Hypoplastic amelogenesis imperfecta, bilateral nephrolithiasis and FGF-23-mediated hypophosphataemia: a triad of FAM20A-related enamel renal syndrome

  1. Neeti Agrawal ,
  2. Avivar Awasthi ,
  3. Partha Pratim Chakraborty and
  4. Animesh Maiti
  1. Endocrinology & Metabolism, Medical College and Hospital, Kolkata, West Bengal, India
  1. Correspondence to Dr Partha Pratim Chakraborty; docparthapc@yahoo.co.in

Publication history

Accepted:19 Oct 2022
First published:08 Nov 2022
Online issue publication:08 Nov 2022

Case reports

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Abstract

Enamel renal syndrome (ERS) due to loss of function (LOF) mutation of FAM20A gene typically consists of hypoplastic amelogenesis imperfecta (AI) and bilateral nephrolithiasis/nephrocalcinosis. Recent evidence suggests that FAM20A interacts with FAM20C and increases its activity; thus LOF mutation of FAM20A leads to impaired FAM20C action. FAM20C, a golgi casein kinase, phosphorylates fibroblast growth factor (FGF)-23, prevents its glycosylation and makes it more susceptible to degradation by furine proteases. Consequently, inactivating mutations of FAM20C lead to increased concentration of bioactive and intact FGF-23 in circulation and resultant hypophosphataemia. LOF mutation of FAM20A, thus, might also be associated with FGF-23-mediated hypophosphataemia; however, such an association has never been reported in the literature. We describe, for the first time, a triad of AI, bilateral nephrolithiasis and FGF-23-mediated hypophosphataemia in LOF mutation of FAM20A. We suggest that serum phosphate should be measured in all patients with ERS to avoid metabolic and skeletal complications of undiagnosed, hence untreated hypophosphataemia.

Background

Amelogenesis imperfecta (AI), an inherited defect in enamel development, is usually encountered as an isolated clinical condition; however, AI at times may have syndromic association.1 The deciduous and permanent teeth, both may be affected and patients with AI typically present with toothache, dental caries, loss of teeth and poor quality of life. The prevalence of AI in the general population varies from 1 in 700 to 1 in 14 000.2 Among various genes reported so far, family with sequence similarity 20, member A (FAM20A) plays a pivotal role in enamel development. FAM20A phosphorylates proteins involved in enamel formation. Patients with homozygous FAM20A mutations often develop renal manifestations at some point of time during the course of their disease, and loss of function (LOF) mutations in FAM20A have been implicated in pathogenesis of the enamel renal syndrome (ERS).3 Nephrolithiasis/nephrocalcinosis seems to be a universal component of the syndrome; however, a wide range of renal abnormalities such as renal cysts and recurrent urinary tract infections including pyelonephritis have also been reported.4 Moreover, defective distal renal tubular acidification secondary to underlying nephrocalcinosis may develop in the long run, and patients might be misdiagnosed as distal renal tubular acidosis (dRTA). Screening for underlying renal abnormalities both at diagnosis and at periodic intervals, thus, is essential to prevent or delay eventual renal failure in these patients. Underlying mechanism(s) of nephrolithiasis/nephrocalcinosis is still unclear and multiple postulations have been put forward.3 The proposed theories include altered metabolism of different stone modulators, increased growth of calcium oxalate crystals, among others. Hypercalciuria due to reduced calcium reabsorption from renal tubule is unlikely to play a role in stone formation as hypocalciuria, and not hypercalciuria has been frequently reported in these patients. Though appears theoretically possible, fibroblast growth factor-23 (FGF-23)-mediated hypophosphataemia has never been reported in ERS due to FAM20A mutation. Hypophosphataemia, an extremely rare component of FAM20A LOF mutation, highlights the role of FAM20A in FGF-23 metabolism.

Case presentation

A woman in her late 20s was referred to us with recurrent bilateral nephrolithiasis and elevated parathyroid hormone (PTH) (104 pg/mL). She had suffered from recurrent episodes of urinary tract infections in the past and detected to have bilateral nephrolithiasis at 18 years of age. She did not have bony fracture or muscle weakness; however, she used to experience diffuse muscle pain at times. A detailed enquiry revealed non-eruption of some permanent teeth while delayed eruption of the others. She had consulted a dentist 3 years prior to her presentation with us. The medical note by the dentist revealed the following: ‘Patient has multiple retained deciduous teeth and microdontia. The erupted permanent teeth are small with yellow-brownish discolouration. There is generalised attrition and wearing out of dentine’ (figure 1A). At that time, the patient underwent extraction of all deciduous teeth and subsequent crown implantation and root canal treatment (figure 1B). The patient was born out of a non-consanguineous union and her family history was non contributory.

Figure 1

OPG before (A) and after (B) dental procedure. OPG, orthopantomogram.

