Magnesium (Mg²⁺) is a pivotal cation and cofactor in maintaining cell function (Li et al., 2011; Jahnen-Dechent and Ketteler, 2012; de Baaij et al., 2015). In the gastrointestinal system, it is reabsorbed in the intestine and in the colon by fine-tuned control of an active transcellular Mg²⁺ channel, followed by extrusion to the blood via the CNNM4 Na⁺/Mg²⁺ exchanger. In the kidney, most of the filtered Mg²⁺ is reabsorbed paracellularly in the proximal tubule (PT) and thick-ascending loop of Henle (TALH) via different claudins (claudins 1 and 2 in the PT, and claudins 10, 14, 16, and 19 in TALH) (Stuiver et al., 2011; de Baaij et al., 2012; Curry and Yu, 2018; Ellison et al., 2021). Like the colon, the distal convoluted tubule (DCT) fine-tunes the reabsorption of tubular Mg²⁺ via the transcellular TRPM6 channel along with basolateral Mg²⁺ extrusion of Na⁺/Mg²⁺ exchanger. Inactivating mutations in claudins 16 and 19 in TALH are responsible for familial hypomagnesemia hypercalciuria nephrocalcinosis (FHHNC types I and II). Defective Mg²⁺ reabsorption in the DCT inevitably causes renal hypomagnesemia because there is no Mg²⁺ reabsorption in the downstream DCT. Gene mutations related to the regulation of Mg²⁺ transport in the DCT can cause renal hypomagnesemia. These genes include SLC12A3 encoding thiazide-sensitive NCC, TRMP6 encoding apical TRPM6 channel, HNF1B encoding HNF1β, PCBD1 encoding PCBD1, EGF encoding EGF, EGFR encoding EGFR, KCNJ10 encoding Kir4.1 (EAST syndrome), KCNA1 encoding Kv1.1, FXYD2 encoding γ-subunit of Na⁺-K⁺ ATPase, and CNNM2 encoding CNNM2 (cyclin M2) (Meij et al., 2003; Groenestege et al., 2007; Adalat et al., 2009; Glaudemans et al., 2009; Reichold et al., 2010; de Baaij et al., 2012; Ferrè et al., 2014; Sponder et al., 2016; Chen et al., 2021).

In 2011 and 2014, mutations in CNNM2 predominantly expressed in the DCT and brain were first reported to cause autosomal-dominant (majority) or recessive (minority) renal hypomagnesemia with seizure and intellectual disability (HSMR) (de Baaij et al., 2012; Chen et al., 2021; Franken et al., 2021; Accogli et al., 2019; Arjona and de Baaij, 2018; Bamhraz et al., 2021). In humans, the cyclin M (CNNM, also formerly known as ancient conserved domain protein, ACDP) family has four member proteins (CNNM1–4) sharing evolutionary homology. As shown in Figure 1, the CNNM2 structure consists of an extracellular domain, transmembrane domain (TMD) containing the domain of unknown function-21 (DFU21), Bateman domain or module containing two functionally essential cystathionine β-synthase (CBS) to bind Mg²⁺-ATP, and cytosolic cyclic-nucleotide-binding homology domain (CNBH). To date, only a few families with CNNM2 mutations have been reported (Arjona et al., 2014; Chen et al., 2021; Li et al., 2021). Although CNNM2 was once thought to be a basolateral Mg²⁺ transporter or Na⁺/Mg²⁺ exchanger, more recent evidence points to its function as an intracellular Mg²⁺ sensor, using a conformational change upon binding of Mg²⁺-ATP (Accogli et al., 2019). However, the precise mechanism by which CNNM2 mutation leads to a conformational change and impaired sensing of Mg²⁺-ATP to affect basolateral Mg²⁺ extrusion and/or apical Mg²⁺ reabsorption remains unknown.

We have encountered an infant with refractory hypomagnesemia and excessive renal Mg²⁺ wasting, seizure, and intellectual disability, consistent with the clinical description of HSMR syndrome. In this study, we aimed to identify the genetic mutation for her phenotype and to assess the functional impact of the identified mutation. Our results indicated that a de novo heterozygous mutation c.G1439T (R480L) in the CBS domain of the CNNM2 gene was identified in this proband. This missense CNNM2 R480L mutation was found to be pathogenic. In vitro studies revealed higher CNNM2-R480L protein expression with proper cellular localization on immunocytochemistry images. The impaired Mg²⁺ efflux with a significant increase in intracellular Mg²⁺ suggests that the CNNM2-R480L mutation blocks Mg²⁺ efflux. In our simulation model, this R480L mutation leads to an attenuated interaction between CNNM2 and ATP-Mg²⁺.

This study was approved by the ethics committee on human studies at Tri-Service General Hospital in Taiwan (IRB2-105-05-136). A trio family including the proband and her parents were enrolled. Written informed consent was obtained from the participants.

In this study, we have identified a novel and de novo heterozygous mutation c.G1439T (R480L) in the CBS domain of the CNNM2 gene in a trio family with typical HSMR. In vitro studies showed that this CNNM2-R480L had a higher expression level than the CNNM2-wild type and proper localization to the plasma membrane. The Mg²⁺ efflux assay in mDCT cells revealed the blockade of intracellular Mg²⁺ efflux under Mg²⁺ depletion. The simulation model also predicted the attenuated interaction of this mutant protein with Mg²⁺-ATP.

