familial hypocalciuric hypercalcemia ( fhh ) is a heterogenic , autosomal dominantly inherited disorder characterized by hypercalcemia with ( relative ) hyperparathyroidism , and hypocalciuria 1 . classically , fhh is caused by inactivating mutations in the gene encoding the calciumsensing receptor ( casr ) on chromosome 3q13 , termed fhh type 1 ( fhh1 ) 2 , 3 , 4 . recently , further two fhh phenotypes have been identified ; fhh2 have been shown to be caused by inactivating mutations in the gene encoding subunit alpha11 of the g protein ( gna11 ) on chromosome 19p13.3 and fhh3 by mutations affecting codon 15 ( p.arg15leu , p.arg15cys , and p.arg15his ) in the gene encoding the adaptor protein 2 sigma subunit ( ap2s1 ) on chromosome 19q13.32 5 , 6 . however , it is an important differential diagnosis to primary hyperparathyroidism ( phpt ) . due to the biochemical similarity to mild phpt , patients are at risk of being referred to parathyroidectomy ( ptx ) which does not benefit patients with fhh1 . new data , however , suggest that fhh3 is associated with hypercalcemic symptoms in > 20% of patients 10 , thus giving rise to suspicion that mutations in ap2s1 have more deleterious effects . at the time of writing , 55 index cases of fhh3 have been reported 6 , 10 , 11 , 12 , 13 . we now report a family with fhh3 among whom two brothers had ptx performed due to suspected phpt with one of the brothers having a concomitant diagnosis of a neuroendocrine tumor ( net ) . the study is conducted in accordance with the helsinki declaration and approved by the health research ethics committee in the central denmark region ( no . the index case , a male born in 1976 , was diagnosed with hyperparathyroid hypercalcemia at the age of 36 ( table 1 ) . despite only moderate hypercalcemia ( ionized calcium 1.48 mmol / l ) , he had numerous symptoms possibly attributable to hypercalcemia including tiredness , a progressive feeling of muscle weakness , and pain in the upper extremities . dxa scans showed a normal bmd at the hip ( tscore 0.3 ) and mild osteopenia at the lumbar spine ( tscore 1.3 ) and forearm ( tscore 1.3 ) 14 . creatinine clearance ratio ( cccr = 0.005 ) , a genetic test was performed for fhh1 , which is in accordance with the diagnostic guidelines for phpt 15 . family screening revealed a father with intermittently elevated serum levels ( total calcium 2.522.62 mmol / l).the oldest brother ( born 1971 ) refused testing , whereas another brother ( born 1973 ) had normocalcemia . biochemical characteristics of the fhh3 family means of the biochemical measurements of affected members in the fhh3 family . cccr , calcium  creatinine clearance ratio . * treated with cinacalcet . * * treated for hypoparathyroidism . besides hypercalcemic symptoms , the index patient also had gastrointestinal symptoms ( 46 loose stools per day ) , leading to a colonoscopy , which showed an intussusception of the terminal ileum . histological examination showed a radically excised intermediategrade net , which was immunohistochemically positive for cd56 , chromogranin a , and synaptofysin . the cooccurrence of hyperparathyroid hypercalcemia and a net leads to suspicion of a multiple endocrine neoplasia ( men ) syndrome , but additional genetic tests did not reveal mutations in cdc73 , ret , protooncogene , or men1 . biochemical testing and scans of the pituitary gland and the pancreas showed no signs of additional tumors . due to the patients young age and the clinical suspicion of a men1 syndrome , the hyperparathyroid hypercalcemia was considered as a state of phpt and he was referred to ptx . prior to surgery , neck ultrasound suggested an enlarged parathyroid gland on the right inferior side of the thyroid gland , but a parathyroid scintigraphy ( pertechnetate/99mtcmibi ) did not support the finding . subtotal ptx was performed in march 2014 with removal of the upper right and lower left glands , as well as half of the upper left gland and the thymus gland . following the second operation , calcium and pth levels remained elevated and the patient had sustained complaints of hypercalcemic symptoms . accordingly , treatment with cinacalcet was initiated causing normalization of serum calcium levels and symptomatic relief . due to the improved wellbeing following normalization of calcium levels , a third ptx was performed 6 months later with removal and partial autoimplantation of the remaining halfgland on the right thigh ( vastus lateralis , m. quadriceps femoris ) . subsequently , the patient developed hypoparathyroidism , but despite consistent needs for treatment , he reported an improved wellbeing . due to the complicated course of the index patient , his brother ( born 1971 ) who initially refused biochemical screening reconsidered and had biochemical tests performed showing hyperparathyroid hypercalcemia ( table 1 ) . he only had weak complains of tiredness . due to the occurrence of a men1like syndrome in the family , he was tested for mutations similar to the index case and underwent colonoscopy without any pathological findings . further examinations revealed a cccr = 0.005 , no renal calcifications , normal bmd ( except for a hip tscore of 1.1 ) 14 . the lower right gland was not identified , but as pth fell from 17.0 to 3.7 pth increased , however , to supranormal levels within 10 days following surgery with persistently elevated ionized calcium levels ( 1.391.46 we therefore screened the two brothers for mutations in ap2s1 and gna11 and found a c.44g > a substitution in ap2s1 resulting in p.arg15his , a mutation previously reported to cause fhh3 6 , 10 , 11 . screening of the remaining family revealed that the brothers had inherited the mutation from their father , whereas the mother , a brother , and three children were mutation negative ( fig . the histopathological analysis of the glands from the index patients showed one gland with a focus of hyperplasia , two glands with diffuse hyperplasia , and an intrathyroidal process diagnosed as a parathyroid adenoma . the hyperplasia in one gland was characterized by dense fibrosis with hyperplasia of both chief and oxyphil cells and loss of lipid cells . in the other glands , hyperplasia was also present and characterized by loss of lipid cells and increased amount of chief cells . the adenoma showed uniform waterclear cells with solid growth pattern and few scattered isles of oxyphil cells . the removed glands from the brother also showed hyperplasia , which in one gland was characterized by loss of lipid cells and hypercellularity of both chief cells and oxyphil cells , whereas the other two glands were characterized by a more normal fat distribution although still with hypercellularity of oxyphil and chief cells ( fig . histological pictures of the parathyroid glands from the index patient ( a  c ) and his brother ( d ) . ( a ) gland from the index patients showing diffuse hyperplasia characterized by loss of lipid cells and increased amount of chief cells . ( b ) gland from the index patient showing a hyperplastic area in an otherwise normal gland . ( c ) intrathyroid parathyroid adenoma characterized by uniform waterclear cells , a solid growth pattern , and with no lipid cells . ( d ) gland from the brother of the index patient showing a mixed oxyphil and chief cell hypercellularity . the histopathological analysis of the glands from the index patients showed one gland with a focus of hyperplasia , two glands with diffuse hyperplasia , and an intrathyroidal process diagnosed as a parathyroid adenoma . the hyperplasia in one gland was characterized by dense fibrosis with hyperplasia of both chief and oxyphil cells and loss of lipid cells . in the other glands , hyperplasia was also present and characterized by loss of lipid cells and increased amount of chief cells . the adenoma showed uniform waterclear cells with solid growth pattern and few scattered isles of oxyphil cells . the removed glands from the brother also showed hyperplasia , which in one gland was characterized by loss of lipid cells and hypercellularity of both chief cells and oxyphil cells , whereas the other two glands were characterized by a more normal fat distribution although still with hypercellularity of oxyphil and chief cells ( fig . histological pictures of the parathyroid glands from the index patient ( a  c ) and his brother ( d ) . ( a ) gland from the index patients showing diffuse hyperplasia characterized by loss of lipid cells and increased amount of chief cells . ( b ) gland from the index patient showing a hyperplastic area in an otherwise normal gland . ( c ) intrathyroid parathyroid adenoma characterized by uniform waterclear cells , a solid growth pattern , and with no lipid cells . ( d ) gland from the brother of the index patient showing a mixed oxyphil and chief cell hypercellularity . ap2s1 mutations causing fhh3 have recently been reported and only sparse data are available on patients with fhh3 . we believe that the disease courses of the patients reported raise several clinical important issues . first of all , the index case represents the first reported association between a net and fhh3 . secondly , our report supports the previously reported notion on some patients with fhh3 having symptomatic hypercalcemia . finally , to avoid unnecessary surgery and risk of postsurgical hypoparathyroidism , our report calls for increased