Clinical examination revealed the following: height: 150.5 cm; weight: 45 kg; body mass index (BMI): 19.9 kg/m2; blood pressure: 120/70 mm Hg. There were no dysmorphic features. The patient had no proximal myopathy. Oral examination suggested full mouth rehabilitation (figure 2). Systemic examination was unremarkable.

Figure 2

Clinical image after full mouth rehabilitation.

Investigations

Baseline investigations were normal. Elevated PTH was secondary to vitamin D deficiency (25OHD: 8.2 ng/mL), which was subsequently treated with oral cholecalciferol. Investigations, performed at first follow-up visit, have been summarised in table 1.

Table 1

Summary of investigations performed 6 months after her initial presentation

Blood parameters
Patient’s value Reference range
RCC, red cell count; WCC, white cell count.
Serum albumin corrected calcium 9.4 8.4–10.2 mg/dL
Serum albumin 4 3.7–4.8 gm/dL
Serum phosphorus 1.5 2.3–4.7 mg/dL
Serum potassium 3.6 3.5–5.1 mmol/L
Serum creatinine 0.58 0.57–1.11 mg/dL
Estimated glomerular filtration rate (eGFR) (chronic kidney disease epidemiology collaboration equation) 126 mL/min/1.73 m2
Serum 25 hydroxy vitamin D 22.68 > 20 ng/mL
Plasma intact parathyroid hormone 70.7 14–72 pg/mL
Serum alkaline phosphatase 100 42–98 units/L
Plasma intact fibroblast growth factor 23 127.60 23.20–95.40 pg/mL
Serum 1,25-dihydroxy vitamin D (calcitriol) 108 47.76–190.32 pmol/L
Arterial blood gas analysis
pH 7.44 7.35–7.45
pCO2 32.7 35–45 mm Hg
pO2 98 75–100 mm Hg
Bicarbonate 21.9 22–26 mmol/L
Urine parameters
Urine pH 6.7 4.7–7.5
Urine nitrite Detected Absent
Urine WCC 20–30 0–5/high power field
Urine RCC 0–1 Absent
Urine casts Not detected
Urine crystals Not detected
Urine culture and sensitivity Klebsiella species
(>100 000 CFU/mL)
Spot urine phosphorus (second morning void sample) 9 mg/dL Not established
Spot urine creatinine (second morning void sample) 46 mg/dL Not established
Tubular reabsorption of phosphate 92.5 85%–95%
Ratio of tubular maximum reabsorption of phosphate to GFR by nomogram from Walton and Bijvoet 1.6 2.2–3.4 mg/dL
24 hours urine volume 54 < 50 mL/kg/day
24 hours urine creatinine 26.24 ≥ 10 mg/kg/day
24 hours urine calcium 0.75 1.5–4 mg/kg/day
Imaging
Orthopantomogram (OPG) (figure 1) Figure 1A:Mal-aligned and deformed teeth in both upper and lower jaws. Few deciduous teeth were retained. Presence of multiple impacted permanent teeth in all quadrants and obliteration of pulp chamber with root canal sclerosis was seen. There was incomplete enamel capping over multiple impacted teeth. Short stubby roots along with multiple pulp stones were visible.
Figure 1B: OPG after extraction of unerupted teeth and crown implantation
Straight X-ray abdomen (figure 3) Bilateral nephrolithiasis
Ultrasonography kidneys Mulitple stones were visible in both kidneys. There was no evidence of nephrocalcinosis and hydronephrosis.
Bone mineral density (BMD) assessment by dual energy X-ray absorptiometry scan
BMD (g/cm2) T score Z score
Left distal one-third radius 0.6 - 1.6 - 1.5
Lumbar vertebrae (L1–L4) 0.87 −1.6 −1.4
Femoral neck 0.539 −2.8 −2.8
Total hip 0.68 −2.1 −2.1

