Journal of Endocrinological Investigation (2023) 46:829–840 https://doi.org/10.1007/s40618-022-01948-7 ORIGINAL ARTICLE Novel germline variants of CDKN1B and CDKN2C identified during screening for familial primary hyperparathyroidism I. Mazarico‑Altisent1 · I. Capel1 · N. Baena2 · M. R. Bella‑Cueto3 · S. Barcons4 · X. Guirao4 · L. Albert1 · A. Cano1 · R. Pareja1 · A. Caixàs1 · M. Rigla1 Received: 18 March 2022 / Accepted: 22 October 2022 / Published online: 5 November 2022 © The Author(s) 2022 Abstract Purpose CDKN1B mutations were established as a cause of multiple endocrine neoplasia 4 (MEN4) syndrome in patients with MEN1 phenotype without a mutation in the MEN1 gene. In addition, variants in other cyclin-dependent kinase inhibitors (CDKIs) were found in some MEN1-like cases without the MEN1 mutation. We aimed to describe novel germline mutations of these genes in patients with primary hyperparathyroidism (PHPT). Methods During genetic screening for familial hyperparathyroidism, three novel CDKIs germline mutations in three unre- lated cases between January 2019 and November 2021 were identified. In this report, we describe clinical features, DNA sequence analysis, and familial segregation studies based on these patients and their relatives. Genome-wide DNA study of loss of heterozygosity (LOH), copy number variation (CNV), and p27/kip immunohistochemistry was performed on tumour samples. Results DNA screening was performed for atypical parathyroid adenomas in cases 1 and 2 and for cystic parathyroid adenoma and young age at diagnosis of PHPT in case 3. Genetic analysis identified likely pathogenic variants of CDKN1B in cases 1 and 2 and a variant of the uncertain significance of CDKN2C, with uniparental disomy in the tumour sample, in case 3. Neoplasm screening of probands showed other non-endocrine tumours in case 1 (colon adenoma with dysplasia and atypical lipomas) and case 2 (aberrant T-cell population) and a non-functional pituitary adenoma in case 3. Conclusion Germline mutations in CDKIs should be included in gene panels for genetic testing of primary hyperparathy- roidism. New germline variants here described can be added to the current knowledge. Keywords Parathyroid · Primary hyperparathyroidism · Multiple endocrine neoplasia type 1 · Multiple endocrine neoplasia type 4 · Cyclin dependent kinase inhibitors Introduction hyperparathyroidism (PHPT) affects 95% of MEN1 patients and is its most common clinical feature [1, 2]. This syn- Multiple endocrine neoplasia type 1 (MEN1) is a syndrome drome is caused by loss-of-function variants of the MEN1 characterized by the pituitary, parathyroid and entero- tumour-suppressor gene (11q13), which encodes menin, pancreatic neuroendocrine tumours (NETs). Primary a protein responsible for genome stability, transcription, 3 * I. Mazarico‑Altisent Pathology Department, Parc Taulí University Hospital, isamazarico@gmail.com Institut d’Investigació i Innovació Parc Taulí (I3PT), Medicine Department, Universitat Autònoma de Barcelona, 1 Endocrinology and Nutrition Department, Parc Taulí Parc Taulí 1, 08208 Sabadell, Barcelona, Spain University Hospital, Institut d’Investigació i Innovació Parc 4 Surgery Department, Parc Taulí University Hospital, Institut Taulí (I3PT), Medicine Department, Universitat Autònoma d’Investigació i Innovació Parc Taulí (I3PT), Medicine de Barcelona, Parc Taulí 1, 08208 Sabadell, Barcelona, Department, Universitat Autònoma de Barcelona, Parc Taulí Spain 1, 08208 Sabadell, Barcelona, Spain 2 Genetic Department, Parc Taulí University Hospital, Institut d’Investigació i Innovació Parc Taulí (I3PT), Medicine Department, Universitat Autònoma de Barcelona, Parc Taulí 1, 08208 Sabadell, Barcelona, Spain 13 Vol.:(0123456789) 830 Journal of Endocrinological Investigation (2023) 46:829–840 and cell division [3–6]. Germline mutations of MEN1 gene Material and methods have been found in around 85% to 90% of clinically sus- pected MEN1 patients [7]. Thus, in a meaningful number of Phenotype patients with MEN1-like phenotype, around 10%, germinal MEN1 mutations are not detected [4, 8–10]. This lack of We re-evaluated data from three patients with a history of detection could possibly be explained by genetic abnormali- PHPT in whom we performed genetic screening for sus- ties in non-coding regions or whole-gene deletion of MEN1, picion of FHPT. Clinical information about probands and however, large deletions of MEN1 appear to be uncommon their first-degree relatives (including clinical features, bio- [5, 11–14]. Therefore, other predisposing genes, such as chemical results, and radiological findings) was obtained cyclin-dependent kinase inhibitors (CDKIs), have been sug- from medical records and routine visits in our hospital gested by different authors to play a role in patients with (Endocrinology and Nutrition Department). Table 1 pre- MEN1-like phenotype without the MEN1 mutation [12, 15, sents the clinical findings. 