Autosomal dominant hypocalcemia type 1 (ADH1) (OMIM # 601198; Hypocalcemia, Autosomal Dominant, HYPOC1) is a rare genetic endocrine disorder, characterized by hypocalcemia, hyperphosphatemia, with low or inappropriately normal parathyroid hormone (PTH) level (1–4). It is caused by an inherited or de novo heterozygote activating mutation of the calcium-sensing receptor (CaSR) gene, located on chromosome 3q13.3–q21 (5). The encoded protein, CaSR, is a G-Protein-coupled cell membrane receptor that is mainly expressed in the parathyroid glands, renal tubules, and the brain (6–9). The CaSR is the key sensor in maintaining extracellular calcium homeostasis, it detects small changes in the serum ionized calcium (iCa²⁺) level and modifies PTH synthesis and release accordingly, to maintain the serum calcium level within the normal range (10–12). In ADH1, the CaSR is activated, even in the presence of normal or low serum calcium levels, resulting in the suppression of PTH synthesis and release, as well as a decrease in renal Ca⁺² reabsorption (13–15), causing hypocalcemia with relative hypercalciuria (11). As a consequence of the increased risk of hypercalciuria, renal complications are not uncommon in patients with ADH1, namely, nephrocalcinosis, nephrolithiasis as well as renal insufficiency. Moreover, since CaSR can affect the calcium and magnesium handling in the kidneys, also the presence of hypomagnesemia is not uncommon in patients with CaSR mutations (16).

ADH1 prevalence was estimated at 3.9 per 100,000 in a recent study of 51,289 subjects from a single US healthcare system (17). Over 100 distinct germline-activating mutations of the CaSR gene have been described in association with ADH1 (8, 18). The mode of inheritance is autosomal dominant; however, sporadic mutations have also been reported and the diagnosis can be confirmed by DNA analysis (5, 19, 20).

The clinical presentation of ADH1 is heterogeneous and may vary from mild hypocalcemia which is not diagnosed until late adulthood, to more severe presentations during infancy or childhood with recurrent seizures (16, 21–24). Patients with ADH1 may have the following clinical characteristics: carpopedal spasms, paresthesia, arthralgia, muscle fatigue, and seizures (24, 25). Nephrocalcinosis has been observed in more than 10% of patients with ADH1, with a higher prevalence of intracerebral calcifications accounting for more than 35% of affected cases (23). The clinical implication of basal ganglia calcifications (BGC) in patients with ADH1 is yet to be explored. Existing observational data from individuals diagnosed with chronic hypoparathyroidism has revealed a noteworthy correlation between the presence of BGC and two factors: a low calcium -to-phosphorus ratio and hypocalcemia. These findings suggest that there may be an association between BGC and imbalances in calcium and phosphorus levels in patients with chronic hypoparathyroidism (26, 27). Conventional therapy with calcium and active vitamin D has been reported to be associated with increased hypercalciuria, which may eventually increase the risk of nephrolithiasis, nephrocalcinosis, and renal insufficiency (3, 5, 8, 22, 28). Several other treatment modalities have been used in treating symptomatic patients with ADH1 including thiazide diuretics, recombinant human PTH (1-84), recombinant human PTH (1-34) (3, 29, 30), and most recently the use of calcilytix (negative allosteric modulators of the CaSR), which are currently in phase 2b open-Label, dose-ranging study (31, 32).

In this study, we aimed to evaluate the existence of a possible phenotype-genotype correlation in patients with ADH1 through the clinical comparison of our 6 ADH1 cases with previously described cases bearing the same genetic variants. We also aimed to determine the severity of the disease in relation to any specific CaSR mutation variant in our patients compared to previously identified variants.

The clinical presentations and characteristics of our ADH1 patients are summarized in Table 1 . Of our cases, 37.5% are of a confirmed familial cause (cases 1, 7, and 8), and 25% are highly likely secondary to de novo mutation due to a lack of family history of hypocalcemia or parathyroid disorder (cases 4 and 5). The remaining 37.5% are cases with a strong family history of hypocalcemia/hypoparathyroidism but with no confirmed molecular diagnosis, which we can infer are familial, making 75% of our cases familial. The lack of molecular diagnosis in family members of our cohort is multifactorial and predominantly related to access issues or living in a different part of the world. All of our mutation variants are heterozygous. Table 2 summarizes our identified CaSR variants, and their comparison with previously published cases.