awareness of the presence of mutations in other genes than the casr , which may cause fhh . the cooccurrence of a net and hyperparathyroid hypercalcemia in a relatively young patient leads to the suspicion of a men1 syndrome . men1 is autosomal dominant inherited and is characterized by phpt , pancreatic tumors , pituitary tumors , and more seldom nets typically located to the adrenal gland , or derived from the embryonic foregut 16 , 17 . our index patient presented with assumed phpt , which is typically the first manifestation of men1 and occurs in 95% of cases 18 , 19 . in addition , he had a net ( formerly known as carcinoid ) , which have been reported to occur in 4% of men1 patients 16 , 19 , 20 . in retrospect , the clinical men1 diagnosis was not clearcut , with several lines of evidence suggesting that our index patient was most likely a socalled phenocopy , that is , a phenotype resembling men1 without being it 21 , 22 . the net found in our index patient was located to the hindgut , which is very rare in men1 . in addition , the patient was negative for all mutations known to cause men1 , although about 515% of considered men1 cases are genetically unverified 17 , 23 , 24 . furthermore , only the index patient had affection of more than one organ , and as the penetrance of men1 is close to 100% in patients older than 50 years , it is unlikely that the 71yearold father should be unaffected 20 , 25 . the low cccr is also not typical for phpt , but can occur due to low vitamin d. according to thakker et al . , men1 phenocopies are seen in up to 10% of cases with the causative mutations found in other genes including cdc73 and casr . consequently , the absence of mutations in the men1 gene should lead to analysis of these genes 26 . our case adds ap2s1 to the genes that need to be considered , when examining a clinical , but genetically unverified men1 patient . in addition to the biochemical findings , the presumed diagnosis of phpt was supported by symptoms , which could be attributable to hypercalcemia . reports of coexisting fhh1 and phpt have previously been published 27 , 28 , 29 . it may therefore be considered whether the index patient had developed phpt superimposed on his inherited fhh3 , which is supported by the histopathological findings showing multiple gland affection with both adenoma and hyperplasia . no previous reports on histopathological findings in parathyroid glands from fhh3 patients report adenomas , but only diffuse hyperplastic or normal glands 10 , 11 , 30 . however , the findings of similar serum calcium levels , pth levels , and low cccr in the index patient and his brother ( table 1 ) do not support that the index patient had developed phpt on top of his inherited fhh3 . rather , our findings support previous reports on hypercalcemic symptoms in approximately 20% of fhh3 patients . ap2s1 mutations are associated with different phenotypes , where p.arg15his give cause to the mildest phenotype , that is , the lowest degree of elevated calcium levels , and also the least degree of hypocalciuria 10 . interestingly , no correlations have been found between genotype and symptoms , and it can be speculated whether the reported symptoms are induced by iatrogenic factors as proposed by marx et al . who found a similar level of hypercalcemic symptoms among fhh1 patients in 1981 31 . however , further characteristics of fhh3 patients need to be performed in order to clarify whether fhh3 is indeed associated with a more severe course , as well as whether there is any correlation to neuroendocrine tumors . in conclusion , we have reported the first case with cooccurrence of fhh3 and a net in the distal ilium . although our index case was most likely a phenocopy , resembling an atypical men1like syndrome , we believe that further attention should be paid to whether fhh3 may be associated with other endocrine tumors . furthermore , our cases underline the importance of considering fhh3 in families with hyperparathyroid hypercalcemia , including patients with a clinical suspicion of men1 who are mutationnegative , as this may spare patients for the worry of being diagnosed with a cancer susceptibility syndrome and ptx . the authors declare that there is no conflict of interest regarding the publication of this manuscript .
key clinical messagefamilial hypocalciuric hypercalcemia type 3 should be considered as differential diagnosis in patients with suspected primary hyperparathyroidism and/or suspected multiple neoplasia syndrome , as correct diagnosis will spare the patients for going through multiple futile parathyroidectomies and for the worry of being diagnosed with a cancer susceptibility syndrome .