16] since it is known that menin affects gene transcription of some cell cycle regulators, such as CDKN1B/p27 and CDKN2C/p18 [17]. In particular, CDKN1B germline path- Gene panel sequencing and variant interpretation ogenic variants have been reported in some patients with MEN1-like phenotype without a MEN1 gene mutation, a DNA was extracted from blood and other tissues samples condition termed MEN4 syndrome (OMIM 610755) [12, using the Gentra Puregene DNA reagent (Qiagen, Valen- 14, 18–20]. cia, CA). The customized gene panel of FHPT screen- PHPT is the most common clinical feature of both MEN1 ing in our centre includes sequence analysis and copy and MEN4 syndromes. It is known that 10% of cases of number variation (CNV) analysis of the following genes: PHPT are hereditary due to germline mutations of different AIP, AIRE, AP2S1, CASR, CDC73, CDKN1A, CDKN1B, genes, a condition called familial PHPT (FHPT) [1]. FHPT CDKN2B, CDKN2C, GCM2, GNA11, MEN1, PTH, RET, involves syndromic forms, such as multiple endocrine neo- and TRPV6. plasia types 1 to 4 and hyperparathyroidism-jaw tumour In the analysis, the mean sequencing depth was > 150 syndrome. Besides, FHPT may present as non-syndromic times, and > 99% of target nucleotides were covered PHPT, which includes familial hypercalcemia hypocalciu- with > 20 × sequencing depth for all assays. The target ric forms, neonatal severe hyperparathyroidism, and others, nucleotides included all protein-coding exons of the genes that are associated with several genes and are all classified on the panels in addition to 20 base pairs (bp) inside each as familial isolated hyperparathyroidism [21]. Until now, intron/exon boundary. The panel was also customized by a consensual systematized genetic diagnosis for FHPT has adding oligonucleotides that targeted deep intronic vari- not been implemented due to a lack of firm evidence about ants (≥ 20 bp from the intron/exon boundary) and non- which individuals have to be tested and which mutations coding variants (promoter region, 5′ or 3′ untranslated should be determined. Even so, most authors agree on the regions [UTR]), which have been reported as disease caus- recommendation of testing for germline mutations in indi- ing in association with hyperparathyroidism. Sequence viduals with PHPT plus some specific situations: (1) PHPT reads of each sample were mapped to the human refer- occurring before the age of 45 years, (2) recurrent or per- ence genome (GRCh37/hg19). Burrows–Wheeler Aligner sistent PHPT, (3) multi-gland disease (MGD), (4) atypical (BWA–MEM) software was used for reading alignment. parathyroid adenoma (APA), (5) parathyroid carcinoma, (6) Duplicate read marking, local realignment around indels, existence of other tumours related to syndromic PHPT, and/ base quality score recalibration, and variant calling were or (7) being a first-degree relative of a known mutation car- performed using genomic analysis toolkit (GATK) algo- rier or having a first-degree relative with PHPT [22, 23]. rithms (Sentieon) for nDNA. Variant data were annotated Germline mutation testing for possible FHPT may include using a collection of tools (VcfAnno and VEP) with a MEN1, CASR, CDC73, CDKN1A, CDKN1B, CDKN2B, variety of public variant databases, including but not lim- CDKN2C, RET, PTH, GNA11, AP2S1, GCM2, and AIP ited to Genome Aggregation Database control population genes according to recent data [1, 23, 24]. cohorts (gnomAD), ClinVar, and Human Gene Mutation Minimal information concerning MEN1-like patients due Database (HGMD). Duplicate read marking, local realign- to CDKIs germline mutations have been reported to date, ment around indels, base quality score recalibration and and this lack of reporting suggests that this type of FHPT variant calling were performed using GATK algorithms probably remains underdiagnosed. This report describes [25]. three individuals with a history of PHPT who were evaluated The sequence variant analysis pipeline was validated in based on a suspicion of FHPT and carried novel germline a Clinical Laboratory Improvement Amendments (CLIA) variants of CDKIs genes. 13 Journal of Endocrinological Investigation (2023) 46:829–840 831 Table 1  Clinical characteristics of patients Mutation LOH/UPD PHPT history Screening of related tumors after genetic Additional diagnosis Medical History of Ca max PTH max Histology Pituitary CT thorax Abnormal lab Endocrine (mmol/L) (pmol/L) diagnosis MRI and abdo- tests or Cancer men Disorders (or remarkable) Case 1 CDKN1B No 3.39 4,950 APA Lack of Enlarged CgA: 493.9 μg/L Colon tubule- c.280_281delinsG, neurohy- right adre- villous p.(Pro94Alafs*25) pophyseal nal gland adenoma bright Bilateral with signal renal cysts low-grade dysplasia Atypical lipomas Obesity T2DM Primary hypothy- roidism Case 2 CDKN1B c.169C>T, No 3.37 33,963 MGD: 3 Normal No tumours CgA: Subclinical p.(Gln57*) glands 1,947.1 μg/L hypothy- para- Gastrin: roidism thyroid 132.6 pmol/L Obesity hyperpla- Prl: Aberrant sia and 1 6,127.2 pmol/L T-cell APA FSH: 106 UI/L population LH: 79.4 UI/L Chronic T(t): 4.23 nmol/L idiopathic T(f): 0.01 nmol/L axonal IGF1: polyneu- 5.85 nmol/L ropathy Case 3 CDKN2C c.319T>G, Yes 2.84 846 MGD: 1 Nonfunc- No tumours CgA 110.3 μg/L Uterine leio- p.