Our six unrelated ADH1 probands showed distinct clinical presentations, while the three ADH1 cases within the same pedigree (cases 1, 7, and 8) shared similar presentation and clinical characteristics, see Table 1 . The biochemical profile differed amongst the cases in terms of the degree of hypocalcemia, associated hypercalciuria, or other biochemical abnormalities, such as hypomagnesemia and hypokalemia, as well as response to therapy. Individualized biochemical profiles are summarized in Table 3 .

In patients with ADH1 (5), there is an established correlation between the presence of an activating mutation in the CaSR gene and the presence of hypocalcemia. However, the literature is lacking in identifying other clinical areas of phenotype-genotype correlation in ADH1 patients (5). In our ADH1 case series, we observed a potential correlation between the presence of specific CaSR variants, affecting specific domains of the CaSR protein, and the clinical characteristics of the patients. In addition, to better evaluate possible association between CaSR variants and ADH1 clinical manifestations, we also clinically compared our cases with the ones, published to date, bearing the same or similar CaSR variants, to identify the presence of common clinical characteristics.

The degree of hypocalcemia and clinical presentation in patients with activating mutation of the CaSR gene have been previously evaluated (18), showing that there is no association between values of serum calcium with the three different mutation sites in the CaSR protein (ligand-binding extracellular domains, 7 transmembrane domains, or C-terminus intracellular domain) (18). However, in a recent systematic review, it was noted that patients with more severe disease had lower serum calcium levels in comparison to asymptomatic patients (7.6 ± 0.7 mg/dL; p<.0001) or moderately symptomatic (7.4 ± 0.5 mg/dL;p< 0.01) patients with ADH1 (8). In our cohort, Case 2, bearing the p.Glu191Lys variant in the ligand-binding 2 extracellular domain, had the lowest mean value of serum-corrected calcium but did not show a severe clinical presentation, in terms of the presence of seizures, renal impairment, recurrent hospital admissions with hypocalcemia and requirement of frequent dose adjustments (43). By contrast, in our case series, we observed a correlation between the severity of hyperphosphatemia and the occurrence of complications affecting both the renal and neurological systems. However, it is important to note that this study is not powered to establish a causal relationship.

The search on the database of human mutations and Pubmed allowed us to identify a total of 10 published cases, in addition to our 3 cases, bearing the p.Phe788Cys mutation. This mutation is located in the fifth transmembrane (TM5) domain encoded by exon 7 of the CaSR gene. All but one case manifested the disease in early childhood, with two undisclosed times of diagnosis, accounting for 77% of the cases presenting in early childhood. The patient with the pPhe788Cys variant who had an adult presentation of ADH1 also bore another mutation in the CaSR gene, thought to be an activating mutation, which may have influenced the clinical presentation and made it less prevalent (36). Moreover, the neurological involvement, such as hypocalcemic seizures and basal ganglia calcifications was more prevalent in patients carrying this mutation compared to others. Seizures were reported at diagnosis in 7 of the 13 cases with the p.Phe788Cys mutation, accounting for 54% of the cases; the remaining 6 cases did not indicate or deny the presence of seizures. Basal ganglia calcification was reported in 5 out of the 13 cases (38.5%). Nephrolithiasis was present in 15% (2/13 cases) and nephrocalcinosis was present in 38.5% of the cases (5/13). Patients with this mutation shared the presence of at least three complications associated with ADH1, which could be a marker of severity. Although family history was positive in three pedigrees, accounting for 61.5% being of familial inheritance, the remaining 38.5% were confirmed to be of de novo inheritance. In our three genetically related cases with the p.Phe788Cys variant (cases 1, 7, and 8), seizures were described in two cases (66.7%), while basal ganglia calcifications were reported to date only in the 25-year-old mother.

It was previously reported that activating mutations in the fifth transmembrane (TM5) domain of the CaSR protein cause severe familial hypoparathyroidism (21). This is also evident in our three patients bearing the p.Phe788Cys variant, as well as in published cases with the same mutation. The individuals with this mutation, including our cases, shared a unique feature consisting in the fact that calcium levels had to be maintained within the range of 1.75- 1.9 mmol/L (7–7.6 mg/dL) in order to avoid symptoms and complications similar to those experienced by patients with frank hypercalcemia. Mutations in the TM5 domain render the CaSR protein more sensitive than normal to activation by Ca⁺². A previous report documented another mutation at the codon 788, the p.Phe788Leu, in two individuals who had ADH1 with basal ganglia calcifications and nephrocalcinosis (44). Both these missense mutations involving the codon 788 demonstrated a leftward shift in the concentration-response curve in relation to extracellular calcium change and intracellular calcium increase (21, 44).

