Heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance

Finding new causes of monogenic diabetes helps understand glycaemic regulation in humans. To find novel genetic causes of maturity-onset diabetes of the young (MODY), we sequenced MODY cases with unknown aetiology and compared variant frequencies to large public databases. From 36 European patients, we identify two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants are enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio = 131, P = 1 × 10−4). We find similar results in non-Finnish European (n = 348, odds ratio = 43, P = 5 × 10−5) and Finnish (n = 80, odds ratio = 22, P = 1 × 10−6) replication cohorts. RFX6 heterozygotes have reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27, 70 and 55% at 25 years of age, respectively). The hyperglycaemia results from beta-cell dysfunction and is associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.

F inding the genetic cause of rare familial diabetes (monogenic diabetes) provides new biological insights into human pancreas development and function, as well as potentially novel therapeutic targets with important treatment implications 1 . Maturity-onset diabetes of the young (MODY) is monogenic diabetes resulting from beta-cell dysfunction which usually present before the age of 25 years in non-obese patients who are non-insulin-dependent and have an autosomal dominant inheritance of diabetes 2 . Mutations in HNF1A, HNF4A and GCK are the commonest causes of MODY responsible for~60% of MODY aetiology 1 .
There has been limited recent success in finding new MODY genes. WFS1 heterozygous variants and loss-of-function variants in the APPL1 gene were shown to be a rare cause of MODY 3,4 . The reason for this limited success is the difficulty of distinguishing monogenic diabetes patients from those with type 1 diabetes 5,6 , or from the increasing number of patients with earlyonset type 2 diabetes due to rising rates of obesity. Another important reason is the lack of large pedigrees with an autosomal dominant pattern of inheritance of diabetes which would allow classical linkage analysis to be performed and which was used to discover the most common forms of MODY such as GCK, HNF1A and HNF4A 7-10 .
Rare-variant association testing is an important step to confirm the pathogenicity of novel variants in monogenic disease 11 . Rarevariant association testing particularly for comparing the frequency of novel protein-truncating variants (PTVs) in monogenic cases with unknown aetiology to the frequency in large control cohorts is now possible because of the availability of resources such as ExACa database of protein coding variants in large control populations 12 . This allows burden testing of the frequency of novel or rare coding variants in diseases of interest and a comparison to rates in controls to identify new genetic causes of monogenic disease.
In this study, we have undertaken next-generation sequencing of MODY cases with unknown aetiology and compared the frequency of PTVs to large publicly available control cohorts to identify new MODY genes. Our study shows that heterozygous RFX6 PTVs are associated with MODY.

Results
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 diabetes 13 (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 proteintruncating) are a known cause of syndromic neonatal diabetes 15 , but heterozygotes were not previously known to have any phenotype.
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 ExAC 12 . 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.Arg377-Ter) (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).  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 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00895-9 ARTICLE NATURE COMMUNICATIONS | 8: 888 | DOI: 10.1038/s41467-017-00895-9 | www.nature.com/naturecommunications 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/m 2 ). 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). 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-cells 21 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).