(Leu107Val) cystic tioning Prl myomas adenoma cystic 1,358.6 pmol/L Bilateral and 1 par- microad- UFC ovarian athyroid enoma 225.5 pg/24 h cysts adenoma LOH: loss of heterozygosity; UPD: uniparental disomy; RV: reference value; Ca max: higher blood calcium levels before parathyroid surgery (RV: 2.1–2.55); PTH max: serum higher parathyroid hormone before parathyroid surgery (RV: 95–618); APA: atypical parathyroid adenoma; MGD: multiglandular disease; CgA: chromogranin A (RV < 100); T2DM: type 2 diabetes mellitus; Prl: Prolactin (RV 212.6–1,034.5); Gastrin RV 6.2–54.8; UFC: Urinary free cortisol (RV 36–137), FSH: follicle-stimulating hormone (RV: 1.5–12.4); LH: luteinizing hormone (RV: 1.7– 8.6); T(t): total testosterone (RV: 6.69–25.6); T(f): free testosterone (RV: 0.02–0.08); IGF1: insulin-like growth factor 1 (RV: 7.54–26.52); MRI: magnetic resonance imaging; CT: computed tomography and College of American Pathologists (CAP) accredited CNVs were confirmed using digital polymerase chain Blueprint Genetics diagnostic laboratory. The series of reaction (PCR). The CNV analysis pipeline was validated selected quality criteria included a variant call quality in the CLIA and CAP-accredited Blueprint Genetics diag- score, variant genomic location, sequence content, and nostic laboratory. integrative genomics viewer visual analysis. This algo- Sanger sequencing of the candidate variants was per- rithm was established based on the outcome of an internal formed on the patient and the family member samples validation performed in the CLIA and CAP-accredited to confirm the presence of the variant and the pattern of Blueprint Genetics diagnostic laboratory. inheritance. Variants were classified following the Ameri- CNV analysis was performed bioinformatically from can College of Medical Genetics and Genomics and the next-generation sequencing (NGS) data using a bioinfor- Association for Molecular Pathology (ACMG/AMP) matic pipeline; one component is a CNV kit and another guidelines [26]. one involves in-house developed proprietary technology. 13 832 Journal of Endocrinological Investigation (2023) 46:829–840 Study of loss of heterozygosity (LOH) and CNV of the adenoma as the PTH dropped from 4950 pmol/L (pre- in tumour excision) to 618 pmol/L (30′ after excision). Histological study evidenced a nodule (size: 4.5 × 3.5 × 2.5 cm; weigh: Tumour tissue samples from formalin-fixed, paraffin-embed- 15.9 g) with histopathological characteristics consistent with ded (FFPE) were used for this analysis. DNA of tumour sam- an atypical parathyroid adenoma (APA), such as fibrous ples from patients who had undergone parathyroidectomy bands associated with haemosiderin deposition and mitotic was extracted using Qiagen DNA extraction kit (QIAGEN, activity (2 mitotic events/10 high-power fields). Proliferation Valencia CA). Genome-wide DNA study of LOH and CNV index (Ki67) was 3%. was performed using OncoScan CNV Assay (Thermo Fisher After surgery, serum calcium nadir was 2.2 mmol/L, Scientific, Inc., Waltham, MA, USA). This assay is designed and during the next 10 years, it oscillated between 2.07 and to cover the entire genome with higher resolution in well- 2.56 mmol/L. Ionic serum calcium remained within normal known cancer drivers (900 cancer genes). Data analysis was limits until now. done using the software Chromosome Analysis Suite 4.3 Given the atypical features of adenoma, genetic testing (ChAS 4.3) for CNV and LOH detection. was performed. Sequence analysis using the panel described above identified a heterozygous frameshift likely pathogenic Immunohistochemistry variant in CDKN1B (NM 004064.4) c.280_281delinsG, p.(Pro94Alafs*25). Immunohistochemical study with antip27/kip1 antibody (clone SX53G8, Ventana) was performed on slides obtained Germline genetic analysis and LOH/CNV study in tumour from formalin-fixed paraffin-embedded parathyroidectomy tissues using the Ventana Benchmark immunostaining sys- This variant deletes two base pairs, inserts one base pair tem (Ventana Medical System, Tucson, USA). Endothelial in exon 1 (of three total exons) and generates a frameshift, cells were used as positive controls. Immunohistochemistry leading to a premature stop codon at position 25 in a new for p18 was not performed due to the lack of a commercially reading frame. This setup is predicted to lead to loss of nor- available monoclonal antip18 antibody. mal protein function, either through protein truncation or nonsense-mediated mRNA decay. This variant is absent in the gnomAD. To our knowledge, this variant has not been Results described in the medical literature or reported in disease- related variation databases, such as ClinVar or HGMD. Loss Case 1 of CDKN1B function is an established disease mechanism, and other truncating variants in the gene have been described Medical history and clinical presentation in patients with phenotypes consistent with CDKN1B-related disease, which is inherited in an autosomal dominant (AD) A 47-year-old man was referred to our department for a manner. This CDKN1B variant is classified as likely patho- cervical node. In his personal history, he presented obe- genic based on the established association between the gene sity, primary hypothyroidism, and obstructive sleep apnoea and the patient’s phenotype, the