ClinVar Genomic variation as it relates to human health

The c.187C>T variant in the hepatocyte nuclear factor 4-alpha gene, HNF4A, causes an amino acid change of arginine to tryptophan at codon 63 (p.(Arg63Trp) of NM_175914.5. This variant is absent in gnomAD v2.1.1 (PM2_Supporting), and was identified in 21 unrelated individuals with non- autoimmune and non-absolute/near-absolute insulin-deficient diabetes (PS4; PMID: 24285859, 25819479, 28693455, internal lab contributors). This variant segregated with diabetes, with at least 7 meioses in 4 families (PP1_Strong, PMID: 24285859, 28693455, internal lab contributors). At least 6 individuals have a clinical history highly specific for HNF4A-MODY (MODY probability calculator result >50%, negative genetic testing for HNF1A, and Fanconi phenotype) (PP4_Moderate; internal lab contributors). Additionally, this variant was identified as a de novo occurrence with both confirmed and unconfirmed parental relationships in 5 individual(s) with a clinical picture consistent with HNF4A-MODY (MODY probability calculator result >50% and negative genetic testing for HNF1A) (PS2_Very Strong; internal lab contributor, PMID: 24285859, 25819479). This variant resides in an amino acid within the HNF4alpha DNA binding domain that directly binds DNA, which is defined as critical for the protein’s function by the ClinGen MDEP (PM1). It is also predicted to be deleterious by computational evidence, with a REVEL score of 0.949, which is greater than the MDEP VCEP threshold of 0.70 (PP3). Lastly, another missense variant, c.188G>A (p.Arg63Gln), has been interpreted as pathogenic by the ClinGen MDEP, and p.Arg63Trp has a greater Grantham distance (PM5). In summary, c.187C>T meets the criteria to be classified as pathogenic for monogenic diabetes. ACMG/AMP criteria applied, as specified by the ClinGen MDEP (specification version 2.0.0, approved 10/11/2023): PS2_Very strong, PP1_Strong, PS4, PP4_Moderate, PM1, PP3, PM2_Supporting, PM5_Supporting.

Not found in the total gnomAD dataset, and the data is high quality. Predicted to have a damaging effect on the protein. Found in multiple individuals with expected phenotype for this gene. Segregation with disease, and data include affected and unaffected individuals from multiple families. One de novo case with parental identity confirmed plus 2 unconfirmed cases.

Published functional studies demonstrate a damaging effect for variant described as R85W, using alternate nomenclature; corresponding variant in transfected cells (Drosophila nephrocytes) showed lipid metabolism defects and mitochondrial dysfunction in a dominant-negative manner; Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect; Reported as R76W using alternate nomenclature; This variant is associated with the following publications: (PMID: 20164212, 22802087, 25819479, 24285859, 27245055, 28693455, 28458902, 30005691, 29493090, 31949432, 31618753, 32960281, 31216405, 33251707, 31875549)

The variant is not observed in the gnomAD v2.1.1 dataset. Functional studies provide strong evidence of the variant having a damaging effect on the gene or gene product (PMID: 31875549). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.95; 3Cnet: 1.00). Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000156152). The variant has been observed in multiple (>3) similarly affected unrelated individuals (PMID: 20164212, 24285859, 27245055, 28458902, 28693455, 30005691, 31875549). A different missense change at the same codon (p.Arg63Gln) has been reported to be associated with HNF4A related disorder (ClinVar ID: VCV000372382 / PMID: 23348805). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

This sequence change replaces arginine, which is basic and polar, with tryptophan, which is neutral and slightly polar, at codon 63 of the HNF4A protein (p.Arg63Trp). For these reasons, this variant has been classified as Pathogenic. Experimental studies have shown that this missense change affects HNF4A function (PMID: 31875549). Algorithms developed to predict the effect of missense changes on protein structure and function are either unavailable or do not agree on the potential impact of this missense change (SIFT: "Deleterious"; PolyPhen-2: "Probably Damaging"; Align-GVGD: "Class C0"). ClinVar contains an entry for this variant (Variation ID: 156152). This variant is also known as R76W and R85W. This missense change has been observed in individual(s) with hypoglycemia with hyperinsulinemia and atypical renal Fanconi syndrome (PMID: 20164212, 25819479, 28458902). In at least one individual the variant was observed to be de novo. This variant is not present in population databases (gnomAD no frequency).

