Gene: HNF1A (HGNC:11621)
Evidence Code: PS3 — Supporting
Modification Type: Gene-specific,Strength
See list of approved functional studies and guidelines for interpretation of data (below).




Luciferase assays for transactivation


“Decreased function” is defined as activity less than 40% of wildtype (WT).


Note: this threshold is not 100% specific for transactivation (TA) activity and is complicated by the fact that TA activity will vary depending on many factors, for instance cell line (HeLa, INS, MIN6, etc.) and reporter construct that is used. Commonly used promoters include rat/human albumin (PMID:12574234) and HNF4A-P2 (PMID: 32910913). Experiments should be designed to include more than one promoter and more than one cell line; any data less than 40% of WT warrants use of PS3_Supporting or PS3_Moderate, with more weight given to assays that have appropriate controls (see below).


Assays should include controls for WT (human HNF1A cDNA (NM_000545.8)), T2DM-risk and known MODY variants, which represent the range of behaviours across the HNF1A allelic spectrum. When calculating an OddsPath or counting the total number of control variants per Brnich et al. 2020 (PMID: 31892348) the following are well-characterized pathogenic HNF1A-MODY variants: c.335C>T (p.Pro112Leu), c.779C>A (p.Thr260Met), c.1340C>T (p.Pro447Leu), c.1556C>T (p.Pro519Leu), c.787C>T p.(Arg263Cys) and c.686G>A p.(Arg229Gln). We also recommend that assays include the following variants to capture dysfunction which increases risk for T2D but is insufficient to cause monogenic diabetes: c.293C>T (p.Ala98Val) and c.1522G>A (p.Glu508Lys) (PMID: 24915262, 26551672). The degree of TA impairment of HNF1A should correlate positively with the severity of the diabetes phenotype: disease-causing HNF1A variants impair TA activity more severely (<30-40% compared to WT) than T2D risk variants (40-75% compared to WT).


 We note that the data presented by the Oxford/Gloyn group in Althari et al (PMID:32910913) have four controls that can be classified as pathogenic based on non-functional data and 10 controls that can be classified as benign based on non-functional data. Using either HeLa or INS1 cells and cutoffs of 0.40/0.75, only ≤2 variants fall outside the expected range, and OddsPath based on the recommendations by Brnich et al 2020 (PMID: 31892348) can be achieved (>4.3) that enable the use of PS3_Moderate and BS3_Supporting. The data presented by the Bergen group in Althari et al (PMID: 32910913) used six controls that can be classified as pathogenic based on non-functional data and 18 controls that can be classified as benign based on non-functional data. Using either HeLa or INS1 cells and cutoffs of 0.40/0.75, the pathogenic variants in the Bergen data consistently fell into the expected TA range (<40%). However, two benign variants (not including T2D-risk variant p.Glu508Lys) fell into the pathogenic range and 14 out of 16 of the benign variants fell into the indeterminate range. We hence recommend applying PS3_Supporting and BS3_Supporting only at this time to data from this group. 


Validation of luciferase assay performed by Bergen and Oxford groups reported in Althari et al, with cutoffs of <40% of WT for functionally abnormal and ≥75% of WT for functionally normal










EMSA for DNA binding


“Decreased function” is defined as activity less than 40% of WT.


Note: the effect of the variant on DNA binding will be highly dependent on whether the variant is located within the DNA binding domain. Our experience is that for HNF1A-MODY causal variants located in the DNA binding domain, we observe DNA binding from 0-35% compared to WT. The following variants can be used as positive controls for reduced DNA binding ability (<40%): c.335C>T (p.Pro112Leu), c.608G>A (p.Arg203His), c.787C>T (p.Arg263Cys) and c.686G>A (p.Arg229Gln) (PMID: 11162430, 12574234, 24915262); we recommend the assay contain at least two. We also recommend that the assay include variants whose DNA binding ability has previously been assessed (e.g., c.298C>A (p.Gln100Lys) (70% of WT) and c.392G>A (p.Arg131Gln) (58% of WT) (PMID: 27899486).






Western blotting and indirect immunofluorescence for protein expression & localization


Determining appropriate thresholds for protein expression is more difficult due to variability in results between different experimental protocols. Sample preparations, gel loading, transfer efficiency, antibody specificity, choice of internal control and quantification method are some of the factors that can contribute to varying and inconsistent results between labs. Altered protein expression can be indirectly captured through the read-out from a transactivation assay and reduced protein expression can provide an explanation for reduced transactivation.


When exploring protein mis-localisation we recommend that the c.589_615del (p.Leu197_Leu205del) variant is included as a positive control for impaired nuclear localization (cytosolic retention) (PMID: 16274290).






Studies performed on a cell line generated from a patient sample (which will be heterozygous and also contain other variants in the patient’s genome which could modify function) will not count as PS3 but instead will count toward PP4_Moderate.


For canonical splice site variants, do not use PS3 for RNA studies demonstrating abnormal splicing, since PVS1 will already be used at some level. Per SVI recommendations, we will apply PS3 at the Strong level to non-canonical splice site variants that have RNA and in silico evidence of aberrant splicing.


 References


Najmi et al (2017) (PMID: 27899486)


Bjorkhaug et al (2003) (PMID: 12574234)


Brnich et al (2020) (PMID: 31892348)


Althari et al (2020) (PMID: 32910913)