variant’s absence in control syndrome. In addition, he had undergone surgery for two populations, and the variant type (frameshift). The genetic atypical lipomas and had an endoscopic polypectomy of a analysis of tumour DNA did not show LOH or CNV. tubule-villous adenoma with low-grade dysplasia. He had no family history of parathyroid disorders. p27/kip1 immunohistochemistry On examination, he had no obvious neck masses or goiter. A neck ultrasound scan (US) showed a homoge- Immunohistochemical analysis of the excised parathyroid neously hypoechoic nodule of 3 cm posterior to the thy- adenoma demonstrated loss of nuclear expression of p27/ roid gland, suggesting a parathyroid lesion. Blood tests kip1 in neoplastic cells. This result is consistent with a revealed severe hypercalcemia (3.39 mmol/L; reference lower expression of the tumour suppressor protein due to value [RV] 2.1–2.55), hypophosphatemia (0.58 mmol/L, the described pathogenic variant (Fig. 1a). RV 0.87–1.45), and elevated parathyroid hormone (PTH) levels (4950 pmol/L, RV 95–618), consistent with PHPT Clinical evolution after the genetic result (Table 1). No clinical history of fractures or nephrolithi- asis was present. Treatment was initiated with cinacalcet Specific guidelines for MEN4 management are lacking as and oral hydration, and he underwent surgical removal of a few cases have been reported until now, however, most solitary abnormal parathyroid gland at the age of 48. Intra- authors recommend following MEN1 guidelines in these operative PTH (ioPTH) monitoring confirmed the excision cases [7, 27]. Thus, we performed a complete radiological 13 Journal of Endocrinological Investigation (2023) 46:829–840 833 Fig. 1  Immunostaining with p27/kip1 of parathyroid adenomas from cases 1 (a) and 2 (b). Absence of nuclear expression in adenoma cells. × 400 and serological study (Table 1). Lab tests did not show obesity, and subclinical hypothyroidism. As the only family alterations in pituitary hormones, and pituitary magnetic history of interest, his mother had had renal colic. He was resonance imaging (MRI) manifested a lack of a neurohy- diagnosed with tertiary hyperparathyroidism six years after pohyseal bright signal, which was attributed to a normal presenting with CKD. Lab tests showed corrected calcium physiological occurrence in the absence of any clinical of 3.37 mmol/L and PTH of 33,963 pmol/L. A bone min- suspicion [28]. A computed tomography (CT) of the chest eral density (BMD) scan was not obtained. He had a previ- and abdomen indicated enlarged right adrenal gland and ous history of symptomatic kidney stones and complicated bilateral renal cysts without clinical significance. Urinary urinary infections. Thus, the possibility of having a PHPT free cortisol was normal. Screening for asymptomatic NETs prior to CKD could not be excluded. Neck US revealed a showed a non-specific mild elevated chromogranin A ([CgA] hypoechoic nodule (1.3 cm in size) posterior to the thyroid 493.9 μg/L, RV < 100) that has almost normalized over time. gland. Conversely, scintigraphy study with technetium 99 m sestamibi (TcMIBI) did not show high radiotracer uptake. Family screening after genetic results Treatment with cinacalcet was initiated but yielded a poor response, so he finally underwent bilateral neck exploration A complete family history was taken after the genetic diag- and total parathyroidectomy without autotransplantation. nosis was obtained. No first-degree relatives were still alive. Histology diagnosis showed MGD due to three hyperplastic Even so, we were able to analyse a DNA sample from a parathyroid glands plus one APA (1.7 g weight). surgical specimen from the mother (I.2, Fig. 2a), who had Post-operatively, lab tests showed a nadir PTH of died years before from a colon neoplasia. In addition, she 561 pmol/L and nadir serum-corrected calcium level of presented hypercalcemia on some occasions, which had not 1.75 mmol/L, suggesting post-surgical hypoparathyroidism. been evaluated. She carried the same variant in the CDKN1B Treatment with calcium supplements and calcitriol was initi- gene as her son. ated, and normal serum levels were recovered. Considering his young age at diagnosis and the identifica- Case 2 tion of APA and MGD with three hyperplastic parathyroid glands, a screening test of the most likely genetic causes of Medical history and clinical presentation FHPT was performed. Sequence analysis of DNA obtained from blood identified a heterozygous nonsense variant of We report a case of a 57-year-old man with a history of CDKN1B c.169C>T, p.(Gln57*) (NM 004064.4). chronic kidney disease (CKD) due to interstitial nephropa- thy diagnosed at the age of 28, which progressed to end- Germline genetic analysis and LOH/CNV study in tumour stage renal disease (ESRD) and was treated with haemo- dialysis since he was 43. As additional significant medical This CDKN1B variant is classified as likely pathogenic based history, he was diagnosed with chronic idiopathic axonal on the established association between the gene, patient’s polyneuropathy, aberrant T-cell population at the age of 46, phenotype, variant’s absence in control populations and type 13 834 Journal of Endocrinological Investigation (2023) 46:829–840 B 1 2 A I 2 ? ? 