sources

Reasoning

NM_003467.3:c.1013C>G is a confirmed nonsense variant, NM_003467.3 is the MANE Select transcript, and the change produces NP_003458.1:p.(Ser338Ter) in exon 2 of 2, removing the final 15 amino acids of the 353-residue protein. The variant therefore satisfies the listed null-variant consequence, but PVS1 requires a gene in which loss of function is a known disease mechanism. Variant-specific studies show that this terminal-tail truncation retains and enhances CXCR4 signaling, impairs receptor internalization/desensitization, and causes enhanced chemotaxis, i.e. a gain-of-function mechanism rather than loss of function (PMID:15536153; PMID:18436740); the clinical cohort paper likewise describes S338X as a C-terminal truncation associated with WHIM syndrome (PMID:23009155). Because this is a terminal exon truncation with preserved receptor function and established gain-of-function biology, PVS1 is not met; no PVS1 strength tier is assigned.

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

• Variant normalization and transcript evidence identify NM_003467.3:c.1013C>G (MANE Select) as NP_003458.1:p.(Ser338Ter), a nonsense variant in exon 2 of 2; the reference protein ends at residue 353 and the predicted protein ends at residue 338, removing the final 15 residues.
• PMID:15536153 directly studied C1013>G/CXCR4 S338X in affected siblings and experimental models; the truncated receptor showed impaired CXCR4 internalization and desensitization, enhanced CXCL12 signaling and chemotaxis, and transdominant-negative effects on wild-type CXCR4, supporting gain-of-function biology rather than loss of function.
• PMID:18436740 directly studied CXCR4 S338X and reported loss of the final 15 C-terminal residues with enhanced CXCL12-induced chemotaxis and prolonged beta-arrestin2-dependent ERK1/2 phosphorylation in patient cells and engineered models.
• PMID:23009155 reported C1013>G/S338X in three patients from one pedigree and described it as a nonsense mutation causing C-terminal-tail truncation.
• The governing PVS1 framework was applied only to the null-variant and loss-of-function-mechanism requirements; the terminal-tail truncation's demonstrated retained/enhanced function prevents assignment of PVS1.

Sources

Reasoning

PS2 is not met. The exact CXCR4 c.1013C>G (p.Ser338Ter) variant was reported in affected siblings who inherited the autosomal-dominant haplotype from their father, and in three patients from one pedigree. This is evidence of familial transmission rather than a de novo occurrence, and the required combination of confirmed maternity and paternity with no family history is not satisfied.

Rule

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

Evidence considered

• Balabanian et al. (PMID:15536153) reports that affected siblings P1 and P2 carried the heterozygous CXCR4 c.1013C>G-related mutation and inherited the autosomal-dominant haplotype from their father.
• Beaussant Cohen et al. (PMID:23009155) reports the C1013→G/S338X variant in three patients from one pedigree.
• These reports support familial inheritance and do not satisfy PS2's requirement for a de novo variant with confirmed maternity and paternity and no family history.

Sources

Reasoning

PM6 is not met. The exact variant was reported in affected siblings with inheritance of the autosomal-dominant haplotype from their father and in three patients from one pedigree. The evidence therefore indicates familial transmission, not an assumed de novo variant lacking parental confirmation.

Rule

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

Evidence considered

• Balabanian et al. (PMID:15536153) reports that affected siblings inherited the autosomal-dominant haplotype from their father and carried the CXCR4 mutation.
• Beaussant Cohen et al. (PMID:23009155) reports the exact C1013→G/S338X variant in three patients from one pedigree.
• The available evidence describes familial inheritance rather than an assumed de novo occurrence without parental confirmation.

Sources

Reasoning

The exact variant is classified Pathogenic in ClinVar, but the case audit reports zero expert-panel submissions. The available assertions are ordinary laboratory or non-expert submissions, so the generic exact-variant expert-panel requirement for PP5 is not met.

Rule

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

Evidence considered

• ClinVar variation 14023 exactly matches NM_003467.3:c.1013C>G, but the submission audit records expert_panel_submissions = 0 and five clinical-laboratory Pathogenic submissions.
• The ClinVar Pathogenic label and ordinary laboratory submissions were not used as PP5 evidence.

Sources

Reasoning

BA1 is not met. NM_003467.3:c.1013C>G (CXCR4 p.Ser338Ter) is absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer subsets, so no filtering allele frequency or qualifying population with at least 2000 observed alleles reaches the generic stand-alone threshold of >=0.05. The absence also provides no basis for a high-frequency benign call despite the pathogenic ClinVar consensus.

Rule

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

Evidence considered

• gnomAD v2.1: variant absent; no allele count, allele frequency, or grpmax FAF reported.
• gnomAD v4.1: variant absent; no allele count, allele frequency, or grpmax FAF reported.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records also report the variant as absent.
• The generic BA1 threshold supplied for this case is >=0.05, and no qualifying frequency is present.

Sources

Reasoning

BS1 is not met on the available population data. The variant is absent from gnomAD v2.1 and v4.1 and from the available non-cancer subsets, so the required filtering allele frequency is not greater than expected for the disorder and does not reach the generic default of >=0.01. No ancestry-specific elevated frequency is available that would require a maximum-credible-frequency analysis.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: variant absent; no filtering allele frequency or grpmax FAF reported.
• gnomAD v4.1: variant absent; no filtering allele frequency or grpmax FAF reported.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records also report the variant as absent.
• The generic BS1 default supplied for this case is >=0.01; absence from the population datasets does not support BS1.

