Novel SLC12A3 mutation in Gitelman syndrome

Abstract

Gitelman syndrome (GS) is an autosomal recessive disease characterised by the presence of hypokalaemic metabolic alkalosis with hypomagnesaemia and hypocalciuria. The prevalence of this disease is 1–10/40 000. GS is usually associated with mild and non-specific symptoms and many patients are only diagnosed in adulthood. The disease is caused by mutations in the SLC12A3 gene. We present the case of a 49-year-old man referred to a nephrology appointment due to persistent hypokalaemia and hypomagnesaemia. Complementary evaluation revealed hypokalaemia, hypomagnesaemia, metabolic alkalosis, hyperreninaemia, increased chloride and sodium urinary excretion, and reduced urinary calcium excretion. Renal function, remainder serum and urinary ionogram, and renal ultrasound were normal. A diagnosis of GS was established and confirmed with genetic testing which revealed a novel mutation in SLC12A3 (c.1072del, p.(Ala358Profs*12)). This novel mutation extends the spectrum of known SLC12A3 gene mutations and further supports the allelic heterogeneity of GS.

Background

The concurrent presence of hypokalaemic metabolic alkalosis with hypomagnesaemia and hypocalciuria is the hallmark of Gitelman syndrome (GS), also referred to as familial hypokalaemia–hypomagnesaemia. The prevalence of this disease is 1–10/40 000, while heterozygote patients constitute approximately 1% of Caucasian populations. It is one of the most common inherited renal tubular disorders.^1–5 Most cases, often asymptomatic or mild, are diagnosed during adolescence or adulthood in routine evaluation.⁶ We present a case of GS with a novel mutation in SLC12A3 gene.

Case presentation

We report the case of a 49-year-old man with history of impaired fasting glucose and dyslipidaemia, with regular medical appointments with his general practitioner. There was no personal or family history of kidney disease. In April 2019, he was referred to a nephrology appointment to evaluate persistent hypokalaemia and hypomagnesaemia, known since 2012. There was no history of vomiting, use of prescription drugs (such as diuretics or laxatives) or over-the-counter medicines. The physical examination was unremarkable, with normal blood pressure.

The aetiological study showed: K+ 3.3 mmol/L (3.5–5.0 mmol/L); Mg2+ 1.4 mg/dL (1.6–2.6 mg/dL); Ca2+ 9.1 mg/dL (8.6–10 mg/dL); Cl− 94 mmol/L (98–107 mmol/L); Na+ 139 mmol/L (135–145 mmol/L); serum creatinine 0.92 mg/dL (0.7–1.2 mg/dL); blood urea nitrogen 32 mg/dL (13–43 mg/dL). Arterial blood gas analysis showed metabolic alkalosis (pH 7.49; HCO₃− 32.2 mmol/L; pCO₂ 45 mm Hg). Further investigation revealed elevated plasma active renin (14.16 μU/mL; 1.6–3.2 μU/mL), normal aldosteronaemia (133.5 mg/mL; 10–160 mg/mL), hypocalciuria (92 mg/24 hours; 100–300), increased urinary excretion of sodium (315 mmol/24 hours; 40–220) and chloride (299 mmol/24 hours; 110–250) and inappropriately normal urinary excretion of potassium and magnesium. Estimated glomerular filtration rate [Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)] was 102 mL/min/1.73 m². Renal ultrasound was normal, with no signs of lithiasis.

Based on the association of hypomagnesaemia, hypokalaemia, metabolic alkalosis and hypocalciuria, the diagnosis of GS was assumed. A genetic study was performed at Genomed laboratory after extraction of DNA from peripheral blood. Genetic analysis was conducted using oligonucleotide-based target capture (SureSelect, Agilent) followed by parallel massive sequencing (next-generation sequencing (NGS)) and confirmation by Sanger sequencing for regions not accurately analysed by NGS. The gene sequence used was NM_00039.2, with codon 1 corresponding to ATG. Variants already described in literature (NCBI database) without clinical significance were not reported. The study showed pathogenic variants in SLC12A3 gene in heterozygosity (c.2581 C>T, p.(Arg861Cys) and c.1072del, p.(Ala358Profs*12)). These findings confirmed our provisional diagnosis.