We identified in one ADH1 case the p.Thr828Pro variant that has not previously been reported, despite the fact that three publications were listed on ClinVar, none of which reported this specific mutation in the manuscript (16, 23, 45). Another missense mutation (p.Thr828Asn), affecting the same codon, has been described as associated with ADH1, however, the published study did not offer additional clinical information (9). Therefore, it could be inferred that alteration at codon 828 may result in an activating mutation of the CaSR gene leading to the development of ADH1. Interestingly, our p.Thr828Pro-bearing patient (case 4) shared similar features to the three patients with the p.Phe788Cys variant (cases 1, 7, and 8), in which the disease presentation is early in life (10 days old), there are recurrent seizures and multiple hospital admissions with hypocalcemia, as well as the presence of nephrocalcinosis and renal insufficiency. Moreover, hypomagnesemia was present in all these four cases.

Time to ADH1 diagnosis was variable in our eight ADH1 cases, presumably reflecting the severity of the disease. Indeed, more severe cases tend to declare themselves early in life, as for our cases 1, 4, 7, and 8, while milder forms tend to present later in life with mild symptoms. Both the p.Phe788Cys and the p.Thr828Pro mutations are located in the exon 7-encoded TM5 domain of the CaSR protein. This suggests that the alteration of this domain could be causative for the shared clinical characteristics in our 4 ADH1 cases and for the earlier presentation of the clinical phenotype. In addition, another missense variant (p.Leu773Arg) located at the TM5 domain, was associated with an ADH1 clinical phenotype characterized by an early onset hypocalcemic seizure, hyperphosphatemia, and basal ganglia calcifications (46), strengthening the hypothesis that single amino acid substitutions at the TM5 domain could be predictive of an early onset disease with neurological involvement.

By contrast, another novel missense mutation (p.Met734Thr) in the TM5 region was described, from a German survey, in an asymptomatic patient with hypocalcemia and low PTH level. However, this mutation lacked further familial and functional characterizations to be classified as “damaging or pathogenic” and was believed to be benign (23).

Based on data from our patients and results from the published studies, we hypothesize that mutations affecting the TM5 domain of the CaSR protein could be responsible for a more severe form of the disease, and they may be associated with a higher rate of neurological manifestations, such as a history of hypocalcemic seizures and the presence of basal ganglia calcifications, as well as of renal involvement, including nephrocalcinosis, nephrolithiasis, and renal insufficiency.

In this study, we also described two novel mutations of the CaSR gene: c.2468T>A; p.Ile823Asn and c.1756G>A; p.Glu586Lys. Neither of these variants has been reported in ClinVar, Human Gene Mutation Database (HGMD), GnomAD, or PubMed. In silico analysis of these two missense mutations with PolyPhen-2 (a tool for the prediction of the functional effect of a non-synonymous single amino acid substitution on a human protein) predicted the Ile to Asn substitution at position 823 as “probably damaging” with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) and the Glu to Lys substitution at position 586 as “benign” with a score of 0.095 (sensitivity: 0.93; specificity: 0.85). Interestingly, another CaSR variant affecting the same codon 586 (c.1758G>C, p.Glu586Asp) has been previously reported, by a single entry on ClinVar, as a VUS variant for the familial hypocalciuric hypercalcemia type 1 (HHC1); a clinical phenotype, mirroring the ADH1, characterized by a lifelong elevation of serum calcium concentrations, an inappropriately low urinary calcium excretion and a normal or mildly elevated PTH, and caused by heterozygous loss-of-function mutations in the CaSR gene. However, there were no citations or functional evidence of this mutation.

In a recent systematic review, it was found that 93% of patients with ADH1 had an incomplete diagnosis of idiopathic hypoparathyroidism or hypocalcemia at the time of presentation, and 7% of the cases were misdiagnosed with seizures or respiratory/cardiovascular disorders (8). This is reflected in our cohort, as 37.5% of our patients’ family members were diagnosed with idiopathic hypoparathyroidism and hypocalcemia.

In conclusion, our data and the comparison with previously published ADH1 cases suggested the existence of a genotype/phenotype correlation regarding the presence of specific ADH1-related clinical manifestations and the early onset and/or severity of the disease. These findings, however, require further evaluation and assessment with a systematic review.

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

Ethical review and approval was obtained. This study uses subjects enrolled to the CALIBRATE study: a Phase 3, Randomized, Open-Label Study that evaluated the efficacy and safety of Encaleret compared to the standard of care in participants with ADH1 (NCT05680818). Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Design and conceptualization of the project: DA, AK, MB. Analysis and interpretation of patients’ clinical and biochemical data: DA, FM. Review/editing of the manuscript: DA, FM, FA, HA, AA, AK, MB. All authors contributed to the article and approved the submitted version.