Phenotypic Spectrum of HNF4α-Associated Fanconi Renotubular Syndrome

Abstract

Introduction

Fanconi renal tubular syndrome 4 (FRTS4) is a very rare, autosomal dominant disease caused by heterozygous variants affecting the arginine residue at position 85 of the HNF4A gene.

Methods

We conducted an international, physician-based, anonymous data collection within the European Rare Kidney Disease Reference Network and beyond, focusing on clinical features, treatment, and the progression of chronic kidney disease (CKD) in patients with FRTS4.

Results

Overall, we collected data on 25 patients. Twenty-four of these carried the classic p.R85W variant, and 1 carried a new p.R85L variant. Prematurity and macrosomia at birth were frequently observed. Congenital hyperinsulinism with postnatal hypoglycemia was documented in 14 of 21 patients. Renal Fanconi syndrome was diagnosed at a median age of 1.5 (interquartile range: 0.1–4.3) years. Common symptoms included failure to thrive (13/23), rickets (9/25), nephrocalcinosis (12/25), and CKD before age 10 years (13/19). All tested patients exhibited low-molecular-weight proteinuria, aminoaciduria, and glycosuria. Hyperphosphaturia, metabolic acidosis, and hypercalciuria were present in most patients. The main extrarenal symptoms were liver abnormalities and cardiac septal defects. During a median follow-up period of 8.1 years kidney function deteriorated slowly. All adult patients progressed to CKD stage 3. Four patients developed maturity-onset diabetes of the young (MODY).

Conclusion

These findings provide a comprehensive picture of FRTS4. The disease should be suspected in children presenting with renal Fanconi syndrome, especially if there was fetal macrosomia and if they have experienced neonatal hypoglycemia. Progression of CKD is invariably observed, and liver and cardiac involvement should always be investigated.

Graphical abstract

In 1996, Yamagata et al.¹ reported for the first time that some patients with MODY carry heterozygous variants in the HNF4A gene, which encodes the transcription factor, HNF4α. Variants in this gene account for approximately 10% of MODY cases in western countries and may cause fetal macrosomia and neonatal hyperinsulinism.^2,3

After the initial description, several HNF4A pathogenic variants have been reported in the literature, including in 2010, a p.R85W (c.253C>T) variant observed in 1 patient with congenital hyperinsulinism who developed diabetes mellitus at the age of 12 years.⁴ Soon after, Stanescu et al. described in 2012, 1 patient with this same variant that, in addition to MODY, had overt renal Fanconi syndrome.⁵ This condition is now referred to as “Fanconi renotubular syndrome 4 with maturity-onset diabetes of the young” (FRTS4) (OMIM #616026, ORPHA #544628).

In the past 2 decades, other cases of FRTS4 that affect the same arginine residue of the HNF4α protein have been reported in the literature.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Because the HNF4A mRNA undergoes multiple alternative splicing, this variant has been reported as p.R63W [NM_175914.5] or p.R76W [NM_000457.4], depending on the reference sequence used.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 In addition, a p.R76Q variant that substitutes arginine for glutamine instead of tryptophan has also been described in 1 patient with FRTS4.²⁰

Due to the rarity of the disease, our knowledge of the p.R85W HNF4A phenotype remains incomplete. In the present study, we conducted a global survey to better define the clinical characteristics of the disease. This work was conducted under the auspices of the European Rare Kidney Disease Reference Network.

Methods

Patient and Data Collection

Clinical and biochemical data at diagnosis and during follow-up were collected using a standardized questionnaire (Supplementary Figure S1). The survey was distributed via the European Rare Kidney Disease Reference Network mailing list and extended to those of the European Society for Pediatric Nephrology, the International Pediatric Nephrology Association, and the Gene and Kidney Working Group of the European Renal Association. Two patients were previously reported as single cases,^19,21 and 2 familial cases (mother and son) were previously described.¹¹ This report includes additional longitudinal data on these patients.

Variables

The estimated glomerular filtration rate was assessed using the FAS equation across the full age spectrum.²² When presented in graphical format, reference data for age was plotted in a gray shaded area in the background.

Serum phosphate and calciuria depend heavily on age. Reference data from Pott et al.²³ were used to normalize these data. Serum phosphate was expressed as SDS, and urinary calcium was expressed as a relative value to the 97.5th percentile for age. Therefore, values > 1 indicate hypercalciuria. For consistency, we also used the data of Pott et al.²³ to normalize adult data using as reference value, the value of phosphate and urinary calcium of normal subjects aged 18 years.