The p.R63W pathogenic mutation (also known as c.187C>T, p.R85W, and p.R76W), located in coding exon 2 of the HNF4A gene, results from a C to T substitution at nucleotide position 187. The arginine at codon 63 is replaced by tryptophan, an amino acid with dissimilar properties. This mutation has been detected in individuals with diazoxide responsive hyperinsulinemic hypoglycemia (HH), Fanconi syndrome, nephrocalcinosis, congenital hyperinsulinism (CHI) and hypoglycemia (Flanagan SE et al. Eur. J. Endocrinol., 2010 May;162:987-92; Hamilton AJ et al. J. Med. Genet., 2014 Mar;51:165-9; Numakura C et al. Diabetes Res. Clin. Pract., 2015 Jun;108:e53-5; Walsh SB et al. BMC Nephrol, 2017 Jul;18:230). In addition, this mutation has been detected as de novo occurrences (paternity not confirmed) in one individual with hyperinsulinism, hepatomegaly, macrosomia, and renal Fanconi syndrome (Stanescu DE et al. J. Clin. Endocrinol. Metab., 2012 Oct;97:E2026-30), in another with hyperinsulinaemic hypoglycaemia and renal tubulopathy (Clemente M et al. Endocrinol Diabetes Metab Case Rep, 2017 Mar;2017:), and in one with Fanconi syndrome, infantile hyperinsulinemic hypoglycemia, transient cholestasis, and bilateral severe hearing loss. (Liu J et al. J Med Case Rep, 2018 Jul;12:203). A different alteration located at the same position, p.R63Q, has been detected in two families with maturity-onset diabetes of the young (MODY); however, specific phenotypic info was not provided (Colclough K et al. Hum. Mutat., 2013 May;34:669-85). In addition, based on internal structural analysis, this variant is more disruptive than known pathogenic variants nearby. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

ACMG classification criteria: PS2, PS3 supporting, PS4 moderate, PM2, PP1, PP3

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0103 - Loss of function and dominant negative are known mechanisms of disease in this gene. Loss of function through a reduction of DNA binding is associated with MODY, type I (MIM#125850) and diabetes mellitus, noninsulin-dependent (MIM#125853) (OMIM; PMID: 31875549). Dominant negative is associated with Fanconi renotubular syndrome 4, with maturity-onset diabetes of the young (MIM#616026) (PMID: 31875549). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0112 - The condition associated with this gene has incomplete penetrance (PMID: 36257325). (I) 0200 - Variant is predicted to result in a missense amino acid change from arginine to tryptophan. (I) 0251 - This variant is heterozygous. (I) 0301 - Variant is absent from gnomAD (both v2 and v3). (SP) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0603 - Missense variant in a region that is highly intolerant to missense variation (high constraint region in DECIPHER). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has frequently been reported as pathogenic in ClinVar and has been observed as heterozygous in multiple families and unrelated individuals with Fanconi syndrome (PMID: 24285859; PMID: 30005691). (SP) 1002 - This variant has moderate functional evidence supporting abnormal protein function (PMID:31875549). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

This patient is heterozygous for the c.187C>T (p.Arg63Trp) variant in the HNF4A gene. This variant has been previously described in multiple patients with Fanconi renotubular syndrome (Hamilton et al 2014 J Med Genet; 51: 165-169).

DNA sequence analysis of the HNF4A gene demonstrated a sequence change, c.187C>T, in exon 2 that results in an amino acid change, p.Arg63Trp. This sequence change has been described previously in a patient with hyperinsulinism, where it was inherited from a parent affected by diabetes (Flanagan et al., 2010). The c.187C>T sequence change has not been described as a known benign sequence change in the HNF4A gene. The p.Arg63Trp change affects a highly conserved amino acid residue located in a domain of the HNF4A protein that is known to be functional. The p.Arg63Trp substitution appears to be deleterious using several in-silico pathogenicity prediction tools (SIFT PolyPhen2, Align GVGD, MutationTaster).