Overlapping Presentations and Diverse Genetic Defects Characterize Neuroendocrine Neoplasms in a Mexican Cohort

Abstract

Context

Genetic tests are part of the routine clinical approach to syndromic and nonsyndromic phenotypes of neuroendocrine neoplasms (NENs). Current data on phenotype–genotype associations in NENs, however, do not accurately represent all populations.

Objective

To describe the frequency, inventory, and clinical associations of germline defects associated with multiple types of NENs in a Mexican cohort.

Methods

Blood DNA from Mexican adults with NENs was analyzed with a 53-gene next-generation sequencing panel developed ad hoc (n = 90) or Sanger sequencing (n = 2). Single nucleotide variants, indels, and structural variants were identified, classified, and subjected to orthogonal confirmation. When possible, tumor samples and blood DNA from additional family members were tested using Sanger sequencing.

Results

Ninety-two probands (70.7% women, 51.5% sporadic) were included; 16 carried pathogenic or likely pathogenic (P/LP) variants and were significantly younger at disease onset than the rest (29.6 ± 10.7 vs 40 [21.5-51.5] years, P = .0384). Likely driving variants were identified in three-quarters of Von Hippel Lindau syndrome cases, one-third of multiple endocrine neoplasia (MEN) type 1, one-quarter of early-onset acromegaly/gigantism, and individual cases of Cushing's disease, MEN2A, and medullary thyroid carcinoma. One patient with clinical MEN1 associated with an SDHA variant and 1 with a pituitary tumor and neurofibromatosis type 1 were also identified. Probands with familial disease were more likely to carry P/LP variants than sporadic cases (26.7 vs 8.5%, P = .0282).

Conclusion

P/LP variants were identified in 17.4% of individuals with NENs. Our research provides a view of the landscape of NEN drivers in a population not previously characterized.

Neuroendocrine neoplasms (NENs) comprise a heterogeneous group of lesions derived from cells with the capacity for producing hormones (1-4). These tumors may arise from various tissues, presenting either individually or as part of syndromes of multiple endocrine neoplasia (MEN) (5). Their clinical behavior is quite variable, spanning from indolence to hormone oversecretion, compromise of surrounding structures, frank aggressiveness, and malignancy (6-8). Although traditionally considered rare, the frequency of NENs has recently increased, due to improved clinical awareness and novel diagnostic methods (9-11).

NENs entail a very strong heritable component: around 40% of pheochromocytomas and paragangliomas (PPGLs), 10% to 20% of gastrointestinal NENs (GI-NENs), pancreatic NENs (panNENs), bronchopulmonary, and thymic NENs, 16% to 25% of cases of medullary thyroid carcinoma (MTC), up to 15% of parathyroid NENs, and 5% to 10% of pituitary neuroendocrine tumors (PitNETs) are due to germline defects (12-19). The genetic drivers involved include MEN–associated genes (CDKN1B, MAX, MEN1, PRKAR1A, and RET), defects leading to inherited isolated NENs (loss of function [LOF] variants in AIP and SDHx or GPR101 gene amplification), alterations causing syndromes of both inherited NENs and cancer (DICER1, TP53, and VHL), and genes causing phakomatoses where NENs are often a manifestation (NF1, PTEN, TSC1, and TSC2) (18, 20-27). In addition, somatic genetic changes, such as loss of heterozygosity (LOH) and hotspot variants (affecting BRAF, GNAS, SF3B1, USP8, and USP48 in PitNETs) might serve as biomarkers for particular tumor phenotypes, while others (RET in MTC) represent potential therapeutic targets (28-31).

In the last decade, the widespread use of powerful tools for genetic analyses has resulted in a dramatic increase in the known genetic causes of NENs. These approaches have also yielded instances of unexpected genetic diagnoses for particular phenotypes that include NENs, uncovering a great overlap in their clinical presentation (32-36). Genetic tests are key for early diagnosis, tailored clinical management, and genetic counseling in patients with NENs (23, 37-39). Yet, published data on the frequency and type of genetic disease drivers do not accurately represent all human populations. In particular, genetic tests are not widely available in Mexico and are almost nonexistent in its public hospitals, which serve the majority of the country's ∼129 million population. Precision medicine strategies require population-specific information, but Mexicans, one of the most genetically diverse human groups, are poorly represented in international genetic databases (40, 41). Therefore, we aimed to explore the type and frequency of germline tumor drivers in a prospective cohort of Mexican individuals with NENs and to define the phenotypes and clinical outcomes associated with specific genetic defects.

Materials and Methods

Patients and Samples

Eligible participants were adults (≥18 years, with no maximum age limit) with a new or previous diagnosis of NENs under medical care at either of 2 participating reference hospitals in Mexico City: Hospital de Especialidades de Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. Individuals with previously established genetic diagnosis were not excluded. The research protocol was approved by the internal review boards of both institutions (protocol IDs R-2022-785-011 and 4090, respectively; ClinicalTrials.gov: NCT06523582). All participants provided written informed consent and a peripheral blood sample at recruitment. Relatives of probands with positive genetic analyses and 3 apparently healthy controls from the same population were also included under informed consent.

Three groups of diagnoses were considered for recruitment: (1) isolated NENs with sporadic presentation (bronchopulmonary NENs, GI-NENs, or panNENs, MTC, PPGLs, PitNETs, and primary hyperparathyroidism [PHPT]); (2) familial isolated NENs (familial isolated pituitary adenoma [FIPA], familial MTC [FMTC], familial PPGLs, familial PHPT, familial gastrointestinal stromal tumors [GISTs], and X-linked acrogigantism); and (3) clinical syndromes encompassing NENs and other manifestations with familial or sporadic presentation (Carney complex, Carney–Stratakis syndrome, Carney triad, Cowden syndrome, DICER1 syndrome, Li–Fraumeni syndrome, Lynch syndrome, MEN-like syndrome [MEN-like], MEN1, MEN2A and 2B, neurofibromatosis type 1 [NF1], Pacak–Zhuang syndrome, PPGL and pituitary adenoma syndrome, tuberous sclerosis complex, and von Hippel–Lindau syndrome [VHL]). Definitions for clinical diagnoses and family history within each group are detailed in supplemental material (42). Exclusion criteria were age <18 years, unavailable clinical data, unwillingness to provide a blood sample, and inability to provide informed consent.

Recruitment started in August 2022 and, as of September 2024, 198 individuals (169 probands and 29 additional family members) had been included. Ninety-four individuals, including 2 patients with previously established genetic diagnoses via Sanger sequencing, were included in the present analysis. Clinical data were obtained directly from the participants and/or their clinical records. Fresh frozen tumor samples were obtained from patients that underwent surgery during the study. When available, formalin-fixed and paraffin-embedded (FFPE) samples were retrieved from the pathology archives. DNA was extracted from blood using the DNA Isolation Kit for Mammalian Blood (Roche 11667327001) plus RNase (Roche 11119915001) treatment. For DNA extraction from tissues, samples were first mechanically disrupted with magnetic beads (fresh frozen tissues) or deparaffinized (FFPE samples) with HistoChoice (Merck H2779) and then processed with the Maga Zorb DNA Mini-Prep kit (Promega MB1004).