Fetal macrosomia was defined as a birth weight > 4.0 kg²³ or a birth weight above the 90th percentile for gestational age.^24,25

Data Presentation

Data are presented as frequencies (percentages) for categorical variables. Continuous variables are presented as mean ± SD if they are normally distributed variables, according to the Kolmogorov–Smirnov/Shapiro–Wilk test. Otherwise, they are presented as median and interquartile range if nonnormally distributed. Growth data were plotted using the average reference data for Northern and Southern European children.²⁶

Ethical Approval

This study is part of the European Rare Kidney Disease Reference Network registry activities that has received approval by the Ethics Committee of the Medical Faculty of the University of Heidelberg (Berufsrechtliche Beratung) in December 21, 2021.

Results

Genetic Characterization

The study included 25 patients followed up in 14 different institutions. Twenty-four patients carried the canonical heterozygous p.R85W variant, whereas 1 patient carried a p.R85L variant, in which the arginine residue was mutated into leucine. The variant was de novo in approximately two-thirds of patients (Table 1). Three patients were diagnosed through family screening, including 1 patient who was diagnosed prenatally. In 2 siblings, the variant was assumed to be hereditary despite the absence of parental data. A familial history, including cases of CKD and diabetes mellitus, was present in a minority of cases (Table 1).

Table 1.

Patient characteristics and symptoms (N = 25)

Parameter	Units	Available data (n)	Value
General characteristics
Sex	M:F	25	15:10 (60:40)
de novo HNF4A variant	Yes	18	14 (78)
Family history of renal Fanconi syndrome or CKD	Yes	24	4 (17)
Family history of type 1 diabetes mellitus	Yes	22	3 (14)
Neonatal and perinatal history
Prematurity	Yes	24	10/24 (42)
Birth length	Cm	14	51 (50–53)
Birth weight	G	22	3600 (3180–3990)
Birth weight, if premature birth	G	9	3270 (3090–3640)
Birth weight, if born at term	G	12	3750 (3430–4010)
Neonatal hypoglycemia	Yes	21	14 (67)
If yes, minimal serum glucose value	mg/dl	11	20 (16–29)
If yes, serum insulin levels	pmol/l	10	19 (6–49)
Treatment with diazoxide	Yes	14	5/14 (36)
Symptoms at diagnosis
Age	yrs	25	1.5 (0.1–5.3)
Failure to thrive/short stature	Yes	23	13 (57)
Rickets / osteomalacia	Yes	25	9 (36)
Recent bone fractures	Yes	23	3 (13)
Nephrocalcinosis	Yes	25	12 (48)
Urolithiasis	Yes	25	1 (4)
Liver symptoms §	Yes	23	13 (57)
Cardiac symptoms §	Yes	23	6 (26)
Signs and symptoms at the last follow-up
Age	yrs	24	10.8 (6.9–16.2)
Rickets / osteomalacia	Yes	25	8 (32)
Recent bone fractures	Yes	25	0 (0)
Nephrocalcinosis	Yes	25	14 (56)
Urolithiasis	Yes	24	4 (17)
Liver symptoms §	Yes	23	10 (43)
Diabetes mellitus	Yes	25	4 (16)
Glycosuria	Yes	21	21 (100)
Low molecular weight proteinuria	Yes	18	18 (100)
Aminoaciduria	Yes	19	19 (100)

CKD, chronic kidney disease.

Neonatal Course

Data on the neonatal course was available for most of the cohort (Table 1). A significant portion of patients were born prematurely. Fetal macrosomia was observed in approximately one-third of patients. The median birth weight of patients born at term was 3750 g, corresponding to the 90th percentile for children born at term.²⁵ The median birth weight of premature infants was 3270 g, which corresponds to the 50th percentile for birth weight in term infants.²⁵ Hypoglycemia was reported in 13 of 19 patients (68%), 6 of whom had documented high insulin levels (> 13 μIU/ml). Five patients received treatment with diazoxide.