1 I ? + 1 2 3 II 4 5 + ? - - - 1 II + III 1 C 2 I 1 ? - II 1 2 3 4 5 6 7 ? ? - ? - - + - III 1 2 3 4 5 6 7 8 9 10 11 12 - + 4 5 IV 1 2 3 Fig. 2  Pedigreeandtumors of probands (withconfirmatedmuta- member; slashed symbol, deceased; + , mutation positive; −, mutation tion) andtheirrelatives. a Case 1, variant c.280_281delinsG, negative; ?, no mutation test performed; filled upper right quadrant, p.(Pro94Alafs*25) CDKN1B; b case 2, variant c.169C > T, PHPT; filled upper rightspot, history of nephrolithiasis, renal colics p.(Gln57*) CDKN1B; c case 3, variant c.319 T > G, p.(Leu107Val) or hypercalcemia; filled upper left quadrant, pituitary adenoma; filled CDKN2C. Generation number is indicated with roman numerals. lower right quadrant, neuroendocrine tumor; filled lower left quad- Arrows indicate index case; circle, female; square, male; double line rant, tumor unspecified below descent line, no offspring; empty symbol, unaffected family of variant (nonsense). It generates a premature stop codon Of Somatic Mutations In Cancer (COSMIC) in more tis- in exon 1 (of 3 exons) and is predicted to produce a loss of sues (skin, breast, prostate, haematopoietic, and lymphoid normal protein function, either through protein truncation or tissues). These data strongly support the pathogenicity of nonsense-mediated mRNA decay. This variant was detected this variant. This variant is absent in gnomAD and, accord- in 48% (132 out of 276) NGS reads. As the patient had an ing to our knowledge, it has not been described as a germline aberrant T-cell population, and some pathogenic CDKN1B variant in the literature or reported in disease-related varia- variants have been associated with hematologic malignancy, tion databases. It should be mentioned that although it is a the identified variant could represent either a germline or variant that when expressed somatically is associated with somatic variant. Therefore, we performed genetic analyses a significant risk phenotype, in this case the patient devel- on other tissues (colon, thyroid, and saliva) [29, 30], and oped a rather mild tumoral phenotype (PHPT due to APA found that all of them showed the presence of this variant; and three hyperplastic parathyroid glands and aberrant T-cell thus, we concluded that c.169C>T was a germline variant. population without clinical affectation for the moment). The This variant has been reported several times in the Catalogue genetic analysis of tumour DNA did not show LOH or CNV. 13 Journal of Endocrinological Investigation (2023) 46:829–840 835 p27/kip1 immunohistochemistry the cystic nodule showed non-specific cystic content. Lab tests revealed a PHPT pattern with corrected calcium lev- Immunohistochemical analysis of the excised parathyroid els of 2.84 mmol/L and PTH of 846 pmol/L. BMD showed glands (three hyperplastic parathyroid glands and one APA) normal bone mineralization, and abdominal US excluded demonstrated loss of staining for p27 in neoplastic cells. renal lithiasis at that moment. Bilateral neck exploration This result is consistent with a lower expression of the with left cyst excision and right extrathyroidal nodule tumour suppressor protein due to the described pathogenic extraction was performed. The final pathologic diagnosis variant (Fig. 1b). was MGD due to one parathyroid adenoma (0.1 g weight) and one mainly cystic parathyroid adenoma (3.5 cm diam- Clinical evolution after the genetic result eter, unknown weight due to splitting of the cyst). After surgery, PTH levels dropped to 409 pmol/L after We completed the screening for MEN4-related comorbidi- which she remained asymptomatic and normocalcemic ties (Table 1). CT scan of the chest and abdomen did not during the post-surgery years. Genetic screening of FHPT show any abnormalities. Measurement of pancreatic poly- forms was performed given the detection of MGD, the peptide (PP) and plasma vasoactive intestinal peptide (VIP) existence of a large cystic parathyroid adenoma, and her was normal as was a dexamethasone suppression test. Serum young age at diagnosis of PHPT. The patient was hete- CgA (1947.1 μg/L) and serum gastrin (132.6 pmol/L, RV rozygous for c.319T>G, p.(Leu107Val) in CDKN2C (NM 6.2–54.8) were elevated. Since the proband did not indi- 001262.2). cate clinical or radiological signs of NETs, these results were attributed to proton pump inhibitor therapy and ESRD [31]. Concerning pituitary hormones, he showed moderated Germline genetic analysis and LOH/CNV study prolactin elevation, hypergonadotropic hypogonadism, and in tumour low insulin-like growth factor 1 (IGF1) levels, which could be explained by CKD [32]. Pituitary MRI did not show This CDKN2C variant was genetically classified as a variant abnormalities. of uncertain significance (VUS) according to the ACMG criteria. This variant affects a conserved amino acid, and it Family screening after the genetic result is predicted to be damaging by most in silico tools. Only two missense variants in CDKN2C are currently reported in the Family members were referred for genetic counselling. HGMD: (1) one in association with MEN1 and (2) the other In terms of first-degree alive relatives, he had two living in association with parathyroid adenoma [16, 33]. Agarwal sisters and one brother. All of them had undergone predic- et al. reported a missense germline variant in a family with tive testing, which did not detect the CDKN1B c.169C>T parathyroid tumours in which a decrease in protein expres- p.