Targeted Next-Generation Sequencing

A panel for capture-based next-generation sequencing (NGS) was designed using the Twist Biosciences platform, covering all exons, 5′ and 3′ untranslated regions (UTRs), and exon–intron junctions (50 bp outside of exons) of the following genes: AIP (11q13.2, reference transcript: NM_003977.4), ATRX (Xq21.1, NM_000489.6), BRAF (7q34, NM_004333.6), CABLES1 (18q11.2, NM_001100619.3), CDKN1A (6p21.2, NM_000389.5), CDKN1B (12p13.1, NM_004064.5), CDKN2B (9p21.3, NM_004936.4), CDKN2C (1p32.3, NM_078626.3), CDC73 (1q31.2, NM_024529.5), DAXX (6p21.32, NM_001141969.2), DICER1 (14q32.13, NM_177438.3), DLST (14q24.3, NM_001933.5), DNMT3A (2p23.3, NM_022552.5), EGLN1 (1q42.2, NM_022051.3), EPAS1 (2p21, NM_001430.5), FH (1q43, NM_000143.4), GNAS (20q13.32, NM_000516.7), GOT2 (16q21, NM_002080.4), GPR101 (Xq26.3, NM_054021.2), H3-3A (1q42.12, NM_002107.7), KIF1B (1p36.22, NM_001365951.3), KIT (4q12, NM_000222.3), MAFA (8q24.3, NM_201589.4), MAX (14q23.3, NM_002382.5), MDH2 (7q11.23, NM_005918.4), MEN1 (11q13.1, NM_130799.3), MET (7q31.2, NM_000245.4), MLH1 (3p22.2, NM_000249.4), MSH2 (2p21, NM_000251.3), MSH6 (2p16.3, NM_000179.3), MERTK (2q13, NM_006343.3), NF1 (17q11.2, NM_001042492.3), PDGFRA (4q12, NM_006206.6), PIK3CA (3q26.32, NM_006218.4), PMS2 (7p22.1, NM_000535.7), PRKAR1A (17q24.2, NM_002734.5), PTEN (10q23.31, NM_000314.8), RASD1 (17p11.2, NM_016084.5), RET (10q11.21, NM_020975.6), SDHA (5p15.33, NM_004168.4), SDHAF2 (11q12.2, NM_017841.4), SDHB (1p36.13, NM_003000.3), SDHC (1q23.3, NM_003001.5), SDHD (11q23.1, NM_003002.4), SLC25A11 (17p13.2, NM_003562.5), SUCLG2 (3p14.1, NM_003848.4), TMEM127 (2q11.2, NM_017849.4), TP53 (17p13.1, NM_000546.6), TSC1 (9q34.13, NM_000368.5), TSC2 (16p13.3, NM_000548.5), USP8 (15q21.2, NM_005154.5), USP48 (1p36.12, NM_032236.8), and VHL (3p25.3, NM_000551.4). The final design (Twist Custom Panel TE-95598353) includes 2023 probes, spans 208 790 bp, and covers 99.5% of the 209 381 bp of the intended target region.

For library preparation, ∼400 bp fragments were obtained by mechanical fragmentation of 200 ng blood DNA in a Covaris S220 sonicator. End repair and dA-tailing, adapter ligation, and amplification, as well as hybridization and postcapture amplification of 8 × 250 ng sample pools were carried out by means of the Twist Biosciences Mechanical Fragmentation and Universal Adapter and Target Enrichment Standard Hybridization protocols. DNA fragment size analyses after sonication, indexing, and hybridization were performed by means of the DNA 1000 or DNA high sensitivity kits (Agilent 5067-1504 and 5067-4626), as appropriate, in a 2100 Bioanalyzer (Agilent). DNA at these steps was quantified with the Qubit dsDNA HS or dsDNA BR Assay Kits (Invitrogen Q32854 and Q32853), as appropriate. Sequencing of 8 pM library pools with 1.25% PhiX Control v3 (Illumina FC-110-3001) was carried out using the MiSeq Reagent Kit v2 (300-cycles, Illumina MS-102-2002) in a MiSeq (Illumina) instrument to obtain paired-end 150 bp reads with an aimed 300× sequencing depth.

Sequencing Data Analyses

Quality was verified using FastQC and reads were then aligned to the GRCh38/hg38 human genome using Burrows–Wheeler aligner-maximum exact matches (43, 44). Polymerase chain reaction (PCR) duplicates were marked with MarkDuplicates and FreeBayes was used for variant calling (45, 46). Copy number variants were searched for with ExomeDepth (47). The Franklin (Genoox) online platform was used to select medium and high confidence nonsynonymous variants in the regions of interest, with sequencing depth ≥20 (48). Using public online database browsers, variants with frequency <0.1% in The Genome Aggregation Database v.4.0, the indigenous Mexican subset of samples from the Mexico City Population Database, and All of Us were then selected (41, 49, 50). For all samples, a manual screen of NGS raw data for the variants of interest was performed, using the Integrative Genomics Viewer 2.3.72 platform (51). Variants of interest were searched for in ClinVar and their reported classification (according to the criteria of the American College of Medical Genetics and Genomics and Association for Molecular Pathology, ACMG/AMP) was noted (52, 53). For variants with conflicting classification or not listed in ClinVar, functional effects were predicted using the in silico tools linked to the Varsome and Franklin online platforms and available clinical and functional data were retrieved using the Mastermind (Genomenon) online platform (54, 55). Based on those data, ACMG/AMP categories were assigned. All variants are reported according to the Human Genome Variation Society nomenclature (56).

Sanger Sequencing and Droplet Digital PCR

All pathogenic and likely pathogenic (P/LP) variants and selected variants of uncertain significance (VUS) identified by NGS were subjected to orthogonal confirmation, using Sanger sequencing for single nucleotide variants (SNVs) and indels or droplet digital (dd) PCR for structural variants (SVs). The same methods were used to analyze the germline variant loci in FFPE or fresh frozen tumor samples. Sanger sequencing was also used to confirm variants of interest in 2 patients with a previous diagnostic test, as well as to investigate BRAF, USP8, and USP48 hotspot variants in 6 FFPE corticotropinomas. The regions of interest were amplified by end-point PCR, using specific primers and GoTaq Green Master Mix (Promega M7122). Amplicons were column-purified (Wizard SV Gel and PCR Clean-Up System, Promega A9281) and subjected to unidirectional sequencing (BigDye Terminator 3.1 Cycle Sequencing Kit, Applied Biosystems 4337456) in a 3500xL Genetic Analyzer (Applied Biosystems). Sequences were analyzed using the Geneious Prime v.2024.0.5 (Biomatters, Ltd.) software.

Reactions for ddPCR were prepared using 6-carboxyfluorescein-labeled assays targeting the regions of interest, a 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein-labeled RPP30 assay (Applied Biosystems 4403326) as an internal control, HindIII restriction enzyme (New England Biolabs, NEB, R3104), and ddPCR SuperMix for Probes (Bio-Rad 1863024). Droplet generation and reading were done in a QX200 Droplet Digital PCR System (Bio-Rad). Results were analyzed with the Quanta Soft software 1.7.4.0917 (Bio-Rad). Primers and assays for end-point PCR, Sanger sequencing, and ddPCR are listed in Table S1 (42). A commercial predesigned assay (Applied Biosystems TaqMan 6-carboxyfluorescein-labeled Hs02125257_cn) was used for ddPCR for VHL exon 3.

Performance of Sequencing Platform

Peripheral blood DNA samples of 92 probands and 3 controls were sequenced using the custom-designed NGS panel, achieving a median sequencing depth of 243.5x (223-259.9) for the 53 genes analyzed (Fig. S1 (42)). Only DAXX yielded a low mean depth of sequencing, at 60.3 ± 8.1x, probably due to the presence of low complexity regions. Due to quality concerns, samples from 2 probands were not considered for further analyses. All SNVs, indels and SVs of interest passed orthogonal confirmation, except for 2 defects in PMS2 and SDHC, which were considered artifactual. In the first gene, exon 13 variant c.2182_2184delinsG, p.T728Afs*7 was found in heterozygosity in 2 probands and passed all selection filters. This region displays homology with the pseudogene PMS2CL, and PCR using primers in specific regions of PMS2 followed by Sanger sequencing ruled out the variant (57). In a different individual, testing of a deletion in exon 5 of SDHC by ddPCR rendered dubious results (4.6 vs 5.2 copies in a control), also likely due to a pseudogene (58).

Statistical Analyses

Analyses were carried out using Prism 10.2.3 (GraphPad Software). For numerical variables, data distribution was analyzed using the Shapiro–Wilk test. Parametric data are presented as mean ± SD and nonparametric data are presented as median and interquartile range. Age at disease onset and at diagnosis were compared among groups using the Mann–Whitney test. The frequency of P/LP variants among sporadic and familial cases was compared using the Fisher exact test. The odds ratio is presented with 95% confidence interval in brackets, and was calculated using the Baptista–Pike method. P < .05 was considered statistically significant.

Results

Clinical Features

We present data for 92 probands (70.7% women, Fig. 1A), 90 studied via NGS panel and 2 with previous Sanger sequencing–based genetic diagnoses. The mean age at recruitment was 48.8 ± 16.9 years and the median ages at disease onset and diagnosis were 37 (21-49.5) and 40 (23.3-50.8) years, respectively. The clinical presentation was classified into 15 different phenotypes, being the most common MEN1, MEN-like, and early-onset PitNETs without growth hormone (GH) secretion, with 16, 13, and 13 individuals, respectively (Fig. 1B). Most individuals (51.1%, n = 47) had no NEN-related family history. Among nonsporadic cases, the most common familial phenotype was cancer predisposition, in one-fifth of probands (Fig. 1C).

