Germline De Novo Mutations in ATP1A1 Cause Renal Hypomagnesemia, Refractory Seizures, and Intellectual Disability.

Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na+, K+-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na+, K+-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na+, K+-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na+, K+-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity.

Clinical and diagnostic features of Bartter and Gitelman syndromes.

BACKGROUND: Bartter and Gitelman syndromes are autosomal recessive disorders of renal tubular salt handling. Due to their rarity, limited long-term data are available to inform prognosis and management. METHODS: Long-term longitudinal data were analysed for 45 children with pathogenic variants in SLC12A1 (n = 8), KCNJ1 (n = 8), CLCNKB (n = 17), BSND (n = 2) and SLC12A3 (n = 10) seen at a single centre between 1984 and 2014. Median follow-up was 8.9 [interquartile range (IQR) 0.7-18.1] years. RESULTS: Polyhydramnios and prematurity were seen in children with SLC12A1 and KCNJ1 mutations. Patients with CLCNKB mutations had the lowest serum potassium and serum magnesium and the highest serum bicarbonate levels. Fractional excretion of chloride was >0.5% in all patients prior to supplementation. Nephrocalcinosis at presentation was present in the majority of patients with SLC12A1 and KCNJ1 mutations, while it was only present in one patient with CLCNKB and not in SLC12A3 or BSND mutations. Growth was impaired, but within the normal range (median height standard deviation score -1.2 at the last follow-up). Impaired estimated glomerular filtration rate (eGFR <90 mL/min/1.73 m2) at the last follow-up was seen predominantly with SLC12A1 [71 mL/min/1.73 m2 (IQR 46-74)] and KCNJ1 [62 mL/min/1.73 m2 (IQR 48-72)] mutations. Pathological albuminuria was detected in 31/45 children. CONCLUSIONS: Patients with Bartter and Gitelman syndromes had a satisfactory prognosis during childhood. However, decreased eGFR and pathologic proteinuria was evident in a large number of these patients, highlighting the need to monitor glomerular as well as tubular function. Electrolyte abnormalities were most severe in CLCNKB mutations both at presentation and during follow-up. Fractional excretion of chloride prior to supplementation is a useful screening investigation in children with hypokalaemic alkalosis to establish renal salt wasting.

Genetic causes of hypomagnesemia, a clinical overview.

Magnesium is essential to the proper functioning of numerous cellular processes. Magnesium ion (Mg2+) deficits, as reflected in hypomagnesemia, can cause neuromuscular irritability, seizures and cardiac arrhythmias. With normal Mg2+ intake, homeostasis is maintained primarily through the regulated reabsorption of Mg2+ by the thick ascending limb of Henle's loop and distal convoluted tubule of the kidney. Inadequate reabsorption results in renal Mg2+ wasting, as evidenced by an inappropriately high fractional Mg2+ excretion. Familial renal Mg2+ wasting is suggestive of a genetic cause, and subsequent studies in these hypomagnesemic families have revealed over a dozen genes directly or indirectly involved in Mg2+ transport. Those can be classified into four groups: hypercalciuric hypomagnesemias (encompassing mutations in CLDN16, CLDN19, CASR, CLCNKB), Gitelman-like hypomagnesemias (CLCNKB, SLC12A3, BSND, KCNJ10, FYXD2, HNF1B, PCBD1), mitochondrial hypomagnesemias (SARS2, MT-TI, Kearns-Sayre syndrome) and other hypomagnesemias (TRPM6, CNMM2, EGF, EGFR, KCNA1, FAM111A). Although identification of these genes has not yet changed treatment, which remains Mg2+ supplementation, it has contributed enormously to our understanding of Mg2+ transport and renal function. In this review, we discuss general mechanisms and symptoms of genetic causes of hypomagnesemia as well as the specific molecular mechanisms and clinical phenotypes associated with each syndrome.

Autosomal-Recessive Mutations in SLC34A1 Encoding Sodium-Phosphate Cotransporter 2A Cause Idiopathic Infantile Hypercalcemia.

Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.

Generation and validation of a zebrafish model of EAST (epilepsy, ataxia, sensorineural deafness and tubulopathy) syndrome.

Recessive mutations in KCNJ10, which encodes an inwardly rectifying potassium channel, were recently identified as the cause of EAST syndrome, a severe and disabling multi-organ disorder consisting of epilepsy, ataxia, sensorineural deafness and tubulopathy that becomes clinically apparent with seizures in infancy. A Kcnj10 knockout mouse shows postnatal mortality and is therefore not suitable for drug discovery. Because zebrafish are ideal for in vivo screening for potential therapeutics, we tested whether kcnj10 knockdown in zebrafish would fill this need. We cloned zebrafish kcnj10 and demonstrated that its function is equivalent to that of human KCNJ10. We next injected splice- and translation-blocking kcnj10 antisense morpholino oligonucleotides and reproduced the cardinal symptoms of EAST syndrome - ataxia, epilepsy and renal tubular defects. Several of these phenotypes could be assayed in an automated manner. We could rescue the morphant phenotype with complementary RNA (cRNA) encoding human wild-type KCNJ10, but not with cRNA encoding a KCNJ10 mutation identified in individuals with EAST syndrome. Our results suggest that zebrafish will be a valuable tool to screen for compounds that are potentially therapeutic for EAST syndrome or its individual symptoms. Knockdown of kcnj10 represents the first zebrafish model of a salt-losing tubulopathy, which has relevance for blood pressure control.