Discussion
Heterozygous RFX6 PTVs are associated with MODY. We identified RFX6 PTVs predicted to be pathogenic 11,19 in unrelated MODY patients in whom the known causes of monogenic diabetes had been excluded. These variants were enriched in Finnish and non-Finnish European MODY probands but were rare in the control cohorts and in patients with type 2 diabetes. We observed co-segregation within pedigrees albeit with reduced penetrance. Finally, these variants are likely to have a functional effect due to nonsense-mediated decay causing haploinsufficiency 12,22 . This is further supported by the studies that showed that the homozygous RFX6 PTVs cause neonatal diabetes 15, 23, 24 . Among unknown MODY cases, RFX6 PTVs were responsible for 7.5% Finnish cases compared to only~1% of non-Finnish European cases. Large-scale control cohorts such as ExAC in combination with next-generation sequencing of well-characterised cases is a useful strategy for identifying new causes of monogenic disease. The large ExAC database provides sufficient power for reliable burden testing of rare variants in monogenic disease 12 without the need for large pedigrees or linkage analysis. RFX6 PTVs were highly enriched in both discovery and replication cohorts compared to control cohorts, supporting their pathogenicity. The ExAC database has been very useful in identifying benign variants because of an unusually high frequency in the population compared to frequency of the disease in question 11,12,25 . However, caution is required for reduced penetrance variants as their frequency can be higher than estimated disease frequency in the general population. RFX6 PTVs are an example where the reduced penetrance explains the higher frequency in control cohorts compared to the estimated frequency of MODY (0.01%) in the general population 26 . In addition, our study highlights the importance of population specific control and disease cohorts. The frequency of RFX6 PTVs is~10-fold higher in the Finnish population compared to non-Finnish populations due to the well-documented bottleneck in population genetics 27,28 . This observation is not restricted to RFX6 alone. It has been shown that the Finnish population has an overall higher burden of genome-wide PTVs (0.5-5%) in many genes compared to non-Finnish Europeans, and some of these have been associated with disease [27][28][29] . RFX6 PTVs are associated with reduced penetrance MODY. This reduced penetrance explains the lack of complete co-segregation in the RFX6 pedigrees. It also clarifies why diabetes was only reported in the parents or grandparents (obligate heterozygous for functional RFX6 variants) in seven out of the 12 published recessive RFX6 neonatal diabetes pedigrees 15,18,24,[30][31][32][33][34][35][36] . The lack of enrichment of RFX6 PTVs in type 2 diabetes patients compared to controls further supports their association with reduced penetrance MODY rather than type 2 diabetes. Further studies are needed to understand the mechanism of reduced penetrance of diabetes in RFX6 heterozygotes. It could be due to a combination of factors, such as expression patterns of normal alleles, epigenetic modifications and rare or common genetic variant modifiers 37 .
There are differences as well as similarities between RFX6-MODY and HNF1A/HNF4A-MODY. In contrast to HNF1A and HNF4A-MODY patients, RFX6-MODY patients do not show enhanced sensitivity to sulphonylureas 38 . All patients with HNF1A/HNF4A-MODY have significant endogenous insulin 3-5 years post diagnosis 39 . RFX6-MODY patients showed a similar pattern, except for one patient who did not have detectable endogenous insulin. Similar to HNF1A/HNF4A-MODY 6, 38 , RFX6-MODY patients lack islet autoantibodies and have isolated diabetes. This suggests that persistent C-peptide, lack of islet autoantibodies and parental history of diabetes, which are currently used to distinguish common forms of MODY from type 1 diabetes, can also be used to identify RFX6-MODY. However, a similar strategy will not help to distinguish late onset RFX6-MODY from type 2 diabetes.
Our study supports the role of RFX6 in the adult human pancreas. RFX6 is from a family of transcription factors that contains winged-helix DNA-binding domains 15 . RFX6 is expressed almost exclusively in pancreatic islets, small intestine and colon 15 . It acts downstream of NGN3, regulates islet cell differentiation and the development of the endocrine pancreas 15 . The homozygous RFX6 missense and PTVs cause syndromic neonatal diabetes (gall bladder aplasia, gut atresia and diabetes) 15 . RFX6 whole-body null mice show phenotypes consistent with human disease and die soon after birth, but the heterozygous whole-body RFX6 mouse has not been reported to develop diabetes 15 . This is not surprising considering the lack of phenotype in heterozygous null mice of HNF1A and HNF1B 40 . Interestingly, the defect in glucose-induced insulin secretion was present in models that are more akin to haploinsufficiency of RFX6 in adult humans 33,41 . 80% depletion of RFX6 protein in the adult mouse pancreas in vivo as well as in human beta cells in vitro showed that this defect was due to reduced expression of ABCC8, GCK and Ca 2+ channels in beta cells and disruption of Ca 2+ -mediated insulin secretion 33,41 . These data support the role of RFX6 in the physiology of adult beta cells. This along with evidence of impaired of beta-cell function (requirement of insulin to maintain euglycaemia, one patient with C-peptide <200 pmol l −1 , lower C-peptide in heterozygotes with diabetes compared to without diabetes ( Supplementary Fig. 4)), suggest that insulin deficiency is the cause of diabetes in these patients.