Outcome and follow-up

Presently, the patient remains asymptomatic and with mild hypokalaemia and hypomagnesaemia, controlled with diet and magnesium supplements.

Discussion

The presence of unexplained hypokalaemia, metabolic alkalosis, renal potassium wasting and a normal or low blood pressure should raise the suspicion of a salt-wasting tubulopathy, Bartter syndrome or GS, once surreptitious vomiting or diuretic use are excluded. Bartter syndrome and GS can be differentiated through the measurement of urinary calcium excretion, as urine calcium excretion is above normal or elevated in Bartter syndrome and below normal in the latter. Genetic testing can be used to confirm the diagnosis. In our case, surreptitious vomiting and diuretic abuse were excluded by the presence of high urinary chloride excretion (299 mmol/24 hours; 110–250 mmol/24 hours reference value) and by a negative history of diuretic use, respectively. Bartter syndrome was also deemed improbable due to the diagnosis in adulthood, absence of symptoms and low urinary calcium excretion.¹

GS is an autosomal recessive disorder first described by Gitelman, Graham and Welt, which is characterised by hypokalaemic metabolic alkalosis with hypomagnesaemia and hypocalciuria.^1–7 GS is usually associated with mild and non-specific symptoms (muscular weakness, fatigue, salt craving, thirst, nocturia or cramps) that appear during childhood, normally at the end of the first decade of life, but many patients are only diagnosed in adulthood, during routine evaluation.^{1 2 7} The presence of more severe phenotypes and the presence of symptoms earlier in life, such as growth retardation, tetany, rhabdomyolysis, seizures and ventricular arrhythmias have been described.^2–8 It has also been found that GS is associated with self-reported negative impact on quality of life, to a degree comparable to arterial hypertension, diabetes and cardiac conditions. This data questions the benignity of GS.^7–9 Late complications of GS include chondrocalcinosis and sclerochoroidal calcifications due to hypomagnesaemia, which leads to decreased solubility of calcium pyrophosphate (PPi) crystals and reduced hydrolysation of PPi into inorganic phosphate, thus promoting the formation of calcium PPi crystals in the joints and sclera. More severe electrolyte depletion can result in prolonged QT interval in ∼50% of the patients, increasing the risk of ventricular arrhythmias and sudden death.^{1 10}

In spite of the lack of correlation between phenotype and genotype, genetic study is helpful in many cases due to the sometimes challenging clinical diagnosis. This is due to (1) the number of diseases that mimic these symptoms and (2) phenotype variability in patients with the same mutation, resulting from differences in sex, environmental factors and dietary habits.^{1 10}

GS is caused by mutations in the SLC12A3 gene encoding the thiazide-sensitive sodium chloride cotransporter (NCC), expressed in the apical membrane of cells lining the distal convoluted tubule. To date, around 250 mutations, scattered throughout SLC12A3, have been identified in GS patients. The majority of patients are compound heterozygotes for SLC12A3 mutations, but a significant number of GS patients are found to carry only a single SLC12A3 mutation, most likely due to failure to identify the second allele mutation.^11–13 The SLC12A3 mutations can be classified into five different classes according to the mechanisms that lead to diminished or absent cotransporter activity.^{10 12–14}

• Class 1 mutations impair protein synthesis or result in the production of a truncated inactive protein. This is seen in nonsense, frameshift, splice site mutations or premature stop codons.

• Class 2 mutations are characterised by an adequate protein translation but impaired NCC protein processing. These mutations result in misfolding, leading to NCC protein retention in the endoplasmic reticulum and subsequent degradation.

• Class 3 mutations prevent insertion of a functional protein into the plasma membrane due to impairment of protein trafficking.