Renal Fanconi Syndrome and Kidney Function

Most patients were diagnosed after the onset of symptoms related to Fanconi syndrome. However, some were diagnosed during family screening while still asymptomatic. The median age at diagnosis was 1.5 years. The most prevalent findings related to their tubular disease were failure to thrive (57%), nephrocalcinosis (48%), and rickets (36%). At diagnosis, serum phosphate levels were low or in the low-normal range, and serum alkaline phosphatase levels were markedly elevated in nearly all patients (Figure 1). These were only partially corrected by therapy during follow-up. Hypercalciuria was frequently observed (Figure 1). Most patients exhibited metabolic acidosis. Low-molecular-weight proteinuria was always present when tested. However, quantitative values were not always available and were obtained using different low-molecular-weight protein markers, namely α1 microglobulin, β2 microglobulin, or retinol-binding protein. When reported, values always largely exceeded the ranges for normal values (data not shown). Additional features of Fanconi syndrome included hypouricemia, aminoaciduria, and glycosuria (Table 1 and Figure 1). Sodium and potassium levels were usually normal, whereas serum chloride levels were often increased, reflecting metabolic acidosis (Supplementary Figure 1). CKD was present at diagnosis in most patients (Figure 2). At the time of their initial diagnosis, 13 of 19 children (68%) aged < 10 years had CKD stage 2 or 3 (Figure 2b).

Figure 1.

Parameters of renal Fanconi syndrome. The most relevant parameters of renal Fanconi syndrome are illustrated. The shaded areas indicate normal values. Filled triangles represent male patients, and open circles represent female patients. Serum phosphate is expressed as SDS according to reference data by Pott et al.²³ Calciuria is expressed as a percentage of the 97.5th percentile, also according to Pott et al.²³

Figure 2.

Evolution of kidney function. Panel (a) shows the available longitudinal data for all patients. Filled triangles indicate male patients, and open circles indicate female patients. Panel (b) shows only the first (open triangles) and last (filled triangles) available eGFR measurements per patient. The shaded red line shows the regression line through all the data points, along with its 95% confidence intervals. eGFR, estimated glomerular filtration rate.

Extrarenal Symptoms

At the time of diagnosis, extrarenal symptoms primarily affected the liver and heart. Hepatic involvement was documented in 12 of 22 patients (55%). This manifested as hepatomegaly, hyperechogenic parenchyma, nodules, cysts, and elevated liver enzymes. One patient underwent a liver biopsy, revealing periportal fibrosis. Cardiac abnormalities were documented in 6 of 22 patients (27%). These included 2 atrial septal defects, 3 ventricular septal defects, 1 patent ductus arteriosus, and 1 case of moderate aortic valve insufficiency.

Evolution and Treatment

Patients were followed up for a median of 8.1 years. One patient was lost to follow-up. The median age at the final follow-up was 10.6 (interquartile range: 6.7–15.3) years. Most patients were still of pediatric age. Five patients were adults (aged 23, 26, 39, 39, and 53 years).

Nephrocalcinosis was observed in 14 of 25 patients (56%) during follow-up, of whom 4 also developed nephrolithiasis. Over time, proximal tubulopathy remained stable in most patients. However, data were difficult to analyze because they were collected under variable supplementation with alkali, oral phosphate, and vitamin D (Figure 1). Most patients had poor control of their metabolic acidosis, with serum bicarbonate levels usually < 22 mmol/l. Urine analyses at the final follow-up demonstrated persistent low-molecular-weight proteinuria, aminoaciduria, glycosuria, and renal phosphate wasting. Given the limited number of adult patients and the absence of long-term data from childhood to adulthood in single patients, CKD progression was difficult to assess. Assuming homogeneous progression of CKD in this cohort, the disease appeared to progress slowly (Figure 2). At the last follow-up, only 3 patients aged < 10 years had normal kidney function. Most children had stage 2 CKD, and all adult patients had stage 3 CKD (Figure 2b).

The treatment for renal Fanconi syndrome included bicarbonate and/or citrate for 16 of 22 patients (72%), with a median alkali equivalent dose of 2.8 mmol/kg/d. Hypophosphatemia was treated with phosphate supplementation in 15 patients (68%), with a median dose of 0.83 mmol/kg/d. Eleven patients (50%) were prescribed potassium supplements at a median dose of 0.8 mmol/kg/d, mostly in the form of potassium citrate. Active 1,25-dihydroxyvitamin D3 was prescribed to 15 patients (68%) at doses ranging from 0.5 to 2 μg/d. Inactive vitamin D was prescribed to 10 patients (45%) at doses ranging from 800 to 3000 IU/d. Additional medications included ramipril, enalapril, or candesartan in 5 patients (23%), thiazides in 3 patients (14%), levocarnitine in 5 patients (23%), oral calcium supplementation in 3 patients (14%), and indomethacin in 1 patient (5%). Five of 22 patients (23%) had received diazoxide during childhood to prevent hypoglycemia.