Sources

Reasoning

BS3 is not met. The exact variant has not been shown by well-established functional studies to have no damaging effect on protein function or splicing. Instead, the available patient-cell, engineered-cell, and in vivo studies consistently demonstrate abnormal CXCR4 trafficking, signaling, chemotaxis, and disease-relevant tumor phenotypes. No benign functional or splicing result, validated normal control interpretation, or assay showing preserved CXCR4 function was identified.

Rule

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

Evidence considered

• Balabanian et al. 2005 (PMID:15536153), Lagane et al. 2008 (PMID:18436740), and Poulain et al. 2014 (PMID:24711662) were directly evaluated for possible benign functional evidence; their exact-variant results instead demonstrate abnormal CXCR4 function and disease-relevant phenotypes.
• The reviewed studies do not provide a normal-function or normal-splicing assay result for CXCR4 c.1013C>G/p.Ser338Ter.

Still missing

• No well-established functional study demonstrating preserved CXCR4 protein function or normal splicing for this exact variant.

Sources

Reasoning

The exact variant has no ClinVar expert-panel benign or likely-benign classification. ClinVar contains Pathogenic laboratory/non-expert submissions only, which cannot trigger BP6 under the specified generic rule.

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 14023 exactly matches the variant; the submission audit records zero expert-panel submissions and no benign or likely-benign expert-panel assertion.
• Ordinary laboratory submissions, aggregate labels, and non-expert assertions were not used as BP6 evidence.

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 case materials identify the variant and relevant WHIM literature but do not provide this patient's phenotype or family history in sufficient detail to assess specificity.

Rule

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

Evidence considered

• The reviewed papers describe WHIM syndrome features and affected individuals carrying the exact variant, but they do not substitute for the patient's own phenotype or family history.
• PMID:15536153 describes WHIM syndrome with the exact variant in affected siblings; PMID:18436740 describes affected siblings with S338X; PMID:23009155 reports a cohort including three patients with C1013G/S338X.

Still missing

• Patient-level phenotype and family-history details, including whether findings are highly specific for WHIM syndrome, are unavailable.

Sources

Reasoning

BS2 is not assessed. The case evidence contains no documented healthy adult carrying NM_003467.3:c.1013C>G in the relevant zygosity or hemizygous state, and population absence cannot establish the healthy-adult observation required by BS2. The available evidence also does not provide a curated penetrance and age-of-onset assessment sufficient to substitute for that observation.

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

• The case bundle provides population-database absence but no healthy-adult carrier observation for this exact variant.
• No qualifying homozygous, heterozygous, or hemizygous healthy-adult observation with age and phenotype context was identified.

Still missing

• A reliable observation of the exact variant in a healthy adult, with relevant zygosity and age/phenotype information, is unavailable.

Sources

Reasoning

BS4 cannot be assessed from the available evidence. The reviewed reports document affected carriers and familial transmission of the exact variant, but do not provide an affected non-carrier or other documented lack-of-segregation observation. The absence of such evidence is not sufficient to establish BS4.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• PMID:15536153 reports affected siblings carrying the variant and does not report an affected non-carrier.
• PMID:23009155 reports three affected patients from one pedigree carrying the variant but does not provide sufficient affected-relative genotype data to evaluate lack of segregation.

Still missing

• Affected-family-member genotypes and phenotypes sufficient to evaluate non-segregation are not reported.

Sources

Reasoning

BP2 cannot be assessed from the available evidence. The reports establish the queried CXCR4 variant in affected individuals and describe a dominant WHIM context, but no report documents a second pathogenic variant in trans or a pathogenic variant in cis with c.1013C>G, and the evidence does not establish that the relevant dominant disorder is fully penetrant as required for the trans pathway. Parent/offspring testing or equivalent phased allele data and explicit penetrance information are needed.

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

• Balabanian et al. 2005 (PMID:15536153): reports the queried variant as heterozygous in affected siblings and describes dominant inheritance, but does not report a second pathogenic variant or cis/trans phase with one.
• Lagane et al. 2008 (PMID:18436740): reports the S338X variant in siblings with WHIM syndrome and functional studies of the single variant, but no second pathogenic allele or phase result.
• Beaussant Cohen et al. 2012 (PMID:23009155): reports C1013→G/S338X in three patients from one pedigree, without a second pathogenic variant or documented cis/trans phase.
• Richards et al. 2015 (PMID:25741868): ACMG/AMP source for the BP2 definition applied here.

Still missing

• No parent/offspring or equivalent testing documents whether another pathogenic variant is in cis or trans.
• The available evidence does not establish full penetrance for the dominant disorder, which is required for the BP2 trans pathway.

Sources

Reasoning

No case-specific evidence establishes that this variant was found in a case with an alternate molecular basis for the relevant disease. The available literature describes the exact variant in WHIM or somatic WM contexts but does not document an alternate molecular cause for the case being interpreted.

Rule

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

Evidence considered

• The reviewed evidence does not document a second pathogenic variant or another molecular diagnosis explaining the relevant case.

Still missing

• Case-level testing and diagnostic information needed to establish an alternate molecular basis are unavailable.