• Class 4 mutations alter functional properties of the cotransporter, despite normal processing and insertion into the plasma membrane.

• Class 5 mutations are associated with accelerated degradation of the cotransporter.

In our case, the patient had two pathological variants in heterozygosity (c.2581 C>T, p.(Arg861Cys) and c.1072del, p.(Ala358Profs*12)). The first mutation has been described in rare patients (ClinVar, Human Gene Mutation Database; minor allele frequency in Genome Aggregation Database of 0.0085%) and leads to a NCC with reduced activity, classified as pathogenic (American College of Medical Genetics and Genomics (ACMG) 2015: PS3, PM1, PM2, PM3, PM5, PP3 and PP5).¹⁵ The latter one, which had not been described in disease or populational databases, results in a deletion in complementary DNA (cDNA) position 1072, which leads to the substitution of an arginine with a proline amino acid and generates a frameshift with a premature stop codon at position 12. This type of mutation falls into class 1, resulting in the impossibility of producing a functional NCC protein. Since this is a recessive inherited disorder and the patient has the disease phenotype and a class 1 mutation, the authors consider this new mutation as possibly pathological (ACMG 2015: PVS1 and PM2). It would be useful to perform functional studies in order to evaluate protein structure and function, and to confirm its pathogenicity. No family members were studied. It could be useful to perform genetic testing in the patient’s pedigree in order to guide future genetic counselling.

The treatment mainly addresses the correction of the hydroelectrolyte abnormalities. Oral potassium and/or magnesium supplementation is the basis of treatment for patients with GS. As hypomagnesaemia can lead to hypokalaemia, magnesium supplementation should be considered first.10 14 16 17 Reasonable targets for serum potassium and magnesium may be 3.0 and 1.46, respectively. In our clinical report, as the patient has acceptable serum potassium without supplementation, only magnesium supplements were given. In order to achieve the target goals, some patients need higher doses of medication, which may result in adverse effects, including peptic ulcers, vomiting or diarrhoea, with worsening biochemistries. Potassium chloride supplements should be given, because other potassium salt formulations, such as aspartate or gluconate, are poorly absorbable and do not correct hypokalaemia and can even worsen the metabolic alkalosis. Potassium-rich foods should be recommended. Intravenous infusion of magnesium or potassium should be reserved for patients who cannot take oral drugs or who have severe and life-threatening hypomagnesaemia and/or hypokalaemia. If supplements are not enough to achieve the recommended serum levels or patients have side effects which hinder the maintenance of the treatment, the use of potassium-sparing diuretics, renin–angiotensin system blockers, non-steroidal anti-inflammatory drugs, such as indomethacin, or a combination of these have been used, though with limited supporting evidence.1 14 16 17 A series of drugs should be avoided or used with caution in patients with GS, including drugs slowing sinus rhythm or influencing the QT interval (eg, negative chronotropic drugs), drugs potentially exacerbating hypomagnesaemia (eg, proton pump inhibitors, gentamicin and antiviral drugs) and acetazolamide.¹

Although the prognosis is excellent in the majority of patients, a few may be at risk of developing cardiac arrhythmias, mostly those with severe hypokalaemia, severe hypomagnesaemia and alkalosis, highlighting the importance of maintaining electrolytes at adequate levels.^{1 17} Progression to end-stage disease is rare and only described in one case report to date.¹⁸

Learning points.

• The presence of unexplained hypokalaemia and metabolic alkalosis should raise suspicion of a salt-wasting tubulopathy, Bartter or Gitelman syndrome (GS).

• Genetic testing can be used to confirm the diagnosis. To date, around 250 mutations in the SLC12A3 gene have been identified in GS patients. The majority are compound heterozygotes for SLC12A3 mutations.

• A novel mutation in SLC12A3 gene, namely c.1072del, p.(Ala358Profs*12), can lead to GS.

• GS adversely affects the quality of life of patients to a degree compared with diseases like hypertension, diabetes and cardiac conditions.