Introduction

IL-12Rβ1 deficiency, also known as immunodeficiency 30 (IMD30, OMIM 614891), is an autosomal recessive disorder caused by biallelic mutations in IL12RB1. To date, 156 patients have been described [1, 2]. Interleukin-12 (IL-12) plays an important role in the interaction between the innate and adaptive immunity. Phagocytic cells and dendritic cells produce this cytokine after an encounter with pathogens. IL-12 is involved in the cytotoxic activities of T cells and NK cells and is important for the production of cytokines, especially interferon (IFN) gamma [3, 4]. The receptor for IL-12 on NK- and T-cells is composed of two chains, IL-12 receptor beta-1 (IL-12Rβ1) and IL-12 receptor beta-2 (IL-12Rβ2). IL-12Rβ1 is primarily responsible for binding, while IL-12Rβ2 is essential for signaling through the JAK–STAT pathway [5, 6]. Patients with biallelic inactivation of IL-12Rβ1 are extremely susceptible to severe infections caused by otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis BCG) and Salmonella spp. [7, 8].

Three relatives of a patient with IL-12Rβ1 deficiency, caused by a homozygous truncating mutation in IL12RB1 [8], developed gastric cancer (GC). In the Netherlands, the incidence of non-cardia GC is only 14 per 100,000 person years for men and 7 per 100,000 person years for women [9]. In its early stages GC is often asymptomatic or causes only nonspecific symptoms. By the time symptoms occur, the cancer has often reached an advanced stage, which is one of the main reasons for the low average 5-year survival [9].

According to the Laurén classification, GC can be roughly divided into three histopathological types; diffuse, intestinal and mixed/indeterminate type [10]. Another commonly used classification of GC is the classification of the World Health Organization (WHO), that recognizes five main types of GC, namely tubular, papillary, mucinous, poorly cohesive (including signet-ring cell type) and mixed carcinomas [11]. Gastric cancer is a multifactorial disease, both genetic alterations and environmental factors play a role in GC development. The main environmental factor involved is infection with Helicobacter pylori (H. pylori) and this pathogen has been recognised as a carcinogen by the WHO in 1994 [12, 13].

Familial aggregation of GC is estimated to occur in 8–30 % of the patients [14–16]. The most important GC susceptibility gene is CDH1, which accounts for 1–3 % of all GC [17]. CDH1-associated GC is mainly of the diffuse-type. The criteria to test patients/families with GC for mutations in this gene include: (1) 2 or more GC cases in family, one DGC <50 years; (2) 3 or more DGC cases in 1st- or 2nd- degree relatives, regardless of age; (3) DGC <40 years and (4) personal or family history of DGC and lobular breast cancer, one diagnosed <50 years [18]. CDH1 mutations have been encountered in up to 50 % of strictly selected families [19–21]. The remaining families are still genetically unexplained and may carry mutations in other, still to be identified, GC susceptibility genes.

To date, there is no literature on subjects with heterozygous and/or homozygous mutations in IL12RB1 and IL12RB2 and gastric cancer. A few studies have been reported about mutations in these genes in esophageal cancer. Cardenes et al. reported a subject with a homozygous splice site mutation in IL12RB1 who developed esophageal squamous cell carcinoma at the age of 25, which is extremely young for this type of cancer. Therefore, the authors speculate on the possible role of a defective IL-12Rβ1 protein underlying this malignancy [22]. This case is also mentioned in an extensive study of 141 patients with IL-12Rβ1 deficiency, in which this patient is the only patient who developed cancer. However, it is unclear whether systematic analysis for the occurrence of tumors was performed [2, 22]. Tao et al. [23] also reported an association between IL12RB1 and esophageal cancer. In their study, they found that the CC genotype of a single nucleotide polymorphism (SNP) in IL12RB1 (rs401502, indicated in the article as 378 C/G) was associated with increased IL-12p40 levels and protection from esophageal cancer susceptibility. Airoldi et al. [24] describe the consequence of lack of Il-12 signaling in mice, they observed that Il12rb2 homozygous knock-out mice are prone to develop tumors of the lung epithelia.

To determine whether IL12RB1 and IL12RB2 can be considered candidate genes for GC susceptibility, we analyzed whether the GC patients in the family with IL-12Rβ1 deficiency were carriers of this mutation and tested 29 GC patients that were suspected for a genetic predisposition based on their personal and/or familial GC history for germline mutations in these genes.