Differential diagnosis

AI and bilateral nephrolithiasis (figure 3) led to a possibility of ERS, however presence of severe hypophosphataemia even after adequate vitamin D repletion was somewhat perplexing. Reduced ratio of tubular maximum reabsorption of phosphate to glomerular filtration rate (TmP/GFR) indicated renal phosphate wasting. Elevated plasma FGF-23 along with normal calcitriol (‘inappropriately normal’ for the degree of hypophosphataemia), normal PTH and normal 25OHD confirmed FGF-23-mediated hypophosphataemia. Low bone mineral density was probably due to hypophosphataemic osteomalacia. Recurrent bilateral nephrolithiasis prompted us to consider the possibility of dRTA, as mutation of WDR72 can be associated with both AI and nephrolithiasis/nephrocalcinosis secondary to inherited dRTA.5 Metabolic acidosis in WDR72 defect is mild and variable in intensity; hence systemic acid loading is often required to unveil defective distal H+ secretion. This patient had urinary tract infection due to Klebsiella species, and measurement of urine pH could lead to inappropriate interpretation; hence, provocative testing with ammonium chloride for assessment of distal tubular acidification system was not performed. Renal loss of phosphate and resultant hypophosphataemia secondary to reversible proximal renal tubular dysfunction is well known in dRTA; however, elevated FGF-23 and hypocalciuria was inconsistent with primary dRTA due to WDR72 mutation. Moreover, WDR72 mutation is commonly associated with hypomaturation AI and not hypoplastic AI, as encountered in this woman.1 We could not trace any serum phosphate concentration prior to her presentation with us. FGF-23-mediated hypophosphataemia in an adult led us to consider the possibility of tumour induced osteomalacia (TIO). However, AI and nephrolithiasis (in a treatment naïve adult) were not consistent with TIO.

Treatment

The patient received nitrofurantoin 100 mg two times per day for 7 days for urinary tract infection. She was put on oral phosphate granules at a dose of 20 mg/kg/day in three divided doses. Calcitriol was added at a daily dose of 1 µg (22 ng/kg/day) to avoid hyperparathyroidism secondary to phosphate repletion and to enhance intestinal phosphate absorption. The patient was counselled about monitoring of serum calcium, phosphate, PTH, renal function test and urinary spot calcium: creatinine ratio, along with ultrasonography kidneys at regular intervals.

Discussion

An increasing number of genes involved in pathogenesis of AI have recently been identified. Distribution and expression of these genes vary not only among different organs, but also within a particular tissue, giving rise to varying clinical presentations.1 Patient described here had multiple retained deciduous teeth, microdontia, dental malocclusion and gingival hyperplasia. Furthermore, the erupted teeth had rough surfaces with yellow brownish discolouration. Multiple impacted permanent teeth with incomplete enamel capping along with multiple pulp stones were evident on X-ray (figure 1). All these features, taken together, were consistent with the hypoplastic form of AI. Patients with ERS due to FAM20A mutation characteristically demonstrate hypoplastic form of AI.6 In addition, bilateral nephrolithiasis in this woman was diagnosed at 18 years of age. Nephrolithiasis/nephrocalcinosis in ERS usually develops during second and third decade of life.4 Presence of hypocalciuria was also consistent with findings from the previous studies.

Interestingly, this patient had hypophosphataemia secondary to renal phosphate wasting as indicated by low TmP/GFR. Presence of normal serum 25OHD, serum calcium and plasma PTH ruled out secondary hyperparathyroidism due to vitamin D deficiency as an aetiology of hypophosphataemia. Elevated plasma FGF-23 in the background of hypophosphataemia indicated FGF-23-mediated hypophosphataemia in this case.

The presence of FGF-23-mediated hypophosphataemia in LOF mutation of FAM20A gene underscores the role of FAM20A in FGF-23 regulation. Activity and circulatory concentration of FGF-23, a phosphatonin, are regulated by phosphorylation, glycosylation and proteolysis. FGF-23 is cleaved and inactivated by subtilisin like proprotein convertase furin (SPC). Family with sequence similarity 20, member C (FAM20C), a golgi casein kinase, phosphorylates FGF-23 at serine residue (Ser180), which neighbours the SPC motif (R176H177T178R179/S180). Ser180 phosphorylation inhibits O-glycosylation by polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) of threonine residue (Thr178) of the SPC motif, rendering the molecule more susceptible to cleavage into C and N terminal fragments by SPC leading to its inactivation.7 Thus, LOF mutation of FAM20C causes hypophosphataemia by increasing intact and biologically active FGF-23 in circulation (figure 4). Depending on the extent of mutation in FAM20C gene, phenotype ranges from lethal Raine syndrome to non-lethal form having hypophosphataemic rickets and osteomalacia.8 9 In addition to enhanced degradation of FGF-23, FAM20C also negatively regulates FGF-23 indirectly through DMP1.

Figure 4

Simplified pictorial depiction of post-translational modification of FGF-23 by phosphorylation, glycosylation and proteolysis. Unprocessed FGF23 is O-glycosylated by GALNT3 preventing its cleavage by proprotein convertase 1 (furin) leading to formation of active FGF-23 (upper panel). FAM20C phosphorylates FGF-23 and prevents O-glycosylation by GALNT3. Phosphorylated FGF-23 is cleaved by furin leading to its inactivation (lower panel). FAM20A (green circle) forms a heterotetrameric complex with FAM20C (purple circle) in the endoplasmic reticulum and allosterically increases FAM20C activity. FAM20, family with sequence similarity 20; FGF-23, fibroblast growth factor-23; GALNT3, N-acetylgalactosaminyltransferase 3; PC1, proprotein convertase 1. (Disclaimer: The figure has been created by Avivar Awasthi, one of the coauthors).