To date, nearly 20mutations including ours in CNNM2 have been reported responsible for HSMR (Stuiver et al., 2011; de Baaij et al., 2012; Arjona and de Baaij, 2018; Accogli et al., 2019; Bamhraz et al., 2021; Franken et al., 2021). Autosomal-dominant inheritance is the most common type with the majority of mutations being de novo and missense as shown in this patient (Stuiver et al., 2011; de Baaij et al., 2012; Accogli et al., 2019; García-Castaño et al., 2020; Chen et al., 2021; Franken et al., 2021). The clinical features including profound hypomagnesemia, epilepsy, and neurodevelopmental delay in our proband were in line with phenotypes reported by a previous large cohort study (Franken et al., 2021).) Epilepsy might be a result of disturbed brain development rather than impaired Mg²⁺ efflux. Arjona et al. had shown that knockdown of CNNM2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments (Arjona et al., 2014). Although the correlation between phenotype and genotype was poor, patients with homozygous mutation usually have more severe forms of structural brain abnormalities (Stuiver et al., 2011; Accogli et al., 2019; Franken et al., 2021).

R480L mutation involved a highly conserved residue in the CBS domain of CNNM2 protein and was pathogenic based on a higher score of pathogenicity by different software prediction and ACME criteria (Richards et al., 2015). We first examined in vitro studies for this R480L mutant. A higher CNNM2-R480L protein expression with proper cellular localization on immunocytochemistry images was found. In contrast to the previous findings, the membranous expressions of mutant CNNM2-R480L were higher than those of the CNNM2-wild type (Franken et al., 2021). The exact mechanism of increased expression of CNNM2-R480L on the plasma membrane remained to be elucidated. We speculate that it is secondary to a compensatory effect of impaired function of mutant or different unique mutations in the CBS domain. We confirmed that the R480L mutation did not alter protein trafficking or membrane localization. Both arginine (R) and lysine (K) are basic amino acids with similar physiochemical properties. The difference between the membranous expression of R480L and R480K was minimal in this study. However, the charged R containing the guanidino head group, as opposed to the amino head group in the charged K, is solvated by more oxygen atoms and can form more H-bonds. However, the charged R containing guanidino head group interacts with individual polar molecules more weakly due to its delocalized charge (Meloni et al., 2020). Furthermore, the positively charged residues R480 in CNNM2 plus the threonine residue of T568 are essential for stabilizing a phosphate moiety (Corral-Rodríguez et al., 2014).

CNNM2 contains an extracellular N-terminal domain, a transmembrane domain of the unknown function (DUF21), and a large cytosolic region including a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain (Chen et al., 2020). The mutation R480L is located in the CBS domain, and this Arg480 has been reported to interact with Glu570 to form a salt bridge and with the acidic aspartate residue D583 for strengthening CBS dimerization (Corral-Rodríguez et al., 2014; Chen et al., 2020). Previous studies have demonstrated the importance of CBS dimerization for binding Mg²⁺-ATP by the mutation of hydrophobic residues lining the interface of dimerization (Baykov et al., 2011; Chen et al., 2020). In line with this finding, our simulation model predicts that R480L alters the binding force between CNNM2 and Mg²⁺-ATP. Therefore, R480L might diminish its binding with Mg²⁺-ATP by impairment of the formation of CBS dimerization (Figure 7).

We conducted a cellular Mg²⁺ efflux assay to evaluate the effect of R480L on CNNM2 transport activity. Consistent with previous studies, R480L impaired the Mg²⁺ efflux activity in the presence of significantly high intracellular Mg²⁺ under Mg²⁺ depletion. It has been shown that Mg²⁺-ATP binding is required for cellular Mg²⁺ efflux since mutations that abolished Mg²⁺-ATP binding prevent Mg²⁺ efflux (Arjona and de Baaij, 2018). Of note, our simulation model predicts the reduction of the binding energy of Mg²⁺-ATP in the CBS1 of CNNM2 by R480L mutation. However, we did not find a significant impairment of Mg²⁺ efflux in CNNM2 T568I in CBS2, unlike a previous report (Corral-Rodríguez et al., 2014; Hirata et al., 2014). This discrepancy might be due to the following reasons. First, the cell line (mDCT cell) we utilized was different from that (HEK293) used by the previous study (Hirata et al., 2014). Second, we presented the relative intensity of intracellular Mg²⁺-green by calculating the mean fluorescence of all cells over 300 gray levels. Hirata et al. presented the relative intensity as the mean fluorescence of 10 cells. In fact, we found that the relative intensity of CNNM2 T568I on the Mg²⁺ efflux assay was higher than the wild type, although the difference was not statistically significant. Altogether, our study demonstrated that this R480L mutation resulted in the diminished binding with Mg²⁺-ATP and consequently led to impairment of Mg²⁺ efflux. This defective Mg²⁺ efflux might account for the hypomagnesemia in this patient with CNNM2 R480L mutation.

There were a few limitations in our study. First, we did not provide direct evidence for the R480L mutation in causing impairment of CBS dimerization by the crystal structure. Second, isothermal titration calorimetry was not performed to confirm the diminishment of the interaction of Mg²⁺-ATP and the CBS-pair. However, the simulation model showed the reduction of the binding energy of ATP-Mg²⁺ in the CBS module of CNNM2 by R480L mutation.

We identified a novel and de novo heterozygous R480L mutation in the CBS domain of the CNNM2 gene in a trio family with severe HSMR. This highly expressed CNNM2-R480L properly localizes to the plasma membrane but impairs Mg²⁺ efflux likely through the attenuated interaction with Mg²⁺-ATP, resulting in the clinical manifestation of refractory hypomagnesemia.