ClinVar Genomic variation as it relates to human health

The variant NM_000551.4(VHL):c.376G>A (p.Asp126Asn) is a missense variant predicted to cause substitution of Aspartic acid by Asparagine at position 126. The GroupMax Filtering Allele Frequency (95% CI) in gnomAD v4.1.0 is 0.00003260 (50/1111878 from European, Non-Finnish Population). This is higher than the ClinGen VHL VCEP threshold of >=0.0000156 (0.00156%) threshold expected for VHL disease (BS1). However, this variant fulfills several pathogenic evidence codes as follows. This variant has been reported in 1 proband in literature, a 53yo male with right jugulotympanic paraganglioma (nonspecific = 0.25 points) (PMID: 22566194). This variant has been seen 12 times in testing at Invitae with 3 cases bearing VHL tumors, 3 times at GeneDX with no cases presenting VHL tumors, and 19 times at Ambry with 2 cases including VHL tumors. For the observed cases without VHL spectrum tumors, they either had no personal history of cancer or non-VHL cancers. None were >65yo and lacked VHL-spectrum tumors. In total, the case points calculated are 2.5 points which sums to PS4_Moderate. One HIF-a degradation assay using this variant showed modest / intermediate inability to degrade HIF-a (PMIDs:21454469), but does not meet the criteria for PS3_Supporting. This variant resides within a region of VHL that is defined as a critical functional domain (the Beta Domain) (PM1). The computational predictor REVEL gives a score of 0.716, which is above the threshold of >0.664, evidence that correlates with impact to VHL function (PP3). In summary, due to the prevalence of this variant in the gnomAD and numerous cases lacking VHL tumors (though not above the threshold of 65yrs to fulfill the benign criteria) as well as weak/intermediate functional evidence, this variant meets the criteria to be classified as Uncertain for autosomal-dominant von Hippel Lindau syndrome (VHL syndrome) based on the ACMG/AMP criteria applied, as specified by the ClinGen VHL VCEP Version 1.0 (Specifications approval date: 02/26/2024. Variant Approval Date 06/25/2024).

The p.D126N pathogenic mutation (also known as c.376G>A) is located in coding exon 2 of the VHL gene. This alteration results from a G to A substitution at nucleotide position 376. The aspartic acid at codon 126 is replaced by asparagine, an amino acid with highly similar properties. The p.D126N mutation has been shown to cause autosomal recessive polycythemia, a condition characterized by increased red blood cell mass and high risk of pulmonary hypertension, peripheral thrombosis and cerebrovascular events (Bond, J et al. Blood. 2011 Mar 31;117(13):3699-701). This alteration has been reported in the homozygous state in a 7-month-old boy with polycythemia, developmental delay, and severe early onset pulmonary hypertension (Sarangi S et al. Pediatr Blood Cancer 2014 Nov;61(11):2104-6). This alteration was also identified in a child with congenital pulmonary arterial hypertension, along with a second VHL variant, p.S183L; functional studies on both variants found that they both impair the ability of VHL to regulate hypoxia-inducible factors (HIF) (Bond, J et al. Blood. 2011 Mar 31;117(13):3699-701). To date, this variant has not been associated in the literature with VHL disease, although it has been identified in three families with a history of pheochromocytomas and in an individual with a history of renal cell carcinoma (Ambry internal data). Internal structural analysis reveals that this variant is anticipated to result in a decrease in structural stability (Van Molle I et al. Chem. Biol., 2012 Oct;19:1300-12). Based on protein sequence alignment, this amino acid position is well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Based on the available evidence, p.D126N is interpreted as a disease-causing mutation. At this time, individuals who are heterozygous for the p.D126N alteration can be interpreted as carriers for autosomal recessive polycythemia, however, the clinical implications of this alteration with respect to von Hippel-Lindau disease have not been determined.

Variant summary: VHL c.376G>A (p.Asp126Asn) results in a conservative amino acid change located in the von Hippel-Lindau disease tumour suppressor, beta domain of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 2e-05 in 251494 control chromosomes. c.376G>A has been reported in the literature in individuals affected with congenital polycythemia and pulmonary arterial hypertension. These data indicate that the variant is likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function. D126N exhibited an intermediate effect in increasing media pH, imparing the ability of VHL to regulate HIF, and destabilizing VHL protein. Eight clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Multiple laboratories reported the variant with conflicting assessments (pathogenic n=2, likely pathogenic n=2, VUS n=4). Based on the evidence outlined above, the variant was classified as likely pathogenic for recessive familial erythrocytosis. However, due to lack of evidence, this variant was classified as VUS for von Hippel-Lindau syndrome.

ACMG evidence PP3, PP5

This sequence change replaces aspartic acid, which is acidic and polar, with asparagine, which is neutral and polar, at codon 126 of the VHL protein (p.Asp126Asn). This variant is present in population databases (rs104893831, gnomAD 0.004%). This missense change has been observed in individual(s) with autosomal recessive erythrocytosis and pulmonary hypertension, and/or polycythemia and pulmonary hypertension (PMID: 21454469, 30338240, 35734542). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. This variant has been reported in individual(s) with clinical features of autosomal dominant von Hippel-Lindau syndrome, as well as in unaffected individuals (Invitae; external communication); however, the role of the variant in this condition is currently unclear. ClinVar contains an entry for this variant (Variation ID: 141044). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt VHL protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects VHL function (PMID: 21454469). In summary, this variant has been classified as Pathogenic for autosomal recessive erythrocytosis. However, the risk conferred by this variant is uncertain for autosomal dominant von Hippel-Lindau syndrome.

This missense variant replaces aspartic acid with asparagine at codon 126 of the VHL protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have reported that this variant results in unstable VHL protein and partial degradation of HIF1alpha and HIF2alpha protein compared to wild-type VHL when expressed in ex vivo cells (PMID: 21454469, 30338240). This variant has been reported in a heterozygous carrier affected with sporadic right jugulotympanic paraganglioma without any other features suggestive of von Hippel Lindau syndrome (PMID: 18551016) and in a compound heterozygous carrier with VHL p.Ser183Leu who was affected with severe erythrocytosis and pulmonary arterial hypertension (PMID: 21454469). This variant has been identified in 5/251494 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

While this variant is considered likely pathogenic for autosomal recessive familial erythrocytosis, it is considered a variant of uncertain significance regarding increased risk for von Hippel Lindau disease; Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Observed in individuals with papillary renal cell carcinoma and isolated paraganglioma in the published literature (PMID: 28043156, 35198402, 35441217); Observed in the homozygous and compound heterozygous state in association with congenital polycythemia, severe erythrocytosis and pulmonary arterial hypertension in individuals referred for genetic testing at GeneDx and in published literature (PMID: 21454469, 24729484, 35734542); Published functional studies demonstrate: intermediate effect on VHL functions (PMID: 21454469, 30338240); This variant is associated with the following publications: (PMID: 21454469, 24969085, 15177666, 24729484, 28043156, 30338240, 35198402, 35767051, 23102223, 35734542, 32238909, 18551016, 35205407, 35441217)