Incretins are gut hormones released in response to meals that potentiate glucose-stimulated insulin secretion. GIP is secreted from enteroendocrine K-cells in the duodenum and upper jejunum and mediates the bulk of the incretin effect in healthy individuals 42 . The secretion of GIP and GLP-1 is preserved in type 2 diabetic patients 43,44 and in patients with other forms of diabetes, including type 1 diabetes 45 and HNF1A-MODY 46 .
The present identification of GIP deficiency in RFX6 PTV heterozygotes is in keeping with the murine data showing that GIP expression and secretion is regulated by Rfx6 21 , and, importantly, identifies the first human form of diabetes associated with decreased GIP secretion.
In conclusion, heterozygous RFX6 PTVs are associated with reduced penetrance MODY and GIP deficiency.

Methods
Study populations. Discovery MODY cohort: The discovery cohort comprises 38 European probands with strong MODY-like phenotype who did not have mutations in the three most common MODY genes (GCK, HNF1A and HNF4A) (Supplementary Table 1). They were diagnosed <25 years of age, non-obese, had ≥3 generation history of diabetes, non-insulin treated or insulin treated with C-peptide > 200 pmol l −1 (if available) and lacked islet autoantibodies.
Non-Finnish European replication MODY cohort: The replication cohort was derived from 469 non-Finnish European routine MODY diagnostic referrals to the Molecular Genetic Laboratory, Exeter, UK. A monogenic aetiology in a known monogenic diabetes gene was identified in 121 patients and the remaining 348 patients with unknown aetiology comprised the replication cohort (Supplementary Table 1).
Finnish-European replication MODY cohort: This cohort consisted of 80 patients who were routinely referred for MODY diagnostic testing to the Genome Center of Eastern Finland, University of eastern Finland in whom no mutation was found in the common MODY genes (GCK, HNF1A, HNF1B and HNF4A) when assessed by Sanger sequencing (Supplementary Table 1). These 80 patients comprise 78% of the total MODY X Finnish cohort.
Finnish-European control cohort: Individuals of this cohort were part of the METSIM study (n = 7040). They were all males aged 45-70 years, randomly selected from the population register of the Kuopio town, Eastern Finland 16 .
Cohort of people with pathogenic HNF1A and HNF4A variants: This cohort included probands and their family members referred to the Molecular Genetics Laboratory, Exeter, UK for MODY genetic testing and were identified to have a pathogenic HNF1A (n = 1265) or HNF4A (n = 427) variant.
Phenotypic characterisation of RFX6 heterozygotes: In total, we had 47 RFX6 heterozygotes of whom 27 had diabetes. 29/47 were part of the discovery and replication cohorts. 18/47 were identified separately or had been previously reported 18,23 (Supplementary Fig. 2 -Families 3-5, Supplementary Fig. 3). The clinical features of RFX6-MODY were based on 27 individuals with diabetes. 13/27 were part of the discovery and non-Finnish replication cohort (Fig. 1,  Supplementary Fig. 1 -Families 1-6). 9/27 individuals were from the Finnish replication cohort (5/9 individuals were from Supplementary Fig. 2 -Families 1 and 2, Pedigrees were not available for 4/9 individuals). In addition to this, we included five diabetic individuals that were identified separately. This comprised a single Finnish individual ( Supplementary Fig. 2 -Family 3) and four individuals from a previously reported family from Belgium ( Supplementary Fig. 3) 18 .