Materials and methods

Patient samples

DNA was isolated from peripheral blood samples and formalin-fixed paraffin-embedded tumor material of two GC patients from the family with IL-12Rβ1 deficiency to test for the familial mutation at the department of Infectious Diseases of the Leiden University Medical Center, Leiden, the Netherlands.

For mutation analysis of IL12RB1 and IL12RB2, DNA was isolated from peripheral blood samples from GC patients who were tested negative for CDH1 mutations at the department of Human Genetics of the Radboud university medical center, Nijmegen after genetic counseling at the Radboud university medical center or the Maastricht University Medical Center, Maastricht, both in the Netherlands. Because of the relatively high age of the GC patients in the IL12RB1 family, no further selection was made based on age and/or family history. Patient characteristics, including H. pylori status, can be found in Table 1.

Table 1 Characteristics of patients screened for mutations in IL12RB1 and IL12RB2
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LOH analysis by pyrosequencing

To isolate DNA from formalin-fixed paraffin-embedded tissue, thin sections were treated by initial xylol extraction to remove paraffin. The extracted tissue was incubated overnight at 37 °C in 600 µl nuclei lysis solution (Promega) supplemented with 400 µg pronase, followed by addition of protein precipitation solution (Promega), incubation for 5 min on ice, and centrifugation to remove proteins. Supernatant was incubated on ice with an equal volume of isopropanol to precipitate the DNA, the pellet was dissolved in TE and further purified with a QIAquick gel extraction kit (Qiagen). PCR and pyrosequencing was essentially performed according to the protocol described previously [25], primers used for the PCR are ps-F: 5′-CTCCCCTCTCCTTCCAGAAC and ps-R: biotine labeled 5′-TTCCAGGCCATTACCCATT. Pyrosequencing primer ps-seq: 5′-TGGCSGCCTGTGGT.

Mutation analysis of IL12RB1 and IL12RB2

The full coding sequence of IL12RB1 (transcript numbers NM_005535.1 and NM_153701.1) and IL12RB2 (transcript number NM_001559.2) including splice junctions was amplified using polymerase chain reaction (PCR) and screened for mutations using Big-Dye terminator sequencing (BigDye Terminators (v 1.1) Applied Biosystems, USA) and analysis on an ABI 3730 DNA Analyzer (Applied Biosystems). Subsequently, data was analyzed for variants using the Sequence Pilot software (JSI Medical Systems, Germany).

Missense variants were analyzed using the Alamut 2.0 software package (Interactive Biosoftware, Rouen, France), which incorporates SIFT [26], PolyPhen-2 [27], Align GVGD [28] and dbSNP (build 135). We used the Exome Variant Server of the University of Washington [29], which contains sequencing data of approximately 6,500 individuals of European and African descent, and the database of the GoNL project [30] to assess whether variants were present in individuals without GC.

Results

Gastric cancer patients from the family with the truncating mutation in IL12RB1

A 73-year old patient (patient A), recently diagnosed with GC, was referred because of a family history of GC. One of her sisters had been diagnosed with GC at age 72 (patient B) and their mother had died of GC at age 70. Her 62-year old sister was healthy and had a medical history of a few rectal polyps (three tubular adenomas with low grade dysplasia and two hyperplastic). Her niece (daughter of the 62-year old sister) was known to have a biallelic mutation (c.1126C>T, p.(Q376*)) in IL12RB1, inducing a rare inheritable immune disorder (described as Patient 2 in the paper by De Jong et al. [8]). The pedigree of this family is shown in Fig. 1. No information is available for the fathers’ family of the niece with the immune disorder. Germline CDH1 mutation analysis of the index patient as well as analysis of her tumor for microsatellite instability were negative.

Fig. 1
figure 1

Pedigree of the family with an IL-12Rβ1 deficiency and gastric cancer

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To elucidate whether an IL12RB1 mutation could underlie the pathogenesis of GC in this family, we tested both sisters that developed GC. Both were heterozygous for the IL12RB1 mutation. No material from their mother was available for testing.