DMP1, an extracellular matrix protein essential for adequate bone mineralisation, is also a substrate for FAM20C, and FAM20C enhances activity of DMP1 by phosphorylation and promotion of its transcription. DMP1 impairs FGF-23 expression by the osteocytes. LoF mutation of DMP1, thus, results in elevated concentration of FGF-23 mRNA and protein emphasising its role in regulation of FGF-23.10 In FAM20C deficiency, decreased expression and lack of phosphorylation of expressed DMP1 ultimately leads to impaired function of DMP1 with resultant increase in FGF-23 level. FAM20C is widely expressed in different tissues and its mutation is associated with FGF-23 related hypophosphataemia, dental anomalies and ectopic calcification.11

FAM20A, on the other hand, is expressed in specific tissues, such as enamel and dental matrices. In preclinical studies, AI has been found both in FAM20a and in FAM20c null mice. FAM20c null mice, in addition, demonstrates defective dentin and hypophosphataemia, whereas normophosphataemia is seen in FAM20a null mice. Moreover, newborn FAM20c null mice may be normophosphataemic and become hypophosphataemic with age so that severe hypophosphataemia is seen during adult life.12 Similarly, LOF mutation in FAM20C, and not FAM20A, has been found to be associated with FGF23-mediated hypophosphataemia in human.11

However, in contrast to mouse model, serum phosphate in humans may get normalised with age despite persistently elevated FGF23 level. Thus, it may be argued that different mechanisms operate in mice and humans.

Whole-exome sequencing in this patient demonstrated homozygous nonsense variation in exon 11 of the FAM20A gene, that resulted in a stop codon and premature truncation of the protein at codon 478 (p. Arg478Ter), causing ERS. This was in accordance with previous studies, where the most common mutation involved premature stop codon insertion leading to FAM20A protein truncation. Hypoplastic AI, nephrolithiasis and hypocalciuria, encountered in our case, were consistent with earlier reports of ERS in literature. Though, hypophosphataemia secondary to incidental vitamin D deficiency has been described, FGF-23-mediated hypophosphataemia has never been reported in ERS due to FAM20A mutation. To the best of our knowledge, this is the first description of FGF-23-mediated hypophosphataemia in a patient with ERS. Recent evidence suggests that FAM20A, a pseudokinase, forms functional heterotetrameric complex with FAM20C and allosterically increases activity of the latter towards its substrates.13 14 Thus, loss of activity of FAM20A in this woman could have caused impaired function of FAM20C, which in turn led to increased full length and bioactive FGF-23 in circulation and resultant hypophosphataemia due to phosphaturia. Though, the patient did not have overt symptoms of hypophosphataemia, low bone mineral density could be attributed to hypophosphataemic osteomalacia; hence, the patient was given oral phosphate and calcitriol supplements, and was put on periodic surveillance to avoid hypercalciuria and aggravation of nephrolithiasis.

Learning points

  • Possible differential diagnoses in patients with amelogenesis imperfecta and bilateral nephrolithiasis/nephrocalcinosis are inherited distal renal tubular acidosis due to WDR72 mutation and FAM20A mutation. Hypophosphataemia may accompany both these condition; hypophosphataemia is due to secondary proximal tubular dysfunction and is fibroblast growth factor (FGF)-23 independent in the former, while it is mediated through FGF-23 in the latter. Moreover, unlike distal renal tubular acidosis, which is accompanied by hypercalciuria, patients with FAM20A mutation demonstrate hypocalciuria.

  • Amelogenesis imperfecta, bilateral nephrolithiasis/nephrocalcinosis and FGF-23-mediated hypophosphataemia is a triad of loss of function mutation of the FAM20A gene. Patients with FAM20A mutation may present with one or more features of this triad.

  • Inactivating mutation of FAM20C leads to elevated concentration of biologically active FGF-23 in the circulation. FAM20A allosterically increases FAM20C activity; hence impaired activity of FAM20A may also give rise to FGF-23-mediated hypophosphataemia as reported in loss of function mutations of FAM20C.

  • In addition to nephrolithiasis/nephrocalcinosis, FGF-23-mediated hypophosphataemia should also be included in the spectrum of renal manifestations in enamel renal syndrome due to inactivating mutation of FAM20A gene.

Ethics statements

Patient consent for publication

Footnotes

  • Contributors NA, AA, PPC and AM were involved in diagnosis and management of the patient. NA, AA and PPC did the literature search. NA wrote the manuscript and AA created figure 4. PPC finalised the manuscript.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Case reports provide a valuable learning resource for the scientific community and can indicate areas of interest for future research. They should not be used in isolation to guide treatment choices or public health policy.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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