Heterozygous RFX6 PTVs in MODY with unknown aetiology

To identify patients with novel heterozygous PTVs, we first assessed 38 European (non-Finnish) probands with a strong MODY-like phenotype who did not have mutations in the common MODY genes (GCK, HNF1A, HNF4A) by Sanger sequencing (Supplementary Table 1). To exclude the other known/less common causes of monogenic diabetes, these patients underwent comprehensive targeted-next generation sequencing (NGS) for all 29 known monogenic diabetes genes, including genes for neonatal diabetes, MODY and mitochondrial diabetes, lipodystrophy or other forms of syndromic diabetes13 (Supplementary Table 2). We identified two probands with mutations in the known MODY gene HNF1B 13, 14. The analysis of heterozygous PTVs in the 29 genes on the targeted panel identified two unrelated probands with a novel heterozygous nonsense variant in Regulatory Factor X 6 (RFX6) (Family 1 - p.Leu292Ter, Family 2 - p.Lys351Ter) (Table 1, Fig. 1 and Supplementary Table 3). We did not identify any rare (<1%) missense RFX6 variants in this cohort. RFX6 was part of the targeted sequencing panel because recessive RFX6 variants (missense and/or protein-truncating) are a known cause of syndromic neonatal diabetes15, but heterozygotes were not previously known to have any phenotype.

Table 1 Frequency of heterozygous RFX6 protein-truncating variants in all study cohorts and control populations Full size table

Fig. 1 Extended pedigree of non-Finnish European patients identified in the discovery cohort. a Pedigree of family 1 that were identified with heterozygous RFX6 variant (NM_173560.3:c.875-T > G,p.Leu292Ter) from the discovery cohort. b Pedigree of family 2 from the discovery cohort with heterozygous RFX6 variant (NM_173560.3:c.1051-A > T, -p.Lys351Ter). Genotype is shown underneath each symbol; M and N denote mutant and wild-type alleles, respectively. Directly below the genotype is the age of diabetes onset in years, duration in years, BMI and treatment at study entry. Squares represent male family members, and circles represent female members. Black-filled symbols denote patients with diabetes. An arrow denotes the proband in the family. OHA, oral hypoglycaemic agents. *age at recruitment. One of the daughters of patient III.1 in family 2 had a history of gestational diabetes Full size image

RFX6 PTVs are enriched in a MODY discovery cohort

We next compared the frequency of RFX6 PTVs in our discovery cohort to a large control population with whole-exome data from ExAC12. Neither of the RFX6 variants from the discovery cohort were present in the 60,706 individuals in ExAC. There were 15 individuals with RFX6 PTVs in the 33,346 ExAC non-Finnish European control population (Supplementary Table 3). The frequency of the RFX6 PTVs in the MODY discovery cohort was significantly higher (after accounting for the multiple testing of 29 genes) than the ExAC non-Finnish European control population (5.5 vs. 0.045%, odds ratio (OR) 131, 95% confidence interval (CI) 14-595, P = 1 × 10−4) (Table 1).

RFX6 PTVs are enriched in a MODY replication cohort

To replicate the findings of our discovery cohort, we then examined 348 non-Finnish European probands who were routinely referred for MODY genetic testing to the Molecular Genetics Laboratory, Exeter, UK and in whom the common causes of MODY were excluded using targeted-NGS assay (Supplementary Table 1). The analysis of heterozygous PTVs identified four unrelated probands with two novel RFX6 nonsense variants (p.Gln25Ter, p.Arg377Ter) (Supplementary Fig. 1 and Supplementary Table 3). Similarly to the discovery cohort, the MODY replication cohort was enriched for RFX6 PTVs compared to the ExAC non-Finnish European control population (1.15 vs. 0.045%, OR = 26, 95% CI 6–82, P = 3 × 10−5) (Supplementary Table 4). This association was maintained when compared to an independent non-Finnish European control population with whole-genome sequence data from gnomAD (http://gnomad.broadinstitute.org) (Table 1 and Supplementary Table 4). The frequency of RFX6 PTVs in the gnomAD genome data set (0.027%) is not statistically different to that in ExAC (0.045%, P = 0.76).