Histopathological characteristics of gastric tumors

Material of both sisters with GC was available for histological re-evaluation. In the total gastrectomy specimen of the patient A an ulcerating tumor in the gastric body was seen with a diameter of 5.4 cm. The tumor invaded the subserosal tissue and was staged as pT3N0. Review showed a poorly differentiated intestinal-type adenocarcinoma. The surrounding mucosa showed extensive intestinal metaplasia and chronic active gastritis. No quantitative difference indicating loss of the wild type IL12RB1 allele was observed in the tumor by quantitative analysis of both alleles by pyrosequencing from blood as well as normal and tumor tissue sections (data not shown). Her sister had a 12 cm large polypoid tumor with central ulceration in the transitional zone of the stomach. Review of the histology showed an intestinal-type adenocarcinoma, poorly differentiated with focally a few poorly formed glands. According to the seventh edition of the TNM classification the tumor is staged as pT4aN3a. The surrounding mucosa showed intestinal metaplasia and chronic atrophic inflammation. Loss of heterozygosity analysis could not be performed due to insufficient quality of the material. Helicobacter pylori was not identified in the resection specimens of both patients.

Mutation analysis of IL12RB1 and IL12RB2 in 29 patients with gastric cancer

Twenty-nine patients with GC, that were suspected for genetic predisposition, were screened for mutations in the two genes encoding the IL-12 receptor chains, IL12RB1 and IL12RB2. The histological characteristics of GC patients in our cohort, including H. pylori status, are shown in Table 1. Several common polymorphisms in both genes (data not shown) and four rare heterozygous variants of unknown significance (VUS) in IL12RB1 were identified (c.102G>A, c.848G>A, c.1161G>A, c.1619-6C>T), but none of them appear to be pathogenic according to various in silico prediction programs (Table 2). The variants were all identified only once and in different patients. We conclude that no clear deleterious mutations were found.

Table 2 Rare variants of unknown significance identified in IL12RB1 and IL12RB2
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Discussion

In the current study we describe cosegregation of a heterozygous germline defect in IL12RB1 and GC development in a family with IL-12Rβ1 deficiency. This heterozygosity might lead to impaired response of T- and NK-cells to pathogens that increase the risk of GC development. Therefore the gene would not act as a tumor suppressor gene for which loss of the wild-type allele would drive tumorigenesis. Indeed, the wild-type allele was still present in the tumor of one of the patients.

The truncating mutation found in the family, (c.1126C>T, p.(Q376*)), probably does not render a stable protein. However, if a stable truncated protein would be formed, part of the FNIII extracellular domains, which are necessary for the IL-12Rβ1-IL-12Rβ2 dimerization would be missing, as well as the transmembrane domain required for expression of the protein on the cell surface [31]. Although IL-12 can bind with low affinity to IL-12Rβ1 and IL-12Rβ2 separately, presence of a heterodimer is necessary for high affinity binding [31].

To determine whether germline mutations in the IL-12 receptor chains are a common event in GC patients, we sequenced these genes in a cohort of GC patients who were suspected for genetic predisposition. Although we identified several rare heterozygous variants in IL12RB1, none appeared to be deleterious using in silico prediction programs. The variants we identified were found in patients with both intestinal-type (n = 2) and diffuse-type GC (n = 1) according to the Laurén classification. For one patient in whom we detected a variant the histological subtype is unknown. One of these variants, c.848G>A, results in an amino acid substitution of an arginine to a glutamine in the FNIII domain of the IL-12Rβ1 protein. This is the domain required for IL-12Rβ1-IL-12Rβ2 dimerization. Although this substitution is predicted not to affect the function of the protein only experimental evidence, such as obtained with IL12RB1 expression constructs [32], can determine whether this is indeed the case.

The results of our study suggest that germline mutations in IL12RB1 and IL12RB2 do not play a frequent role in GC predisposition. However, in contrast to the two heterozygous carriers with GC of the index family, the majority of patients in our cohort have been diagnosed with diffuse-type GC and therefore mutations in IL12RB1 and IL12RB2 may still play a more prominent role in intestinal-type GC predisposition.

Taken together, we found a heterozygous IL12RB1 mutation to segregate with intestinal-type GC in one family. No additional mutations were found in 29 families with GC. Since only little is known about cancer risks in families with IL12RB1 mutations, the observation in the current study may warrant additional research in other families with this deficiency to determine whether they are at increased risk for developing (gastric) cancer.