Familial Hypocalciuric Hypercalcemia: The Challenge of Diagnosis

Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant genetic disorder classically characterized by lifelong mild-to-moderate asymptomatic hypercalcemia with inappropriately normal to elevated serum parathyroid hormone (PTH) concentrations and hypocalciuria, best expressed by a urine calcium-to-creatinine clearance ratio (CCCR)<0.01[1,2]. FHH prevalence is estimated between 1:10 000 to 1:100 000[3,4]. In 60% of cases, FHH is due to CASR inactivating mutation[5]. More rarely FHH is due to AP2S1 or GNA11 inactivating mutation, both genes encoding for proteins involved downstream of CASR activation[6]. These molecular alterations are found in all parathyroid cells, explaining disease persistence following partial parathyroidectomy and the ineffective surgical management of these patients. FHH phenotypes could however overlap with primary hyperparathyroidism (PHPT). Indeed, even if patients with FHH are currently asymptomatic, some of them present chondrocalcinosis, kidney stones or bone fracture and very high level of PTH or calcemia[7]. Nonetheless, the distinction has to be adressed since the therapeutic approach signicantly differs between these two conditions. Surgery is usually recommended for PHPT[8] while follow-up is preferred in the latter case[9,10]. We report and discuss 7 cases, 6 out 7 being operated for a presumed PHPT. had typical features of PHPT: calcemia>3mmol/L with a marked elevation of serum PTH 1-84 (629pg/ml), hypophosphatemia (0.74mmol/L) and a normocalciuria (234mg /day) with a high CCCR (0.026). Work-up revealed an osteopenia, a stage 3 renal insuciency with normal kidney ultrasound. Neck US and parathyroid scintigraphy were concordant for a left P4-derived adenoma. A large left P4 adenoma, i.e adenoma on left superior parathyroid gland (weight:580mg), was resected via minimally-invasive surgery with a signicant decline in intraoperative PTH 1-84. Genetic testing revealed an unknown CaSR variant in exon 3(c.347C>T,p.(Ala116Val)) classied as VUS. Phosphocalcic imbalance was normalized following surgery. Although, we could consider in the present case that CASR may be not involved in the patient’s pathology, physicians should be aware that this rare variant may be not always pathogenic.


Introduction
Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant genetic disorder classically characterized by lifelong mild-to-moderate asymptomatic hypercalcemia with inappropriately normal to elevated serum parathyroid hormone (PTH) concentrations and hypocalciuria, best expressed by a urine calcium-to-creatinine clearance ratio (CCCR) < 0.01 [1,2]. FHH prevalence is estimated between 1:10 000 to 1:100 000 [3,4]. In 60% of cases, FHH is due to CASR inactivating mutation [5]. More rarely FHH is due to AP2S1 or GNA11 inactivating mutation, both genes encoding for proteins involved downstream of CASR activation [6]. These molecular alterations are found in all parathyroid cells, explaining disease persistence following partial parathyroidectomy and the ineffective surgical management of these patients. FHH phenotypes could however overlap with primary hyperparathyroidism (PHPT). Indeed, even if patients with FHH are currently asymptomatic, some of them present chondrocalcinosis, kidney stones or bone fracture and very high level of PTH or calcemia [7]. Nonetheless, the distinction has to be adressed since the therapeutic approach signi cantly differs between these two conditions. Surgery is usually recommended for PHPT[8] while follow-up is preferred in the latter case [9,10]. We report and discuss 7 cases, 6 out 7 being operated for a presumed PHPT.

Methods
We performed a retrospective analysis of patients with hyperparathyroidism managed in the department of Endocrine Surgery that were found to carry CASR variants between 2016-2020. Our surgery department is a high volume endocrine surgery deparment with approximately 300 parathyroid interventions per year. The study was approved by the Assistance Publique-Hôpitaux de Marseille ethics advisory committee (PADS21-46).