Figure 1.

Clinical characteristics of the study population and general description of genetic findings. (A) Sex distribution of the probands (n = 92). (B) Categories of clinical presentation of the study probands (n = 92). The categories were determined according to the clinical features observed at diagnosis and during follow-up. (C) Family history in probands (n = 92). Patients with no relevant family history were classified as sporadic and the rest were grouped into 10 categories, according to the phenotypes described in the family members. (D) All variants (n = 303) identified in the study, according to their predicted effect. (E) ACMG/AMP categories for all variants identified in the study (n = 303). (F) Comparison of the age at disease onset and diagnosis between individuals with a detected pathogenic or likely pathogenic variant (P/LP) and the rest of probands (Rest). The horizontal bar represents median, and the error bars depict interquartile range. CS, Cushing's syndrome; del, deletion; dup, duplication; FIPA, familial isolated pituitary adenoma; FMTC, familial medullary thyroid carcinoma; FPPGLs, familial pheochromocytomas/paragangliomas; gig/EO acro, gigantism and early onset acromegaly; ins, insertion; ins/del, insertion/deletion; LB, likely benign; LFL, Li–Fraumeni-like; LP, likely pathogenic; MEN-like, syndrome similar to multiple endocrine neoplasia; MEN1, multiple endocrine neoplasia type 1; MEN2A, multiple endocrine neoplasia type 2A; MTC, medullary thyroid carcinoma; M GI-NENs, multiple gastrointestinal neuroendocrine neoplasms; M panNENs, multiple pancreatic neuroendocrine neoplasms; NF1, neurofibromatosis type 1; non-GH EO PitNETs, early onset PitNETs not causing GH excess; panNEN, pancreatic neuroendocrine neoplasm; PitNET, pituitary neuroendocrine tumor; PPGL, pheochromocytoma/paraganglioma; VHL, Von Hippel Lindau syndrome; VUS, variants of uncertain significance.

Results of Genetic Analyses

In total, 303 genetic variants were identified, almost half of which (n = 143) were missense (Fig. 1D). One-hundred and eighty-one variants were benign, 67 were likely benign (LB), 35 were VUS, 14 were P/LP, and 6 were not classified (Fig. 1E). All variants were found at the germline level, except for USP8 p.S718P, which was a somatic change in 1 corticotropinoma. The 248 different benign and LB variants identified in the study are listed in Table S2 (42).

The P/LP variants included 12 SNVs or indels and 2 SVs, affecting 8 genes (AIP, MEN1, NF1, RET, SDHA, SDHD, VHL, and USP8) and were found as heterozygous changes in 16 cases (17.4% of those analyzed, Table 1). Two variants, RET p.C618R and VHL p.L89P, were detected in 2 cases each, while the rest affected individual probands. MEN1 was the most frequently affected gene, with 5 P/LP variants in 5 different individuals with MEN1 (3 sporadic and 2 familial), including 3 novel changes. Two variants per gene were found for VHL and AIP, while the rest of genes displayed single variants. Probands carrying P/LP variants had a significantly younger age at disease onset (29.6 ± 10.7 years) than the rest (40 [21.5-51.5] years, P = .0384). These individuals were also diagnosed slightly earlier (32.6 ± 11.3 years), although not at a significantly younger age than other probands (40.5 [24-54.3] years, P = .1250, Fig. 1F).

Table 1.