Growth remained within the normal range for most patients, though nearly all fell within the lower half of that range (Figure 3). It is important to note that the data in Figure 3 were plotted using a reference chart for the average European patient, which may not have been appropriate for all patients included in the study.

Figure 3.

Linear growth, When available, height data were plotted against reference data for the average European population.²⁶ The shaded areas indicate the normal range. Filled symbols show the heights of adult patients at their last follow-up, and their ages are shown next to the symbols.

Four adult patients had diabetes mellitus. The ages of onset of diabetes mellitus were 16, 21, and 39 years, when available. During follow-up, 1 patient developed hypertrophic cardiomyopathy, which was not present at diagnosis.

Discussion

To date, only missense variants that alter the arginine residue at position 85 of the HNF4A gene have been identified as causing FRTS4.³ In this report, we describe a new variant that substitutes arginine at position 85 with leucine. Due to the rarity of the disease, clinical information is limited. In this study, we collected data from a large cohort of 25 patients carrying this variant. Our data show that patients present with symptoms of renal Fanconi syndrome early in life. Most are macrosomic at birth, frequently experience neonatal hypoglycemia secondary to hyperinsulinism, and are often born prematurely. Very few conditions cause neonatal hypoglycemia accompanied by low-molecular-weight proteinuria. Patients with Fanconi-Bickel syndrome may exhibit a phenotype similar to that of the p.R85W HNF4A variant. However, phosphaturia tends to be more pronounced, and most patients are born with low birth weight.^27,28 In our cohort, we observed a significant number of patients presenting with cardiac and liver symptoms.

From a renal standpoint, CKD in patients with FRTS4 typically develops during childhood and progresses over several decades. Because the cohort consisted mostly of children, long-term data were still scarce. However, our results indicate a high prevalence of CKD at early ages. In addition, they suggest that CKD progression is similar to or even slightly slower than that of other proximal tubule genetic diseases, particularly Dent disease and Lowe syndrome.^29,30 Because longitudinal data were limited, estimates of CKD progression are based primarily on a cross-sectional analysis of the data we collected. Preclinical experimental models have demonstrated positive effects of citrate supplementation in models of Dent1 disease, of ketogenic diet in models of cystinosis, and of alpelisib, a phosphatidylinositol 3-kinases inhibitor, in Dent2 disease.31, 32, 33 Unfortunately, similar studies are not available in FRTS4. Until then, medical management relies primarily on providing symptomatic treatment to prevent chronic metabolic acidosis and severe hypophosphatemia, to begin rapid treatment once patients develop diabetes mellitus, and to treat complications of CKD as they develop.

HNF4α is a transcription factor that plays critical roles in organ development, cell differentiation, and cellular metabolism.³⁴ It belongs to the nuclear hormone receptor superfamily, which shares similar ligand- and DNA-binding domains.^34,35 Prior to DNA binding, the protein dimerizes with itself. Functional diversity is achieved through dimerization of different splice variants.^20,34,35 The R85 residue is located in the protein DNA-binding domain and has been shown to bind less tightly to promoters when mutated. However, in some hepatic cell lines, such as the Hep2 cell line, the mutation increases transactivation of the G6PC (glucose-6-phosphatase) gene.^20,35

In the kidney, HNF4α is exclusively expressed in proximal tubules. It is detectable in different stages of renal embryogenesis and in differentiated proximal tubule cells, where it regulates the expression of > 200 genes associated with transmembrane transport, lipid metabolism, and mitochondrial homeostasis.^36,37 FRTS4 however, is only associated with variants affecting the arginine residue at position 85.^16,18

Deletion of Hnf4a in mice decreases the expression of proximal tubule–specific genes and causes proximal tubular dysgenesis, which leads to renal Fanconi syndrome.^36,38 Introducing the p.R85W variant into the orthologous gene of Drosophila melanogaster causes the wild-type protein to aggregate in the cytosol, indicating a dominant-negative effect.³⁷ These findings potentially point to a variant-specific effect in the kidneys beyond haploinsufficiency.