(Gln57*) variant in tested samples (Fig. 2b). Parental sam- sion as a probable cause of sporadic endocrine tumours was ples were not available. found [16]. The c.319T>G variant in CDKN2C has been reported in gnomAD in 9/251,454 alleles with a low allele frequency (3.58 × ­10−5). It should be noted that the gnomAD Case 3 database excludes individuals known to be affected with the severe paediatric disease in addition to their first-degree rela- Medical history and clinical presentation tives [34]. The clinical age-related penetrance in patients with CDKN2C variants could be low or limited due to the We present a 44-year-old woman that noticed a neck lump small number of cases described in the literature. Functional when she was 28. She had no significant medical history. studies would help to a better classification of this variant. No family history of parathyroid known alterations was Sporadic adenomas have not been extensively investigated reported, but it should be noted that her mother had a his- for candidate variants in CDKI genes and specifically for tory of renal colic episodes. Neck US showed an extrathy- CDKN2C. This gene has been examined in a small sporadic roidal hypoechoic nodule of 1.1 cm posterior to the right adenomas series and to date, has no known identified vari- thyroid lobe and a large cystic nodule (3 cm) on the left ant [34, 35]. In this case, the analysis of LOH and CNV side. A neck CT scan revealed the same cystic lesion in a in tumour sample showed a gain of whole chromosome 1 left para-traqueal location, measuring a maximum diame- and the genotype for three copies were identical leading ter of 3 cm. It remained unclear whether it was of parathy- to somatic uniparental disomy of chromosome 1 (Fig. 3). roid or thyroid origin. Scintigraphy using TcMIBI showed The detection of this event may reinforce the pathogenicity avid tracer uptake near the right thyroid lobe, suggesting a of this variant although this result should be viewed with single parathyroid lesion. Fine-needle aspiration biopsy of caution. 13 836 Journal of Endocrinological Investigation (2023) 46:829–840 Fig. 3  Genome-wide DNA study of loss of heterozygosity (LOH) and copy number variation (CNV) in case 3. CNV track shows a gain of chro- mosome 1 and B-allele frequency (BAF) track shows uniparental disomy of whole chromosome 1 Clinical evolution after the genetic result glands. Lab tests showed hypercalcemia (2.97 mmol/L) and elevated PTH (2423 pmol/L). US of the neck revealed After finding the CDKN2C variant, physical, imaging, and a hypoechoic nodule (0.5 × 0.3 cm) posterior to the left lab exams were performed following MEN1 guidelines thyroid lobe and scintigraphy using TcMIBI showed an (Table 1). Concerning dermatological features, physical increased tracer uptake in the same location, so she was examination revealed a preauricular right tag and seborrheic diagnosed with PHPT. Abdominal US confirmed the pres- keratosis. A CT scan of the abdomen and chest showed uter- ence of renal lithiasis. Due to densitometric osteoporosis ine leiomyomas and some ovarian cysts. No adrenal altera- (lumbar spine 0.637 g/cm2 and T score − 4.69 SD; femoral tion was seen. MRI indicated a nodule (0.6 × 0.7 cm), sug- neck 0.593 g/cm2 and T score − 3.23 SD), alendronate was gestive of cystic non-functional pituitary microadenoma, and prescribed. We performed unilateral neck exploration with the only alteration of pituitary lab test was mild elevated the removal of a single parathyroid adenoma. PTH levels prolactin (1358.64 pmol/L, RV 212.6–1034.5) without clin- declined to normal after excision. CT scan of the chest and ical significance. Urinary free cortisol level was elevated abdomen did not show abnormalities. Regarding pituitary twice (225.5 pg/24 h and 156.9 pg/24 h; RV 36–137), but no hormones, lab test showed a low morning serum cortisol clinical features of Cushing syndrome (CS) were detected. level (5.04 µg/dL; RV 6.02–18.4) with low adrenocortico- In addition, a salivary cortisol and dexamethasone suppres- tropin hormone level (4.2 pg/mL; RV 7.2–63.3), pattern sion test showed no alterations, so CS diagnosis was not suggesting secondary adrenal insufficiency. We performed confirmed. Screening for asymptomatic NETs showed only a 250 µg cosyntropin stimulation test, which showed nor- a non-specific mild elevated CgA (110.3 μg/L). mal post-stimulation cortisol levels: (1) 30 min − 26.9 µg/ dL and (2) 60 min 31.9 µg/dL (RV > 14 µg/dL). No other Family screening after the genetic result lab pituitary hormone alterations or radiological abnor- malities of the gland were detected. First-degree relatives underwent genetic counselling. Other family members (proband’s father, sister, grand- Proband’s mother (II.7, Fig. 2c) carried the variant. She mother, and great uncles) did not present the variant and was offered clinical assessment, including biochemical and showed normal phospho-calcium metabolism lab tests imaging tests of the pancreas, the pituitary, and the adrenal (Fig. 2c). 