PPP-Botnia Study: The Prevalence, Prediction and Prevention of diabetes (PPP)-Botnia Study is a population-based study in Western Finland aiming at obtaining accurate estimates of prevalence and risk factors for type 2 diabetes, impaired glucose tolerance, impaired fasting glucose and the metabolic syndrome in the adult population (Isomaa). Altogether 5208 individuals randomly recruited from the national Finnish Population Registry participated in the baseline study in 2004-2008 (representing 6-7% of the population), and 3870 (77%) individuals participated in the follow-up study in 2011-2014. The participants with fasting plasma glucose < 10 mmol l −1 participated in an 75 g OGTT with venous samples taken at 0, 30, 120 min for plasma glucose and serum insulin; at 0 and 120 min for serum C-peptide, GIP and GLP-1. The participants gave their written informed consent and the study protocol was approved by the Ethics Committee of Helsinki University Hospital, Finland.
The RFX6 p.His293Leufs variant was genotyped in 5187 individuals by the Kompetitive Allele Specific PCR genotyping system (KASPTM) on Demand (KOD) assay according to the manufacturer's testing conditions including six positive control samples identified by direct sequencing (LGC Hoddesdon, Herts, UK). Two out of 5180 participants had RFX6 p.His293Leufs (the genotyping failed in 7), which was confirmed by direct sequencing.
DNA analysis. Targeted next-generation sequencing: The analysis of all known monogenic diabetes genes in European cohorts was conducted using targeted-NGS 13 . The panel included 29 genes in which variants are known to cause monogenic neonatal diabetes, MODY, mitochondrial diabetes, lipodystrophy or other forms of syndromic diabetes 13 (Supplementary Table 2). The RFX6 PTVs identified by targeted-NGS were confirmed using Sanger sequencing. The essential splice site, nonsense and frameshift variants excluding the last exon were considered PTVs in this study 12,22 . The targeted-NGS assay covered 100% bases of the RFX6 coding region with >10× read depth for all the samples.
Sanger sequencing: Genomic DNA was extracted from whole blood using standard procedures and the coding region and intron/exon boundaries of the RFX6 gene were amplified by PCR. Amplicons were sequenced using the Big Dye Terminator Cycler Sequencing Kit v3.1 (Applied Biosystems, Warrington, UK) according to manufacturer's instructions and reactions were analysed on an ABI 3730 Capillary sequencer (Applied Biosystems, Warrington, UK). Sequences were compared with the reference sequences (NM_173560.3) using Mutation Surveyor v3.24 software (So Genetics, State College, PA, USA).
The Finnish-European MODY cohort was analysed for p.His293Leufs variant using Sanger sequencing as described above. Family co-segregation analysis was performed in available family members using a Sanger sequencing assay for the specific RFX6 variant identified in that family. DNA analysis of the METSIM study has been described before 23 .
Statistical analysis. Fisher's exact test was used to compare the frequency of RFX6 PTVs. The threshold P-value for association was 1 × 10 −3 as there were 29 genes on the panel (0.05/29). The penetrance of diabetes was assessed using survival time analysis method. The statistical analysis was conducted using Stata 14 (StataCorp, Texas, USA). The comparison of RFX6 heterozygotes to PPP-Botnia controls were conducted using R (3.3.2) with packages for the data manipulation (dplyr) and visualization (ggplot2). Continuous variables were compared with Mann-Whitney U-test and categorical variables with chi-squared test. Single point non-parametric linkage analyses were performed using MERLIN 1.1.2 47 . The Z-score was converted into a LOD score by use of the Kong and Cox exponential model implemented in MERLIN 47,48 .
Ethics. Informed consent was obtained from all subjects. The UK study is approved by the North Wales Research Ethics Committee. The FINNMODY/PPP-Botnia study is approved by the Research Ethics committee for Medicine of the Helsinki University Hospital.
Data availability. The majority of data used in this study are publically available and can be accessed via the studies cited in the text. Considering issues of patient confidentiality and restrictions in IRB permissions, other original data are available through specific request.