Higher frequency of RFX6 PTVs in Finnish population

Finnish individuals had ~10-fold higher frequency of RFX6 PTVs compared to non-Finnish Europeans. The ExAC database showed a relative abundance of RFX6 PTVs in Finnish Europeans (15/3305, 0.45%) compared to non-Finnish Europeans (15/33,346, 0.045%) (Supplementary Table 3). All of the Finnish individuals in ExAC with RFX6 PTVs had the same variant, p.His293Leufs. To further validate this finding in a larger Finnish control population, we analysed RFX6 PTVs in 7040 control individuals from the METSIM study in Eastern Finland16. There were 26 individuals with RFX6 PTVs in this cohort and all had the p.His293Leufs variant. The frequency of p.His293Leufs was not significantly different from the ExAC Finnish population frequency (0.37 vs. 0.45%, P = 0.63) (Supplementary Table 3). The METSIM study has contributed to the ExAC Finnish cohort, so to prevent duplication we used the data from the larger METSIM study for further analysis12.

Enrichment of RFX6 p.His293Leufs in Finnish MODY patients

To assess whether the p.His293Leufs variant is associated with MODY in Finnish patients, we genotyped the RFX6 p.His293Leufs variant in 80 Finnish probands who were routinely referred for MODY genetic testing to Genome Center of Eastern Finland, University of Eastern Finland and did not have mutations in the most common MODY genes (GCK, HNF1A, HNF4A and HNF1B) (Supplementary Table 1). We identified six probands with the p.His293Leufs variant. The frequency of this variant was significantly higher in the Finnish MODY cohort compared to the METSIM controls (7.5 vs. 0.37%, OR = 22, 95% CI 7–56, P = 1 × 10−6) (Table 1). The meta-analysis of the three independent case–control analyses confirmed the strong association of RFX6 PTVs with MODY in the study cohorts (OR = 34, 95% CI 15–80, P = 1 × 10−16) (Table 1 ).

Enrichment of RFX6 PTVs is not due to technical artefacts

To ensure that the association we observed is not due to differences in sequencing technologies or analysis pipelines between cases and controls, we performed a series of sensitivity analyses. This included comparisons to additional whole exome, whole genome and in-house control cohorts and an analysis that removed exon 1 which was the least well covered exon in ExAC. These sensitivity analyses (Supplementary Table 4) show that results are consistent for all these analyses.

RFX6 PTVs co-segregate with diabetes

To further assess the causality of RFX6 PTVs, we conducted a co-segregation analysis in families with genetic data available on more than three affected individuals. We had only one family (family 1) with >3 affected individuals with genetic data (Fig. 1)17. The analysis showed that the RFX6 variant p.Leu292Ter co-segregated in 9 out of 10 individuals with diabetes (LOD score = 0.65, P = 0.04). One individual without the RFX6 variant had diabetes which is likely to be a phenocopy of type 2 diabetes considering the large pedigree, age of diagnosis and obesity (51 years, body mass index (BMI) 30 kg/m2). There were two family members with an RFX6 variant but with normal HbA1c level at the time of study (18 and 57 years) suggesting that RFX6 PTVs may have reduced penetrance.