13 Journal of Endocrinological Investigation (2023) 46:829–840 837 Discussion moderate to severe hypercalcemia was seen in our three cases in contrast to other cases of PHPT associated with We report three novel CDKIs germline variants in three CDKIs mutations [53] (Table 1). Considering that most of different patients with PHPT identified during the course these patients presented remission after surgery, differing of screening for FHPT causes: (1) two likely pathogenic from many MEN1 cases, a minimally invasive parathyroid variants of CDKN1B gene (cases 1 and 2) and (2) one VUS surgery could be contemplated. Regarding endocrine his- of CDKN2C gene (case 3). tology, most of the cases of PHPT-related to CDKIs muta- As stated above, CDKIs germline mutations have been tions described to date were caused by a single parathyroid proposed as a cause of MEN1-like syndromes without adenoma [53], except for a few cases of MGD [16, 39]. To MEN1 mutation. This situation is called MEN4 when a our knowledge, we report for the first time two cases of CDKN1B germline mutation is found. However, the clini- APA and one case of cystic parathyroid adenoma related cal course of patients affected by mutations in CKDIs is to CDKIs germline mutations (Table 1). still poorly understood since few cases have been reported Regarding pituitary neoplasms, up to now, some cases of until now. We reviewed medical literature and genetic these tumours were described in patients with MEN4 (43%) databases (ClinVar, HGMD) and found that a few more [12, 38, 45, 47, 49, 50, 52, 53]. These findings agree with the than 100 cases with MEN4, including 52 different vari- estimated prevalence of pituitary tumours in patients with ants of CDKN1B, have been reported to date [12, 16, 20, MEN1 (40%). Recently, in a large cohort of 211 patients 29, 36–54]. As supplementary material, we added a table (mostly paediatric) with Cushing disease, five germline with the total number of CDKIs pathogenic/likely patho- CDKN1B variants were identified (2.6%) [50]. In addition, genic/VUS germline variants thus far reported along with three germline CDKN1B variants were detected in three the type of parathyroid lesion or other tumours found (if paediatric patients with pituitary adenomas, with no other reported) and pathogenicity level of the variants, which manifestations related to MEN4 [47]. In addition, one case was assessed following the ACMG guidelines [26] (see of macroprolactinoma was described in a case with ger- supplementary material Appendix 1). It should be noted mline CDKN1A mutation [16]. Somatotroph, corticotroph, that 34 of these CDKN1B variants have been reported and non-functioning pituitary adenomas have been found from a single submitter as criteria provided and 10/55 are to be the most frequent pituitary tumours in MEN4 cases. classified as VUS. All these facts support that so very lit- On the contrary, prolactinomas seem less frequent in MEN4 tle knowledge about the clinical implications of CDKIs patients [45, 52]. Non-functioning microadenomas are usual pathogenic variants is available to date. In addition, four in the general population, and it remains challenging to dis- other variants of CDKN1B have been reported in patients tinguish between incidentalomas and MEN-related pituitary with medulloblastoma, paraganglioma, acute lymphoblas- tumours [53]. In this regard, a non-functioning microade- tic leukaemia and familial colorectal cancer [30, 55–57]. noma was detected in case 3. The age of onset of pituitary Germline mutations in three other CDKI genes tumours in MEN4 is still unclear due to the few number of (CDKN2B, CDKN2C, and CDKN1A) were found in cases reported until now. MEN1-like cases without detectable MEN1 mutations Concerning NETs, we did not find any radiological image [16]. Afterall, a reliable prevalence of CDKIs mutations or analytical alteration suggestive of tumour in our cases. in MEN1-like patients or patients with PHPT is difficult to That finding fits with the last data about MEN4 cases, which estimate as genetic screening of these genes has not been indicates a lower prevalence of NETs than in MEN1 [53]. widely established. We report three novel CDKIs germline Other tumours, such as meningiomas, liver haemangio- mutations in three different patients with PHPT. The pos- mas, skin tumours, adrenal nodes, colon cancer, breast can- sibility of including these cases within the term MEN4 or cer, and genitourinary neoplasms, have been described in MEN1-like states should be assessed. patients with germline mutations of CDKIs [12, 16, 20, 36, MEN4–PHPT affects approximately 80–90% of the 38, 53]. We describe a colon adenoma with low-grade dys- reported MEN4 cases to date [7, 53]. This finding is in plasia and atypical lipomas in case 1 and a colon neoplasia accordance with MEN1 classical presentation [58]. On the in her mother who carried