Clinical associations of pathogenic and LP variants in the study cohort

Varianta	Frequency in general population (%)			ACMG/AMP classification		Reported clinical and experimental data	Case	Clinical presentation	Family history and targeted screening
	gnomAD	MCPS IMX	All of Us	ClinVar (accession)	This study
AIP (NM_003977.4)
c.713G>A, p.C238Y (rs267606569) germline	0.0001	n/a	n/a	LP (VCV000041197.9)	LP	Missense variant in exon 5. Results in an unstable protein with loss of multiple molecular interactions. Cosegregation proven in 1 family with FIPA (83)	1	Male, AR: 22 y. Gigantism: AO 14 y (accelerated growth, prognathism, headaches, DKA), Ad 18 y (macroadenoma); TSS (improvement, active tumor remnant), LAN 120 mg monthly (partial control), RT to tumor remnant planned. Lost to follow-up	Apparently sporadic. Family n/a
c.910C>T, p.R304* (rs104894195) germline	0.0014	n/a	0.0006	Pathogenic (VCV000004888.33)	P	Nonsense variant in exon 6. Most frequent germline AIP variant causing FIPA. Results in a truncated and unstable protein with loss of multiple molecular interactions (83)	2	Male, AR: 50 y. Gigantism: AO 15 y (accelerated growth), Ad 18 y (macroadenoma); TSS (18 y, improvement with tumor remnant, then recurrence), BEC (improvement, then tumor growth), and RT (remission with hypopituitarism, empty sella)	FIPA: 1 paternal first cousin (female) with young-onset acromegaly. F and 1 sister are apparently healthy variant carriers, 1 sister is not a carrier
MEN1 (NM_130799.3)
c.(?_-23-27)_(445 + 65_?)del germline	n/a	n/a	n/a	n/a	P	Large deletion spanning intron 1, exon 2, and intron 2. Not previously reported; predicted to cause LOF (truncated protein)	3	Female, AR: 21 y. MEN1: 1. Two panNENs, AO 19 y (recurrent severe hypoglycemia), Ad 19 y (17, 21 mm tumors); subtotal pancreatectomy (21 y, well-differentiated NET grade 1, 2 mitoses/10 HPF, Ki-67 2%, insulin + <1% cells; remission) 2. PHPT (AO/Ad 21 y, targeted biochemical screening); 3½ gland parathyroidectomy (21 y, remission, ddPCR: 1 copy of MEN1 exon 2 in fresh frozen tumor). Underwent uneventful pregnancy (22 y) and delivered a healthy baby	Apparently sporadic. M and brother are not variant carriers. Paternal family n/a
c.496C>T, p.Q166* germline	n/a	n/a	n/a	Pathogenic (VCV000618208.9)	P	Nonsense variant in exon 3. Predicted to cause LOF (truncated protein). Previously reported germline variant in individuals with MEN1 (84, 85) and as a somatic change in 1 porocarcinoma (86) and 1 somatotropinoma (87)	4	Male, AR: 42 y. MEN1: 1. PHPT, AO 25 y (recurrent nephrolithiasis), Ad 37 y 2. Meningioma (AO/Ad 30 y), surgically resected 3. PanNEN (AO/Ad 36 y); subtotal pancreatectomy	Familial MEN1: multiple cousins of F had PHPT. PGF died of prostate cancer, brother with recurrent UTIs. M and 2 sisters are not variant carriers. Paternal family n/a
c.660G>A, p.W220* (rs886039414) germline	n/a	n/a	n/a	Pathogenic (VCV000265235.14)	P	Nonsense variant in exon 4. Predicted to cause LOF (truncated protein). Previously reported in multiple families with MEN1 (88-90), including 1 with 2 thymic NEN cases (63) and 1 with a case of malignant melanoma (91), as well as in 1 panNEN (92)	5b	Male, AR: 43 y. MEN1: 1. Pancreatic head neuroendocrine carcinoma, AO/Ad 34 y (jaundice, 47 × 47 mm tumor); Whipple procedure (34 y, well-differentiated neuroendocrine pancreatic carcinoma, synaptophysin and CgA +, no metastases, Ki-67 20%, positive surgical margins; metastases 2 y later), LAN 90, then 120 mg monthly (since 40 y, improvement, progression 3 y later), 177Lu-DOTATATE (44 y, 7 cycles, stable stage IV). 2. PHPT (AO/Ad: 34 y, targeted screening); 3½ gland parathyroidectomy + thymectomy. 3. Thymic NEN: AO/Ad: 34 y (incidental finding during follow-up, regrowth in image study 10 y after resection) 4. PitNET, AO/Ad: 34 y (targeted screening, 2 mm). Developed CS (41 y), pending localization studies	Familial MEN1: affected M, younger brother (PHPT), 1 maternal uncle (PHPT and panNEN), 1 maternal aunt (PHPT and thymic NEN). Three maternal uncles with recurrent nephrolithiasis. One apparently healthy daughter, affected younger brother, and maternal aunt are variant carriers. M is obligate carrier. One brother and 1 sister tested negative for variant
c.936_938del, p.Y313del germline	n/a	n/a	n/a	n/a	LP	In-frame deletion in exon 7. Not previously reported; predicted to cause LOF	6	Female, AR: 38. MEN1: 1. Gastric NEN (AO/Ad: 36 y, GERD, polyp); endoscopic resection (gastric lesion: well-differentiated neuroendocrine tumor, grade 1 WHO, CKAE1-AE3 +, enolase +, synaptophysin + 100%, chromogranin + 100%, Ki-67 1%) 2. Multiple panNENs (AO/Ad: 37 y, incidental finding in CT scan, n = 7: 27, 16, 16, 6, 5, 11, and 6 mm); subtotal pancreatectomy 3. PHPT (AO/Ad: 37 y, targeted screening); 3½ gland parathyroidectomy (diffuse hyperplasia in 3 glands). LOH at variant locus in fresh frozen parathyroid tissue	Apparently sporadic. M and sister are not variant carriers. Paternal family n/a
c.984_985del, p.H328Qfs*38 germline	n/a	n/a	n/a	n/a	LP	Frameshift variant in exon 7. Not previously reported; predicted to cause LOF (truncated protein). Another variant with the same effect on the protein (c.984_990del, p.H328Qfs*38) was reported in 1 case of MEN1 (93). Other variants in the same residue have been associated with MEN1 (94)	7	Male, AR: 52 y. MEN1: 1. PHPT: AO 36 y (nephrolithiasis)/Ad: 40 y; resection of 1 and 2 glands in 2 surgeries, then resection of remnant gland with implant on forearm (1 gland with adenoma, 1 with hyperplasia) 2. PanNEN, AO/Ad: 40 y (recurrent severe hypoglycemia); subtotal pancreatectomy (multiple panNENs, the largest of 16 mm, glucagon +) 3. Prolactinoma, AO/Ad: 42 y (targeted screening, microadenoma); CBG	Apparently sporadic
NF1 (NM_001042492.3):
c.147C>A, p.Y49* germline	n/a	n/a	n/a	Pathogenic (VCV002114679.2)	P	Nonsense variant in exon 2. Predicted to cause LOF (truncated protein). Reported as a germline change in patients with NF1 (95-97) and at the somatic level in 1 case of ovarian cancer (98) and 1 of synchronous endometrial and ovarian cancer (99)	8	Male, AR: 54. NF1: 1. Multiple cutaneous neurofibromas 2. Café-au-lait spots 3. NF-PitNET (AO/Ad 54 y, headaches, hemianopsia, memory impairment and seizures, 16 × 15 × 15 mm tumor); TSS (54 y, PitNET, 0 mitoses/2 mm2, weak/hypogranular PAS, chromogranin +, ACTH, GH, and prolactin –, Ki-67 < 1%). No LOH at NF1 variant locus. Improved symptoms, tumor remnant not compromising vision or gland function	Familial NF1: café-au-lait spots in F, 8/10 siblings, and daughter. One nephew died (28 y) from complications of epilepsy. Affected daughter and 2 apparently healthy grandchildren are variant carriers.c History of endogamy. From small community with multiple affected individuals
RET (NM_020975.6)
c.1852T>C, p.C618R (rs76262710) germline	0.0001	n/a	n/a	Pathogenic (VCV000013905.89)	P	Missense variant in exon 10. Results in a protein with constitutive oncogenic activity and altered expression in vitro (100, 101). Reported as a germline change in multiple families with MEN2 and FMTC, proven cosegregation (102-105) and in cases of Hirschsprung disease (106). ATA-MOD (39)	9	Male, AR: 64 y. MEN2A: 1. PHEO, AO/Ad 33 y; surgical resection 2. MTC, AO/Ad 61 y; TT and selective neck dissection. Currently in remission	Familial MEN2A: M, sister and 1 son with MTC, 1 niece with PHEO. Affected son and niece, and apparently healthy daughter are variant carriers; 3 sisters and 1 brother are not carriersc
							10	Female, AR 35 y. MTC, AO 30 y (palpable neck mass)/Ad 35 y; TT and selective neck dissection (MTC with lymphatic, perineural, and muscle invasion, calcitonin +++, CEA +++, Tg –, Ki67 +++), radical neck dissection (persistent disease), RT	FMTC: F and 6 half-siblings with unspecified disease who required TT, 1 paternal first cousin (female) with MTC
SDHA (NM_004168.4)
c.2T>A, p.M1? (rs750380279) germline	0.0001	0.0016	0.0020	Pathogenic (VCV000582115.13)	P	Start loss variant in exon 1; predicted to cause LOF. Previously reported at the germline level in 1 patient with carotid body PGL (107). Other variants with the same effect have been reported in PPGLs (108)	11	Male, AR 59 y. MEN1: 1. Acromegaly, AO 36 y (acral growth), Ad 51 y (13 mm PitNET); LAN 120 mg monthly, TSS (active remnant), LAN 120 mg monthly + CBG, RT. 2. PHPT, AO/Ad 52 y (nephrolithiasis); right inferior parathyroidectomy + TT 3. Thyroid nodule, AO/Ad 52 y (finding during studies for PHPT); follicular adenoma	Apparently sporadic. F had nephrolithiasis; brother had liver neoplasm (primary?)
SDHD (NM_003002.4)
c.320T>G, p.L107R (rs876658477) germline	0.0001	0.0022	n/a	Conflicting classifications of pathogenicity: pathogenic, LP, VUS (VCV000230274.27)	LP	Missense variant in exon 4. In silico analyses: deleterious effect. Germline defect in 1 case of carotid body PGL (109). Different missense variant in this residue (p.L107P) reported in familial PPGLs, with proven cosegregation (110)	12	Female, AR 40 y. Bilateral carotid body PPGLs: AO 30 y (palpable neck masses, paroxystic high blood pressure), Ad 42 y (right 47 × 36 × 57 mm, left: 27 × 12 × 31 mm); RT, OCT LAR. Tumor growth in last evaluation	Family with cancer predisposition. Maternal uncle with tongue cancer, paternal aunt with Bc
VHL (NM_000551.4)
c.266T>C, p.L89P (rs5030807) germline	n/a	n/a	n/a	Pathogenic/LP (VCV000182979.12)	P	Missense variant in exon 1. Predicted to disrupt protein function. Previously reported as germline change in multiple individuals and families with VHL (111-123), including 1 family from Mexico (124). Reported in ccRCC as a germline (125) and somatic variant (126-129), and in CNS HB (130)	13	Male, AR 42. VHL: 1. Vermian cerebellar HB (AO/Ad 41 y) 2. Pulmonary nodule (27 × 33 × 24 mm) 3. Uncinate process panNEN (by PET-CT, no biopsy) 4. Bilateral renal cysts 5. Left ccRCC	Familial VHL: F died of bladder cancer; M with renal cyst
							14b	Female, AR 36 y. VHL: 1. Pancreatic adenocarcinoma, AO/Ad 22 y; Whipple procedure, chemo, RT 2. Left ccRCC, AO/Ad 31 y; partial left nephrectomy 3. Right ovary serose cystadenoma, AO/Ad 30 y (incidental finding during follow-up); resection of right adnexa 4. Right retinal hemangioma, AO/Ad 30 y (targeted screening) 5. Signet ring cell adenocarcinoma of gastro-jejunal anastomosis, AO 34 y (weight loss), Ad 35 y (24 × 45 mm); partial gastrectomy + open Roux-en-Y gastric bypass, chemo. Deceased	Familial VHL: MGM died of gastric cancer, affected M, maternal aunt, 4 maternal first cousins, brother, and sister are variant carriers; 1 unaffected maternal aunt and 1 sister are apparently unaffected variant carriers; 2 sisters and 1 brother are not carriersc
c.(?_464-50)_(*50_?)del germline	n/a	n/a	n/a	n/a	P	Large deletion spanning intron 2 and exon 3 up to 3′-UTR. Not previously reported; predicted to cause LOF (truncated protein)	15	Male, AR 38 y. VHL: Retinal angioma (AO/Ad 28 y) PanNEN (AO/Ad 29 y); subtotal pancreatectomy Renal cysts (AO/Ad 29 y); under surveillance Multiple intracranial HBs (AO/Ad 29 y) Pulmonary nodules (AO/Ad 29 y); under surveillance	Familial VHL: 1 sister, F, and paternal uncle with VHL; paternal aunt with renal tumors; paternal aunt with suspected kidney disease; PGM died of renal cancer; 4 brothers of PGM and 4 children of 1 of them with probable VHL. From small community with multiple affected individuals
USP8 (NM_005154.5)
c.2152T>C, p.S718P somatic	0.0001	0.0000	0.0000	Pathogenic (VCV000161992.1)	P	Missense variant in exon 14 hotspot. One of the most common USP8 variants in CD. Leads to loss of interaction with 14-3-3, increased USP8 cleavage/DUB activity, reduced EGFR degradation, and enhanced ACTH suppression with pasireotide in vitro (131)	16b	Female, AR 21 y. CD: AO 15 y (CS), Ad 21 y, (PitNET 8.4 × 4.3 × 3.6 mm). Treated with KTCZ (21 y), then TSS (21 y, densely granulated corticotropinoma, ACTH +, TSH, FSH, LH, prolactin, and GH –, CK1/AE3 +, Ki-67 < 2%; active tumor remnant); currently on KTCZ	n/a (somatic variant)