Reduced penetrance of diabetes with RFX6 PTVs

To assess the penetrance of RFX6 PTVs for diabetes compared to common causes of MODY, we combined data for all six non-Finnish European proband families. There were 18 RFX6 heterozygotes of whom five had not developed diabetes at study entry. 27% (95% CI 11–58) developed diabetes by the age of 25 years and 78% (95% CI 55–95) by 51 years (Fig. 2). Two out of six probands did not have affected parents at study entry (Supplementary Fig. 1). The penetrance of diabetes for RFX6 heterozygotes was substantially lower compared to pathogenic variants of HNF1A (70%, 95% CI 67–72 by the age of 25 years and 97%, 95% CI 96–98 by 50 years) and moderately lower than pathogenic variants of HNF4A (55%, 95% CI 50–60 by the age of 25 years and 91%, 95% CI 88–94 by 50 years) (Fig. 2). Similar to non-Finnish European proband families, the Finnish RFX6 p.His293Leufs variant also showed reduced penetrance in Finnish families (Supplementary Fig. 2). In two previously reported families of neonatal diabetes children with homozygous p.Arg181Gln RFX6 15, 18 or RFX6 p.His293Leufs19, the genetic information available on RFX6 heterozygous family members was also suggestive of reduced penetrance of diabetes (Supplementary Figs 2 and 3).

Fig. 2 Penetrance of diabetes in people with MODY. Heterozygous RFX6 PTV (n = 18), pathogenic HNF1A variant (n = 1265) or HNF4A variant (n = 427) Full size image

RFX6 PTVs are not enriched in type 2 diabetes

The reduced penetrance and later age of onset of diabetes with RFX6 PTVs raised the possibility that these variants may be associated with type 2 diabetes. To assess this, we used freely available data from the Type 2 Diabetes Knowledge Portal which contains whole-exome data on type 2 diabetes patients20. Burden testing of RFX6 PTVs for exome sequencing data from 8373 type 2 diabetes cases and 8466 controls showed no significant association with type 2 diabetes (0.14 vs. 0.083%, OR = 1.79, 95% CI 0.7–4.57, P = 0.22)20 (Supplementary Table 3).

Phenotype of RFX6 heterozygotes with diabetes

We assessed the diabetes phenotype in 27 RFX6 heterozygote individuals with diabetes. The clinical features are shown in Table 2. The median age at diagnosis of diabetes was 32 years (IQR 24–46, range 13–64 years) and median BMI of 25.1 kg/m2 (IQR 23–28). After a median 10 years (IQR 5–22) of diabetes 69% of patients were treated with insulin but there was significant endogenous insulin present in 24/25 patients at recruitment. There was no history of sulphonylurea sensitivity and they did not have islet autoantibodies (GADA/IA2-Ab). All patients had isolated diabetes and there were no reports of the other features of homozygous RFX6 mutations, such as duodenal or gall bladder atresia.

Table 2 Clinical characteristics of patients with RFX6-MODY Full size table

RFX6 haploinsufficiency is associated with reduced GIP

RFX6 is a transcription factor and has been shown to increase expression and secretion of gastric inhibitory polypeptide (GIP) in mouse enteroendocrine K-cells21. We therefore measured the incretin hormone GIP in 17 RFX6 heterozygotes (eight with diabetes) and compared to 26 controls (two with diabetes). The fasting GIP was markedly lower in RFX6 heterozygotes compared to controls (16 (10–24) vs. 49 (28–65) pg ml−1, P = 1.2 × 10−5). Fasting glucagon-like peptide-1 (GLP-1) levels were not different in both groups (23 (12.5–32) vs. 24 (14–32) pg ml−1, P = 0.98). To remove potential confounding factors, we compared the OGTT data for the 11 Finnish RFX6 p.His293Leufs heterozygotes without diabetes to five matched (age, sex and BMI) controls for each heterozygote from the PPP-Botnia Study (Fig. 3 and Supplementary Table 6). This confirmed that both fasting and 120 min stimulated GIP was reduced (18.3 vs. 48.9 pg ml−1, P = 8 × 10−3, 167 vs. 241 pg ml−1, P = 0.029, respectively). In addition, the non-diabetic heterozygotes had higher fasting glucose (5.5 vs. 5.1 mmol l−1, P = 0.02) with a similar fasting insulin level suggesting a beta-cell defect (Supplementary Table 6).