the same variant in CDKN1B. In contrary, in most MEN4 cases, the clinical course of PHPT addition, we report a malignant hemopathy (aberrant T-cell seems to be less severe than in patients with MEN1 syn- population) in a CDKN1B carrier (case 2). drome and exhibits a female predominance. Only one case In the firsts two cases (cases 1 and 2), genetic anal- of MEN4 presented recurrent PHPT after subtotal par- ysis of tumour samples showed nor LOH neither CNV athyroidectomy [39, 53]. All cases that we reported agree for CDKN1B, findings that agree with those in the litera- with this as all of them showed remission of PHPT after ture. We reviewed recent data of MEN4 published cases surgery to date (at least nine years follow-up). However, describing CDKN1B pathogenic variants that have ana- lysed tumour tissue to assess the presence of a possible 13 838 Journal of Endocrinological Investigation (2023) 46:829–840 LOH. In most of the cases, tumour testing failed to dem- Supplementary Information The online version contains supplemen- onstrate a second somatic event in the wildtype allele. tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 40618-0​ 22-0​ 1948-7. Therefore, in contrast to MEN1 syndrome, haploinsuffi- Acknowledgements The authors would like to thank the staff of the ciency for CDKN1B appears to be enough for tumours Endocrinology and Nutrition Department, Pathology Department, to occur in MEN4 syndrome [41, 53, 59]. Conversely, in Genetic Department, and Surgery Departmen of our hospital for guid- case 3, genetic tumour testing showed uniparental disomy ance and suggestions, the Blueprint Genetic laboratory for the clinical support and Elena Aller (Thermo Fisher Scientific) for her help in of chromosome 1, including CDKN2C gene, which could LOH interpretation. support a pathogenic role of this variant. Another ger- mline variant of CDKN2C with LOH was described in a Funding Open Access Funding provided by Universitat Autonoma de patient with parathyroid adenoma [33]. In addition, some Barcelona. This article did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. reports propose the contribution of CDKN2C as tumour suppressor gene in different neoplasms [60–63]. However, Declarations further studies are required to establish whether CDKN2C germline pathogenic variants cause genetic predisposition Conflict of interest The authors declare that there is no conflict of in- to FHPT or multiple endocrine neoplasms. In addition, terest that could prejudice the impartiality of the article. immunohistochemistry testing for p18 (not available in our Ethics approval The protocol for the study was approved by the insti- laboratory) could be of interest in future studies of similar tutional Ethics Committee of Institut d’Investigació i Innovació Parc cases to determine the protein expression in tumour tissue. Taulí I3PT. To summarize, we report two novel likely pathogenic Research involving human participants and/or animals All procedures variants of CDKN1B gene in patients with suspected performed were in accordance with the ethical standards of the institu- familial PHPT. The first one was found in a patient with tion and with the 1964 Helsinki Declaration and its later amendments. atypical parathyroid adenoma, atypical lipomas, and a colon tubule-villous adenoma with low-grade dysplasia. Informed consent Written informed consent was obtained from all patients. His mother, who carried the same variant, died from colon neoplasia and once presented with non-studied hypercalce- Open Access This article is licensed under a Creative Commons Attri- mia. The second one found in a patient with PHPT is asso- bution 4.0 International License, which permits use, sharing, adapta- ciated with aberrant T-cell population and other comor- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, bidities, such as obesity, subclinical hypothyroidism, and provide a link to the Creative Commons licence, and indicate if changes chronic idiopathic axonal polyneuropathy. In addition, we were made. The images or other third party material in this article are describe a novel variant in CDKN2C gene with uniparental included in the article's Creative Commons licence, unless indicated disomy in tumour sample that was classified as uncertain otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not significance in a patient with PHPT and a non-functional permitted by statutory regulation or exceeds the permitted use, you will pituitary adenoma. Although the case 3 variant was clas- need to obtain permission directly from the copyright holder. To view a sified as VUS, we would like to highlight its clinical rel- copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. evance since both the proband and her mother, who carried the same variant, presented PHPT. As a reflection, we would like to point out that genetic References screening of FHPT could be considered in patients with features such as APA or in the presence of other tumours 1. Thakker RV (2016) Genetics of parathyroid tumours. J Intern Med apart from PHPT. Considering all reported cases of 280:574–583 2. 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