Abbreviations: AD, age at diagnosis; AO, age at onset; AR, age at recruitment; ATA-MOD, American Thyroid Association, moderate risk of MTC; BC, breast cancer; BEC, bromocriptine; CBG, cabergoline; ccRCC, clear cell renal cell carcinoma; CD, Cushing's disease; CEA, carcinoembryonic antigen; CNS, central nervous system; CS, Cushing's syndrome; CT, computed tomography; ddPCR, droplet digital polymerase chain reaction; DKA, diabetic ketoacidosis; DUB, deubiquitinase; F, father; FMTC, familial MTC; HB, hemangioblastoma; HPF, high-power fields; KTCZ, ketoconazole; LAN, lanreotide; LOF, loss-of-function; LP, likely pathogenic; M, mother; MEN, multiple endocrine neoplasia; MGM, maternal grandmother; MTC, medullary thyroid carcinoma; n/a, not available; NEN, neuroendocrine neoplasm; NET, neuroendocrine tumor; OCT LAR, octreotide long-acting release; P, pathogenic; panNEN, pancreatic NEN; PET, positron emission tomography; PGF, paternal grandfather; PGL, paraganglioma; PGM, paternal grandmother; PHEO, pheochromocytoma; PHPT, primary hyperparathyroidism; pitNET, pituitary neuroendocrine tumor; PPGL, pheochromocytoma and paraganglioma; RT, radiotherapy; Tg, thyroglobulin; TT, total thyroidectomy; TSS, transsphenoidal surgery; UTIs, urinary tract infections; UTR, untranslated region; VHL, Von Hippel-Lindau syndrome; VUS, variant of uncertain significance.

^a dbSNP ID in parentheses, if available.

^b Variant detection was done with Sanger sequencing.

^c Family screening in a different laboratory.

The 35 VUS were heterozygous, occurred in single cases, and affected 19 different genes (Table 2). Previous publications proposed a pathogenic effect for 4 of these variants. AIP p.R106C was detected in a patient with neuroendocrine gastric carcinoma and multiple gastric type 1 NENs (Case 17). This variant was previously reported in a patient with a prolactinoma and family history of PHPT (59). Also affecting AIP, p.V291_L292del was found in an individual with early-onset acromegaly in the setting of FIPA (Case 18), but the additional affected family member has not been tested. This variant was originally identified in a sporadic case of gigantism in a Mexican patient and is predicted to disrupt the packaging of the C-terminal α-helix of AIP (60). Regarding these cases, it must be noted that PitNETs account for the only known clinical association of germline LOF AIP defects. CDKN1B p.I119T was identified in a patient with sporadic gigantism (Case 19). Previous reports of this defect in the literature include 1 case of acromegaly within a FIPA family and 1 case of pediatric Cushing's disease (CD); CDKN1B LOF is the cause of MEN4 (26). Finally, the MEN1 p.M558del variant, included in ClinVar, was found in 1 case of sporadic MEN1 (Case 28). The region affected by this in-frame deletion is required for multiple molecular interactions and contains variants with a known pathogenic effect. Functional studies and testing of additional family members are required to accurately categorize these genetic changes.

Table 2.

VUS identified via NGS panel in the study cohort

Varianta	Frequency in general population (%)			ACMG/AMP classification		Case	Clinical presentation (family history)
	gnomAD	MCPS IMX	All of Us	ClinVar (accession)	This manuscript
AIP (NM_003977.4)
c.316C>T, p.R106C (rs369414668)	0.0075	0.0027	0.0043	VUS (VCV000485059.10)	VUS	17	Neuroendocrine gastric carcinoma and multiple gastric type 1 NENs (sporadic)
c.872_877del, p.V291_L292del	n/a	n/a	0.0004	n/a	VUS	18	Early-onset acromegaly (FIPA)
CDKN1B (NM_004064.5)
c.356T>C, p.I119T (rs142833529)	0.0706	0.0016	0.0722	Conflicting interpretations of pathogenicity: VUS, benign, LB (VCV000307664.28)	VUS	19	Gigantism (sporadic)
c.482C>T, p.S161F (rs373917399)	0.0007	0.0547	0.0014	Conflicting interpretations of pathogenicity: VUS, LB (VCV000582241.11)	VUS	20b	Insulinoma (familial cancer predisposition)
FH (NM_000143.4)
c.1431_1433dup, p.K477dup (rs367543046)	0.1915	n/a	0.1373	Conflicting interpretations of pathogenicity: pathogenic, LP, VUS (VCV000042095.81)	VUS	21	Acromegaly (suspected FIPA)
GNAS (NM_000516.7)
Upstream from NM_000516.7; c.*42 + 12949T>C, p.? in NM_016592.5	n/a	0.0060	0.0004	VUS (VCV002355759.2)	VUS	22	Bilateral carotid body PGL (Li–Fraumeni-like)
Upstream from NM_000516.7; c.*42 + 13032C>T, p.? in NM_016592.5 (rs1354669859)	0.0006	0.0026	0.0008	n/a	VUS	23	MEN-like: ccRCC, left adrenal tumor, 2 panNENs, interhemispheric lipoma, multiple colorectal adenomas (familial cancer predisposition)
Upstream from NM_000516.7;c.*42 + 13400G>A, p.? in NM_016592.5 (rs777234998)	0.0013	n/a	0.0004	n/a	VUS	24	MEN-like: right testicular non-seminomatous germ cell tumor and left adrenal PHEO (sporadic)
KIF1B (NM_001365951.3)
c.1898G>A, p.R633H (rs1650295604)	0.0001	0.0016	n/a	VUS (VCV001779575.2)	VUS	25	NF-PitNET, PHPT, PTC, uterine fibroids and benign left breast tumor (MEN1, sporadic)
MAFA (NM_201589.4):
c.109G>A, p.E37K (rs529585367)	0.0006	n/a	0.0018	n/a	VUS	26	Gigantism (sporadic)
MDH2 (NM_005918.4)
c.236-4C>G, p.? (rs1554586397)	0.0002	n/a	0.0004	n/a	VUS	27	Multiple gastric NENs (sporadic)
MEN1 (NM_130799.3)
c.1673_1675del, p.M558del	n/a	n/a	n/a	VUS (VCV001777737.2)	VUS	28	MEN1: insulinomatosis, GH and prolactin-secreting PitNET, multiple parathyroid adenomas (familial MEN1)
MERTK (NM_006343.3)
c.1396_1397delinsGA, p.R466E (rs386649210)	n/a	n/a	n/a	VUS (VCV001414697.5)	VUS	20b	See above
c.2590G>A, p.V864I (rs557004700)	0.0025	0.0358	0.0043	VUS (VCV000893540.6)	VUS	29	MEN-like: PHPT and left aldosterone-producing ACA (sporadic)
c.2674C>G, p.P892A	0.0001	0.0011	n/a	n/a	VUS	30b	MEN1: NF-PitNET and PHPT (sporadic)
c.2689A>G, p.I897V (rs775526399)	0.0001	0.0077	0.0004	n/a	VUS	31	MEN-like: meningioma and panNEN (sporadic)
c.2947G>A, p.D983N (rs781065147)	0.0128	0.0463	0.0137	VUS (VCV001504094.6)	VUS	32c	MEN-like: NF-PitNET and multiple lipomas (sporadic)
MET (NM_000245.4)
c.*23C>G, p.? (rs980940721)	0.0008	n/a	0.0055	n/a	VUS	33	MTC (sporadic)
MSH6 (NM_000179.3)
c.30C>A, p.F10L (rs786201869)	0.0001	n/a	n/a	VUS (VCV000628563.10)	VUS	32c	See above
c.1601A>T, p.N534I (rs1572723598)	n/a	0.0097	n/a	VUS (VCV000819644.8)	VUS	3d	See Table 1
c.2331G>T, p.W777C (rs876660037)	0.0001	0.0021	n/a	VUS (VCV001428821.6)	VUS	34	Bilateral carotid body PGL (sporadic)
c.3059A>G, p.N1020S	n/a	0.0005	n/a	VUS (VCV001006155.7)	VUS	32c	See above
PDGFRA (NM_006206.6)
c.-13 + 5029G>C (rs546074770)	n/a	0.0151	0.0002	n/a	VUS	32c	See above
c.1432_1436delinsCCCCA, p.S478_R479delinsPQ	n/a	n/a	n/a	n/a	VUS	35	PHPT, PHEO (MEN2A, sporadic)
c.1631T>C, p.V544A (rs181854060)	0.0084	0.0011	0.0334	Conflicting interpretations of pathogenicity: LP, VUS, LB (VCV000039617.19)	VUS	24	See above
c.2323 + 1243G>T (rs1052140725)	0.0013	n/a	0.0059	n/a	VUS	26	See above
c.3221_3222delinsGC, p.D1074G	n/a	n/a	n/a	VUS (VCV000950280.5)	VUS	36	Early-onset prolactinoma (sporadic)
PMS2 (NM_000535.7)
c.209A>G, p.D70G (rs1554305000)	n/a	0.0003	0.0002	VUS (VCV000484322.15)	VUS	37	Gastrinoma (sporadic)
c.1139T>G, p.V380G (rs748971523)	0.0002	0.0272	0.0008	VUS (VCV000630207.14)	VUS	38	Early-onset acromegaly (sporadic)
PTEN (NM_000314.8)
c.-348G>C	n/a	n/a	0.0002	n/a	VUS	30b	See above
SDHA (NM_004168.4)
c.1396G>A, p.A466T (rs111387770)	0.2992	n/a	n/a	Conflicting interpretations of pathogenicity: VUS, benign, LB (VCV000472327.21)	VUS	2d	See Table 1
SDHC (NM_003001.5)
c.180-3C>T (rs1328389180)	0.0001	0.0027	0.0002	VUS (VCV001331275.7)	VUS	36	See above
TMEM127 (NM_017849.4)
c.*542C>G, p.?	0.0047	n/a	0.0075	n/a	VUS	24	See above
TP53 (NM_000546.6)
c.871A>G, p.K291E (rs1555525126)	0.0001	0.0162	0.0004	VUS (VCV000480951.17)	VUS	39	Right carotid body PGL (Li–Fraumeni-like)
VHL (NM_000551.4)
c.65A>G, p.E22G (rs2125124570)	0.0001	0.0114	0.0004	VUS (VCV001403440.10)	VUS	40	MTC (sporadic)