Fly cells carrying this variant exhibit significant mitochondrial damage and depletion, as well as lipid droplet accumulation in their cytosol.³⁷ Very few human renal biopsies from patients with FRTS4 are available. Electron microscopy performed in single patients included in this study, previously reported,^16,19 as well as in another published case¹⁴ revealed abnormal mitochondrial changes similar to other forms of renal Fanconi syndrome.39, 40, 41, 42 This was associated in 1 case with partial respiratory chain complex IV deficiency.¹⁹

A consistent finding in HNF4a-associated FRTS4 is low-molecular-weight proteinuria. This condition was present in all patients included in the study and represents a surrogate marker for disrupted receptor-mediated endocytosis in the proximal tubules. Because young patients often had limited or no extrarenal symptoms, they may be misdiagnosed with Dent disease, as already reported.¹⁷ Quantification of low-molecular-weight proteinuria does not allow differentiation between different forms of renal Fanconi syndrome. When reported, values in patients with FRTS4 collected in this study were similar to those previously reported in Dent1 disease.⁴³ Due to the broad spectrum of differential diagnoses⁴⁴ HNF4A should be included in genetic panels that screen for hyperinsulinemic hypoglycemia as well as in genetic panels that screen for proximal tubulopathies.

Patients with pathogenic variants in the HNF4A gene often exhibit a biphasic glucose metabolism profile: neonatal hyperinsulinemic hypoglycemia followed by the onset of maturity-onset diabetes of the young (MODY) later in life.^3,18 Hyperinsulinemic hypoglycemia typically ceases in most patients after the first few years of life and disappears by around the age of 10 years, at which point patients begin to develop diabetes.¹⁸ In contrast to other genetic entities, HNF1α- and HNF4α-mediated hyperinsulinemic hypoglycemia in infants and young children respond well to diazoxide.^4,45 Accordingly, several patients included in the present study received diazoxide during early life. The low prevalence of MODY in our cohort was probably related to the small number of adult patients. The switch from insulin excess in early life to insulin deficiency in later life is incompletely understood but might involve changes in HNF4α-dependent gene expression, increased beta cell proliferation in early life, and mitochondrial dysfunction impairing insulin secretion in later life.⁴⁶

To date, 25 other patients with the p.R85W variant have been reported in the literature, as recently summarized by Hudson et al.¹⁶ Their clinical presentation aligns with our observations in this large cohort. According to reports in the literature, patients with FRTS4 with MODY developed diabetes mellitus between the ages of 12 and 23 years.¹⁶ Delayed growth has frequently been reported.¹⁶ On average, we observed that patients were shorter than the general population, although most were still within the normal range. This may be largely due to renal phosphaturia and metabolic acidosis. As highlighted recently in patients with distal renal tubular acidosis, optimization of metabolic control aiming at obtaining bicarbonate level around 24 mmol/l may help improving growth.⁴⁷ In most patients of our cohort, liver disease manifested in a relatively mild form, consistent with previous observations.¹⁶ Clinical presentations included hepatomegaly and hyperechogenic parenchyma, potentially accompanied by nodules and cysts. Elevated liver enzymes were noted.

In addition, we observed a high prevalence of cardiac septal defects. When these were aggregated with data from the literature,^11,16 ventricular septal defects are present in approximately 10% of patients. Furthermore, we observed 3 cases of atrial septal defects. These observations demonstrate that the p.R85W variant causes cardiac abnormalities in a substantial number of patients. Of note, cardiac septal defects observed in mice after trichlorethylene fetal exposure have been linked to an inhibition of HNF4a transcriptional activity.⁴⁸

In conclusion, we assembled the largest cohort of patients with the p.R85W HNF4A variant to date. Combined with the existing literature, this allows delineating a phenotype characterized by fetal macrosomia, neonatal hypoglycemia, and overt Fanconi syndrome. Patients should be monitored for liver and cardiac lesions, as well as for the development of MODY after the age of 10 years. Kidney function is usually abnormal at diagnosis and deteriorates slowly over time.

Disclosure

All the authors declared no competing interests.

Supplementary Material

Supplementary File (PDF)

Figure S1. Serum sodium, potassium, and chloride levels.