Results
Seven index cases (5F/2M, mean age 53.4y) with CaSR variants were identi ed, 6 of which had undergone surgical intervention. Six out of 7 could be classi ed as FHH with identi cation of new pathogenic or likely pathogenic variants, while the 7th case had a parathyroid adenoma (PA) with a CASR variant that was ultimately classi ed as variant of uncertain signi cance (VUS). All had hypercalcemia with inappropriately normal or high PTH 1-84 values ( Table 1).
Three patients presented with a classical FHH phenotype. In the rst case (1:F-45y), the disease occurred in a context of IgA nephropathy with progressive decline of kidney function. Surgical intervention was indicated after a 5 years follow-up in our nephrology tertiary referral center, where hypocalciuria was misinterpreted as being secondary to chronic kidney disease, in the presence of a single parathyroid abnormality visible on both neck ultrasound (US) and parathyroid scintigraphy. Calcemia was 2.64mmol/L(N:2.20-2.55mmol/L) with high PTH level(68.8pg/ml,N:15-65pg/ml). Minimally invasive surgery was converted to open surgical intervention due to insu cient decrease of intraoperative PTH, and a subtotal parathyroidectomy (PTx) (3 glands removed) was performed.
The second patient (2:M-76y) had a previous history of resection of a parathyroid adenoma. His latest follow-up showed a persistent hypercalcemia (2.65mmol/L) with hypophosphatemia (0.75mmol/L,N:0.81-1.45mmol/L). Calciuria was in the normal range limit (103 and 160mg/day,N:100-300mg/day) and his CCCR (0.017) was inconclusive to differentiate between FHH and PHPT. The patient had renal lithiasis, osteopenia and chronic renal failure stade 2. Neck US and parathyroid scintigraphy were negative. Because of the presumed persistent PHPT with negative parathyroid imaging, a genetic screening was performed. Genetic testing for case 2 revealed the same pathogenic CASR variant as case 1(c.893C>T,p.(Ala298Val)) [7], a nding which argued against reoperation.
Three additional cases had undergone surgical intervention due to a less typical clinical picture with marked hypercalcemia and uctuating values of calciuria. All of them were relatively young at diagnosis and had negative parathyroid imaging studies despite signi cant hypercalcemia (2.80,2.90,2.84mmol/L,respectively). Bilateral neck exploration was performed which resulted in resection of two hyperplastic glands in cases 4 and 6, and one hyperplastic gland in case 5. All had persistent hypercalcemia post-operatively. It has to be underlined that the existence of a multiglandular disease, objectivated during cervicotomy, prompted the surgeons to limit the extent of parathyroid resection to prevent the occurrence of post-operative hypoparathyroidism. Genetic counseling enabled the identi cation of relatives with CASR variants.
In another patient (5:F-36y) a CASR variant(c.511A>T,p.(Ser171Cys)) was identi ed. It was present in her mother, her sister, her brother and her niece. followed-up in our institution and has a marked hypercalcemia (2.94mmol/L).
Taken together, these cases (4-6) illustrate that the presence of hypercalcemia in relatives during family screening is very powerful indicator suggesting for the diagnosis of FHH. Negative parathyroid imaging together with marked hypercalcemia and calciuria in the low normal range could also raise the suspicion of FHH.
Finally, the last case (7: F-46y) had typical features of PHPT: calcemia>3mmol/L with a marked elevation of serum PTH 1-84 (629pg/ml), hypophosphatemia (0.74mmol/L) and a normocalciuria (234mg /day) with a high CCCR (0.026). Work-up revealed an osteopenia, a stage 3 renal insu ciency with normal kidney ultrasound. Neck US and parathyroid scintigraphy were concordant for a left P4-derived adenoma. A large left P4 adenoma, i.e adenoma on left superior parathyroid gland (weight:580mg), was resected via minimally-invasive surgery with a signi cant decline in intraoperative PTH 1-84. Genetic testing revealed an unknown CaSR variant in exon 3(c.347C>T,p.(Ala116Val)) classi ed as VUS. Phosphocalcic imbalance was normalized following surgery. Although, we could consider in the present case that CASR may be not involved in the patient's pathology, physicians should be aware that this rare variant may be not always pathogenic.

Discussion
This series illustrates the heterogeneity in FHH biochemical phenotypes with a possible overlap with PHPT that could lead to inappropriate management.
Beyond biochemical characterization, parathyroid imaging also plays an important role in parathyroid disease subtyping. In PHPT, a negative parathyroid imaging study should raise the suspicion of multiglangular hyperplasia [13,14]. This series shows that negative parathyroid imaging is part of the clinical picture of FHH. In France, FHH genetic testing is recommended in all patients presenting with familial PHPT or isolated PHPT before 50 years old. This involves the simultaneous exploration of genes involved syndromic hyperparathyroidism (i.e.MEN1, CDKN1B, CDC73) [15]. Genetic testing should be also performed in case of suspicion of FHH or in doubtful clinical situations or in patients with persistent hypercalcemia following PTx. However, despite technological improvements in genetic sequencing, it does not appear reasonable to submit all hyperparathyroid patients to genetic analysis. In our insitution, we are in agreement with the guidelines that recommend performing genetic testing for patients with one of the following criteria[16]:

Age<50y
Family history of hypercalcemia Hyperparathyroidism with CCCR<0.01 and/or calciuria below 1.33mg/day/kg using 24-hour urine collection However, based on this case series, we would like to emphasize that age at diagnosis has a limited diagnostic value, presence of single gland abnormality on parathyroid imaging is not synonym of PHPT and presence of CASR variant is not synonym of FHH.
Addtionally, we consider that : 1. FHH screening is unnecessary if simultaneous calciuria and CCCR are above 4mg/kg/24h and 0.02, respectively. Indeed, looking speci cally at CCCR and 24h-calciuria, we found very few patients [7,17] with a real FHH (i.e.without single parathyroid adenoma) and simultaneous CCCR>0.02 and 24h-calciuria>0.1mmol/Kg 2. In the presence of serum total calcium ≥2.8mmol/L, a negative parathyroid imaging should raise the suspicion of FHH.
3. In case of doubt regarding the disease, assessement of calcemia in rst degree relatives is of powerful value and may guide genetic testing.