Abbreviations: ACA, adrenal cortex adenoma; ccRCC, clear cell renal cell carcinoma; FIPA, familial isoalted pituitary adenoma; LB, likely benign; LP, likely pathogenic; MEN, multiple endocrine neoplasia; MEN-like, syndrome similar to MEN; MTC, medullary thyroid carcinoma; n/a, not available; NEN, neuroendocrine neoplasm; NF-PitNET, non-functional PitNET; panNEN, pancreatic NEN; PGL, paraganglioma; PHEO, pheochromocytoma; PHPT, primary hyperparathyroidism; PitNET, pituitary neuroendocrine tumor; PTC, papillary thyroid carcinoma; VUS, variant of uncertain significance.

^a dbSNP ID in parentheses, if available.

^b Two coexisting VUS in different genes.

^c Four coexisting VUS in 4 different genes.

^d This patient carries a causative variant in a different gene.

Since the association of CABLES1 variants with CD has been proposed, but not confirmed, an ACMG/AMP category was not assigned to 6 variants identified in this gene (61). Out of them, 3 rare variants were detected in Case 25 (p.S17R), Case 27 (p.A155S), and in a patient with MEN2A and negative RET screening (p.R412W). The variant p.E178 K, which affects the ability of CABLES1 to reduce cell proliferation in vitro, was found in Case 11. This individual carried a different germline variant that could explain the phenotype (see “Genotype–Phenotype associations”). Such variants could represent disease modifiers, although further research is required to test this hypothesis.

Screening in Additional Family Members

For 6 cases with variants of interest, 21 additional family members were recruited for the study. Nineteen of them underwent targeted screening to determine their carrier status, using Sanger sequencing (n = 19) or ddPCR (n = 2). Five variant carriers were identified in 3 families, 3 carrying AIP variants (c.910C>T, p.R304* in 2 individuals and p.V291_L292del in 1), and 2 members of 1 family carrying MEN1 p.W220*. The AIP variant carriers are 77, 75, and 46 years old and are apparently unaffected. Biochemical and imaging testing was advised only for the youngest patient and remains pending. Both MEN1 variant carriers, aged 41 and 56 years at recruitment, underwent biochemical and imaging testing and were found to be affected with PHPT and PHPT and thymic NEN, respectively.

Genotype–Phenotype Associations

Fifteen out of the 16 patients with P/LP variants had clinical presentations previously associated with the affected genes. As expected, three-quarters of patients with clinical VHL (75%) carried pathogenic VHL variants (Table 3). In line with the high penetrance of this entity, all cases had positive family history. The only patient with clinical VHL and negative genetic testing has also a history of hereditary hemorrhagic telangiectasia, but no other relatives with VHL-associated features. A defect in a gene different than VHL (perhaps ELOC) might be the underlying cause of his phenotype (62).

Table 3.

Genetic findings in probands according to clinical categories

Clinical categories	Genetic findingsa			Total, no. (%)
	P/LP, no. (%)	VUS, no. (%)	No VOI, no. (%)
Recruitment groups b
Sporadic isolated NENs	2 (6.9)	10 (34.5)	17 (58.6)	29 (100)
Familial isolated NENs	3 (11.5)	5 (19.2)	18 (69.2)	26 (100)
Familial or sporadic syndromic NENs	11 (29.7)	9 (24.3)	17 (45.9)	37 (100)
Total:	16 (17.4)	24 (26.1)	52 (56.5)	92 (100)
Clinical presentation in probands
Ectopic Cushing's syndrome	—	—	2 (100)	2 (100)
Gigantism or early onset acromegaly	2 (25)	4 (50)	2 (25)	8 (100)
MEN-like	—	5 (38.5)	8 (61.5)	13 (100)
MEN1	6 (37.5)	3 (18.8)	7 (43.8)	16 (100)
MEN2A	1 (33.3)	1 (33.3)	1 (33.3)	3 (100)
MTC	1 (12.5)	2 (25)	5 (62.5)	8 (100)
Multiple GI-NENs	—	1 (50)	1 (50)	2 (100)
Multiple panNENs	—	1 (100)	—	1 (100)
Multiple PPGLs	1 (10)	2 (20)	7 (70)	10 (100)
NF1	1 (100)	—	—	1 (100)
Early onset PitNETs not causing GH excess	1 (7.7)	1 (7.7)	11 (84.6)	13 (100)
PanNEN	—	2 (40)	3 (60)	5 (100)
PitNET	—	1 (25)	3 (75)	4 (100)
Single PPGL	—	1 (50)	1 (50)	2 (100)
VHL	3 (75)	—	1 (25)	4 (100)
Total:	16 (17.4)	24 (26.1)	52 (56.5)	92 (100)
Family history in probands
Sporadic	4 (8.5)	18 (38.3)	25 (53.2)	47 (100)
Cancer predisposition	1 (5)	2 (10)	17 (85)	20 (100)
FIPA	1 (16.7)	2 (33.3)	3 (50)	6 (100)
Familial MTC	1 (100)	—	—	1 (100)
Familial PPGLs	—	—	1 (100)	1 (100)
Li Fraumeni-like	—	2 (100)	—	2 (100)
MEN-like	—	—	5 (100)	5 (100)
MEN1	4 (80)	—	1 (20)	5 (100)
MEN2A	1 (100)	—	—	1 (100)
NF1	1 (100)	—	—	1 (100)
VHL	3 (100)	—	—	3 (100)
Total:	16 (17.4)	24 (26.1)	52 (56.5)	92 (100)

Abbreviations: FIPA, familial isolated pituitary adenoma; FPPGL, familial pheochromocytoma and paraganglioma; GI-NEN, gastrointestinal NEN; MEN, multiple endocrine neoplasia; MEN-like, syndrome similar to multiple endocrine neoplasm; MTC, medullary thyroid carcinoma; NEN, neuroendocrine neoplasm; NF1, neurofibromatosis type 1; P/LP, pathogenic/likely pathogenic; panNEN, pancreatic NEN; PPGL, pheochromocytoma and paraganglioma; VHL, Von Hippel-Lindau syndrome; VOI, variant of interest; VUS, variant of uncertain significance.

^a Individuals with more than 1 type of variant were included in the most clinically relevant category.

^b Clinical presentation in the family (according to available data) was used to classify cases as isolated or syndromic.

Regarding clinical MEN1, 5 out of 16 patients (31.3%) carried P/LP MEN1 variants, and 1 carried a novel VUS, accounting for 37.5% of patients with this phenotype if both P/LP and VUS are considered. All these patients developed panNENs and PHPT and all, except the 2 youngest ones, had also PitNETs. Interestingly, in Case 5, both the proband and another carrier of MEN1 p.W220* were diagnosed with thymic NEN, a rare occurrence that has been previously associated with this variant (63). Out of the 10 patients with clinical MEN1 and negative MEN1 screening, only 1 individual presented with the association of panNEN, PitNET, and PHPT, while the rest developed PitNETs (6 nonfunctioning PitNETs, 1 somatotropinoma, and 1 corticotropinoma) and PHPT, but no panNENs. One of these individuals (Case 11) carried a pathogenic SDHA variant with loss of the start codon. This patient presented with acromegaly and PHPT, a particular combination of components of MEN1 that usually tests negative for defects in MEN1-associated genes (64). To our knowledge, this is the first report of a patient with clinical MEN1 associated with this genetic defect. Two other MEN1-negative patients, Cases 25 and 27, carried VUS in KIF1B (p.R633H) and MERTK (p.P892A), respectively. These genes have not been associated with the genotype before.

P/LP AIP variants were identified in 2 out of 8 individuals with early-onset acromegaly or gigantism (25%). If 2 additional patients carrying a VUS of interest in AIP (p.V291_L292del) and CDKN1B (p.I119T) are considered, the frequency of variants of interest in this group rises to 50%. Out of these 4 cases, 2 presented in the setting of FIPA and 2 had sporadic gigantism. Among the 4 remaining cases, Case 38 (sporadic young-onset acromegaly) carried a PMS2 VUS and Case 26 (sporadic gigantism) had VUS in 2 genes not associated with his phenotype. The remaining 2 patients (sporadic gigantism and early-onset acromegaly with family history of cancer predisposition) had no variants of interest identified. In the subgroup of young onset PitNETs not secreting GH, only 1 out of 13 individuals (7.1%) had a clear tumor driver (somatic USP8 variant). One patient (Case 36, early-onset prolactinoma) carried 2 VUS in genes not previously associated with the phenotype; the rest had negative screening. None of the patients with isolated PitNETs with age at onset >30 years (n = 4) carried P/LP variants but Case 21 carries a VUS in FH, a gene not associated with this phenotype. Interestingly, a pathogenic NF1 variant was found in a single patient with NF1 who developed a PitNET, which is not a typical component of the syndrome

Among patients presenting with MEN2A (n = 3) or MTC (n = 8), 2 carried a pathogenic RET variant, 1 with familial MEN2A and 1 with FMTC. Among the 9 remaining subjects, 8 presented sporadically and 1 had MEN-like family history. Three of these individuals carried VUS in genes not previously associated with their phenotypes: Case 33 (sporadic MTC, MET 3′-UTR variant), Case 35 (sporadic MEN2A, PDGFRA p.S478_R479delinsPQ), and Case 40 (sporadic MTC, VHL p.E22G).

One out of 10 individuals with multiple PPGLs analyzed carried a pathogenic variant in SDHD (p.L107R). Two individuals carried VUS in GNAS and MSH6; these genes are not associated with the phenotype. None of the patients with single PPGLs carried P/LP variants, but 1 patient with a Li–Fraumeni-like family history carried a VUS in TP53 (p.K291E). Unfortunately, in this case no samples from tissues or additional family members were available for study.

Finally, no P/LP variants were identified for isolated single or multiple panNENs (n = 6), multiple GI-NENs (n = 2), MEN-like phenotypes (n = 13), or ectopic Cushing's syndrome (CS) (n = 2). VUS were found in 2 patients with GI-NENs (Cases 17 and 27), including the abovementioned AIP p.R106C, and an intronic MERTK variant. Also, 2 patients with panNENs had VUS. Case 37 (sporadic gastrinoma) carried PMS2 p.D70G and Case 20 carried 2 variants: MERTK p.R466E and CDKN1B p.S161F. Regarding the latter variant, a different missense change in the same residue (p.S161C) was reported as a germline defect in a recently published case of multiple panNENs, but there are no available functional or cosegregation data for this variant (65). Among individuals with MEN-like presentations, VUS were detected in 5 individuals: an intronic GNAS variant was identified in Case 23, Cases 29 and 31 carried MERTK variants, Case 24 had 1 PDGFRA and 1 TMEM127 VUS, and Case 32 carried variants in MERTK, MSH6 (2 variants), and PDGFRA. Additional validation is required to determine the role of the identified variants on this heterogeneous phenotype.

Patients with a familial presentation were significantly more likely to carry a P/LP variant, compared with sporadic cases (26.7 vs 8.5%, odds ratio 3.9 [1.2-11.7], P = .0282). P/LP variants were detected in all patients with familial VHL (n = 3), familial NF1 (n = 1), familial MEN2A (n = 1), and FMTC (n = 1), as well as 4 out of 5 cases of familial MEN1, 1 out of 6 cases of FIPA, and 1 out of 20 cases with cancer predisposition. No P/LP variants were found in cases with family history of MEN-like (n = 5), Li–Fraumeni-like (n = 2), and familial PPGLs (n = 1). Therefore, a positive family history for the specific phenotype present in the proband is a strong predictor of P/LP variants.

Discussion

We developed and validated a custom designed NGS panel that can be applied for the genetic diagnosis of multiple types of NENs. Three main characteristics distinguish this research platform from available routine NGS panels for the different categories of NENs included in the study. First, the selection of genes is designed to cover the most frequent, although not all, germline and somatic defects involved in these tumors. This design allowed for a “medium sized” panel, thus reducing sequencing costs and simplifying data analyses. Second, 2 genes of uncertain significance with a suggested, but not fully proven, role in the pathogenesis of corticotropinoma (CABLES1 and RASD1) were included; genes of uncertain significance are not typically part of routine panels (66). Third, cancer-related genes without a known implication in NENs are not covered, thus reducing the possibility of reportable secondary findings. We believe that this simplified strategy for genetic testing for multiple types of NENs will be useful for widespread routine use.

Variant classification in this population was challenging due to the finding of variants that are extremely rare in other human groups. Fortunately, during the course of the study, a large dataset of genetic variants in general Mexican population, MCPS, became available (41). Also, in the last years, the Hispanic/Latino subset of samples in the All of Us project has considerably grown (50). These data greatly eased our work, thus highlighting the importance of population-matched reference databases for variant interpretation. Our own dataset was also useful for variant classification, even though the cohort is still small. Remarkably, 4 out of 14 P/LP variants (3 in MEN1 and 1 in VHL) have not been reported before. As the number of participants grows, it will become clear whether these population-specific variants display a founder effect. Also, 11 variants not listed in ClinVar were classified as benign due to their high frequency in MCPS and/or Hispanic individuals from All of Us. Four other defects absent from ClinVar were classified as LB based on in silico prediction plus frequency ≥5% in our dataset. Finally, DICER1 c.4206+9_4206+21delinsT, reported in ClinVar as a VUS, was reclassified as LB due to the high allele frequency observed in our samples (18.3%, including 3 homozygous individuals).

Most of the clinical associations of the genetic defects identified in our study are well-known, and in most cases our findings align with previous studies. For instance, LOF MEN1 variants explain 90% of MEN1 cases with positive family history, but only one-third of those with sporadic presentation; therefore, these defects drive 70% to 90% of all MEN1 cases (67-70). We detected P/LP MEN1 variants in one-third of all probands with MEN1: 12.5% (1/8) of those with sporadic presentation and 50% (4/8) of those with positive family history. An additional sporadic MEN1 case was associated with an MEN1 VUS that requires further validation. To our knowledge, there are no previous reports of the frequency of MEN1 variants in Mexico.

In our cohort, all individuals with P/LP MEN1 variants developed panNENs and PHPT, while none of the patients with the combination of PitNET and PHPT carried MEN1 defects. As pointed out by previous studies, such cases are usually not caused by MEN1 defects, suggesting that other genetic drivers might be involved. Interestingly, 1 of these patients carried a pathogenic variant in SDHA. Germline SDHA LOF underlies the syndrome of hereditary PPGLs 5 (MIM # 614165), and less frequently causes GISTs and PitNETs, either isolated or coexisting with PPGLs (71-73). Due to low penetrance, SDHA-driven tumors are usually apparently sporadic (74). The coexistence of PHPT with acromegaly in an SDHA variant carrier has not been described before and causality for PHPT requires further confirmation. Regardless of this atypical presentation, targeted screening for additional SDHA-associated tumors will be required in this individual.

One-quarter of cases of early-onset acromegaly or gigantism, including 16.7% of FIPA, were caused by AIP LOF variants. Previous studies have reported a frequency of 29% among cases of gigantism, 6% to 20% of young sporadic PitNETs, and 15% to 20% of FIPA families (13, 38, 75-77). A single individual with MEN2A, representing one-third or the MEN2A patients in the study as well as the only 1 with familial disease, carried a well-characterized RET variant. The same defect was found in 1 patient with MTC, accounting for 12.5% of all MTC cases in the study and the only one with history of FMTC. Almost all cases of MEN2A and FMTC and around one-fifth of all cases of sporadic MTC are due to RET germline defects; variants in the same gene are found as somatic defects in around 47% of MTCs (12, 19). The 2 probands with MEN2A and negative RET screening presented sporadically. These cases could be explained by a coincidental association of NENs similar to MEN2A, or an unknown genetic syndrome.

The low frequency of SDHx variants among patients with multiple PPGLs is intriguing. Only 1 of these individuals carried an SDHD variant and had a family history of cancer predisposition. Carriers of germline LOF SDHD variants are at risk for familial predisposition to PPGLs (MIM # 168000), GISTs, renal cell carcinoma, PitNETs, and Carney–Stratakis syndrome (MIM #60686) (20, 71-73, 78, 79). The genetic origin of PPGLs involves more than 20 genes (all included in our NGS panel), but around half of cases remain without a known germline defect (23, 25). Additional unrecognized genes could be involved in the origin PPGLs in our study participants, since they belong to a population that has not been genetically characterized before. Also, no P/LP variants were identified for isolated single or multiple panNENs, multiple GI-NENs, MEN-like phenotypes, or ectopic CS. While a fraction of panNENs and GI-NENs are due germline genetic causes, ectopic CS and MEN-like are heterogeneous entities with unclear genetic backgrounds (15, 17). Additional analyses including exome/genome-wide paired sequencing of blood and tumor samples should be useful to shed light on these cases.

Our data confirm previous findings showing that NENs are part of a variety of syndromic and nonsyndromic presentations with overlapping phenotypes and genetic drivers. Nevertheless, in our cohort, three-quarters of patients with positive family history, and therefore with a high likelihood of carrying germline defects, remain without genetic diagnosis. The discovery of genetic drivers of NENs in these cases will likely require a combination of multiple technologies for genetic analyses, such as comparative genomic hybridization, and long-read exome/genome sequencing (80). Given that the number of known drivers of NENs is expected to continue growing, including the name of the affected genes in the nomenclature of these phenotypes would be advisable to improve clarity, when possible (81). This precision will become more important as strategies for personalized medicine in this field are incorporated into the clinical routine toolbox. Furthermore, future research efforts should prioritize VUS reclassification, via clinical evaluation and experimental assessment (82).

The limitations of the study are duly recognized. At this stage, our work sought to optimize the sequencing protocol and to validate the performance of the NGS panel for germline analyses. Therefore, tissue samples were analyzed for only a few cases in which either hotspot variants or LOH at the germline variant loci were investigated. As this prospective study progresses, we expect to obtain both fresh frozen tumor and blood samples from participants to perform paired analyses. This is particularly important for phenotypes that are often caused by well-characterized somatic drivers, such as MTC, PPGLs, and corticotropinomas. We are also actively recruiting additional family members, which will allow for investigating cosegregation as well as for a fine characterization of phenotype–genotype correlations. Finally, based on our results, we expect to refine the list of genes included in the panel, to maximize its diagnostic potential.

Conclusion

An NGS panel for genetic diagnosis in patients with multiple types of NENs has been designed and validated. Using this tool, we determined, for the first time, the frequency and type of genetic defects underlying multiple phenotypes of NENs in the Mexican population, including SNVs, indels, and SVs. Novel P/LP variants and VUS have been described. In addition, we described the association of a pathogenic SDHA defect with MEN1 in a single individual. Our findings will hopefully contribute to improve the access of the general population to platforms for routine genetic testing and simplify the interpretation of genetic tests in ours and other populations. Finally, our ongoing work on this prospective cohort will seek to determine how genetic variability among human populations might shape the clinical phenotypes and outcomes in patients with monogenic causes of NENs.

Abbreviations

CD

Cushing's disease

CS

Cushing's syndrome

dd

droplet digital

FFPE

formalin-fixed and paraffin-embedded

FIPA

familial isolated pituitary adenoma

FMTC

familial MTC

GH

growth hormone

GI-NEN

gastrointestinal NEN

GIST

gastrointestinal stromal tumor

LB

likely benign

LOF

loss of function

LOH

loss of heterozygosity

MEN

multiple endocrine neoplasia

MTC

medullary thyroid carcinoma

NEN

neuroendocrine neoplasm

NF1

neurofibromatosis type 1

NGS

next-generation sequencing

panNEN

pancreatic NEN

PCR

polymerase chain reaction

PHPT

primary hyperparathyroidism

PitNET

pituitary neuroendocrine tumor

P/LP

pathogenic or likely pathogenic

PPGL

pheochromocytoma and paraganglioma

PTC

papillary thyroid carcinoma

SNV

single nucleotide variant

SV

structural variant

UTR

untranslated region

VHL

Von Hippel–Lindau syndrome

VUS

variant of uncertain significance

Funding

Our research was supported by departmental funding from the Coordination of Scientific Research from Universidad Nacional Autónoma de México (UNAM), research grants from UNAM's Support Program for Research Projects and Technological Innovation (UNAM-PAPIIT, projects TA200322 and TA200524) and Sociedad Mexicana de Nutrición y Endocrinología, and an equipment grant from the United Kingdom's Society for Endocrinology.

Disclosures

The authors declare no conflicts of interest concerning the contents of this manuscript.

Data Availability

Sequencing data have been deposited to NCBI BioProject. Original deidentified research data pertaining to this study are available upon reasonable request by contacting L.C.H.R.

Ethics Declaration

Study participants provided written informed consent and were recruited under a protocol approved by the IRBs of Hospital de Especialidades de Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, respectively (protocol IDs R-2022-785-011 and 4090, respectively; ClinicalTrials.gov: NCT06523582).

Clinical Trial Information

R-2022-785-011 (Hospital de Especialidades de Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, approved March 23, 2022), 4090 (Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, approved April 13, 2022), and NCT06523582 (ClinicalTrials.gov, uploaded July 26, 2024).