Germline cytotoxic lymphocytes defective mutations in Chinese patients with lymphoma
- Authors:
- Published online on: September 6, 2017 https://doi.org/10.3892/ol.2017.6898
- Pages: 5249-5256
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Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
The perforin-dependent granule-mediated cytolysis of cytotoxic lymphocytes (CLs), including natural killer cells and cytotoxic T lymphocytes, is the key machinery in the clearance of viral, and intracellular bacterial infections, as well as in the prevention of tumor development (1,2). The proteins encoded by perforin 1 (PRF1), unc-13 homolog D (UNC13D), syntaxin 11 (STX11), and STXBP2 (syntaxin binding protein 2) serve an essential role in this pathway. Mutations in these genes lead to function defects of CLs and are causative of familial hemophagocytic lymphohistiocytosis type 2 (FHL2), FHL3, FHL4, and FHL5 (3–6). The clinical manifestation of X-linked lymphoproliferative disease (XLP), which is caused by mutations in SH2 domain containing 1A (SH2D1A) (7) or X-linked inhibitor of apoptosis (XIAP) (8) genes, resembles hemophagocytic lymphohistiocytosis. Furthermore, XLP2 due to XIAP deficiency has been suggested to be classified as X-linked FHL (9).
A proportion of patients with lymphoma have been reported to harbor mutations in PRF1, UNC13D, STX11, STXBP2 or SH2D1A genes (10–14), indicating that genetic defective function of CLs may increase susceptibility to lymphomagenesis. The aim of the present study was to investigate the association between mutations in genes involved in the cytotoxic function of CLs and the development of lymphoma in Chinese patients.
Patients and methods
Cases and controls
In the present study, 68 and 34 patients with lymphoma were admitted to Hebei Yanda Lu Daopei Hospital (Sanhe, China) and Peking University First Hospital (Beijing, China), respectively, between August 2013 and August 2015; 12/102 were excluded due to poor DNA quality. A total of 90 (61 from Hebei Yanda Lu Daopei Hospital and 29 from Peking University First Hospital) unrelated patients with lymphoma (48 males and 42 females; age range, 3–60 years) were recruited in the present study; 39 were diagnosed with Hodgkin lymphoma and 51 were diagnosed with non-Hodgkin lymphoma according to the World Health Organization classification (15). Healthy donors of Han nationality (n=210) at the Hebei Yanda Lu Daopei Hospital served as controls. The present study was approved by the Ethics Committees of Hebei Yanda Lu Daopei Hospital and Peking University First Hospital. Written informed consent was obtained from all patients and healthy donors or their parents in accordance with the 1964 Helsinki declaration, and its later amendments or comparable ethical standards.
Amplification and sequence analysis
Genomic DNA was isolated from peripheral blood and bone marrow using the TIANamp Blood DNA kit (item no. DP318; Tiangen Biotech Co., Ltd., Beijing, China) or from nails using the TIANamp FFPE DNA kit (item no. DP331; Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. Referenced coding sequences of the PRF1 (NM_005041.4), UNC13D (NM_199242.2), STXBP2 (NM_003764.3), STX11 (NM_006949.2), SH2D1A (NM_002351.3), and XIAP (NM_001167.2) were obtained from the National Center for Biotechnology Information Consensus CDS database (https://www.ncbi.nlm.nih.gov/projects/CCDS/CcdsBrowse.cgi). Primers were designed to amplify the coding exons and the flanking intron sequences by polymerase chain reaction (PCR). The sequences of primers are presented in Table I. The PCR system comprised of 1 µl genomic DNA (10 ng/µl), 1 ml forward primer (20 pmol/µl), 1 ml reverse primer (20 pmol/µl), 10 µl Phusion Flash High-Fidelity PCR Master mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 7 µl distilled water in a total volume of 20 µl. Reaction conditions were 10 sec at 98°C followed by 38 cycles of 10 sec at 98°C, 10 sec at 68°C, 15 sec at 72°C, and then 1 min at 72°C. The amplified PCR products were purified with ExoSAP-IT (USB Co., Cleveland, OH, USA) and followed by cycle sequencing PCR using a BigDye Terminator Sequencing Kit version 3.1 (Thermo Fisher Scientific, Inc.). Fluorescent labeled products were separated using an ABI 3500xL Genetic Analyzer (Thermo Fisher Scientific, Inc.). Variations were analyzed using Variant Reporter software (version 1.1; Thermo Fisher Scientific, Inc.). Genetic polymorphism information from the Single Nucleotide Polymorphism database (dbSNP; http://www.ncbi.nlm.nih.gov/snp/), 1000 Genomes Project (http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/) and the Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org/) were referenced to obtain the frequencies of variants in large populations. Variants with minor allele frequencies >1% in the 1000 Genomes Project and/or ExAC were regarded as SNPs rather than mutations.
 | Table I.Primers used for amplification of the coding exons and the flanking intron sequences of perforin 1, unc-13 homolog D, syntaxin binding protein 2, syntaxin 11, SH2 domain containing 1A and X-linked inhibitor of apoptosis. |
Confirmation of germline derivation of mutations
For patients determined to harbor mutations, the same mutation was detected in the DNA isolated from peripheral blood of their parents. In the absence of one or both parents, the detection of the same mutation in DNA extracted from nails of the patients could be of value. This was performed in order to confirm that the mutations were germline-derived.
In silico analysis
Two bioinformatics tools were used to predict whether an amino acid substitution was benign or deleterious: Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org/) predicts whether an amino acid substitution affects protein function based on the degree of conservation of amino acid residues in multiple sequence alignments derived from closely associated sequences (16); and Polymorphism Phenotyping version 2.0 (PolyPhen-2; http://genetics.bwh.harvard.edu/pph/) predicts the possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative analyses (17). Iterative Threading ASSEmbly Refinement (I-TASSER; http://zhanglab.ccmb.med.umich.edu/I-TASSER/) was also used to predict and simulate the influence of the variants in protein tertiary structures.
Statistical analysis
Comparisons of mutant frequencies as well as genotype distributions between patients with lymphoma and controls were performed using the Chi-square test with SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Analysis of the gene mutations
A total of 18 different mutations were identified in 24 unrelated patients (26.67%) (Fig. 1). A total of 16 patients (17.78%) carried mutations in UNC13D, including 12 with monoallelic mutations, 1 with homozygous mutation and 3 with compound heterozygous mutations. Seven patients (7.78%) had PRF1 mutations, including 4 with monoallelic mutations, 1 with homozygous mutation and 2 with compound heterozygous mutations. One patient (1.11%) was detected to carry STX11 monoallelic mutation (Table II). All mutations were confirmed to be germline-derived.
Sixty unrelated healthy donors were sequenced for these 6 genes with the same methods and 5 of them (8.33%) were detected to harbor mutations. All 5 individuals were heterozygous for UNC13D mutations (c.680G>A/p.R227H; c.3134C>T/p.T1045M; c.3229_3235del/p.Arg1077SerfsTer48; c.2553+5C>G; c.602A>G/p.H201R).
The Chi-square test revealed that the difference between mutant frequencies of patients with lymphoma and healthy donors was of statistical significance (P=0.005). Individuals carrying mutations of these genes were more likely to develop lymphoma compared with those without mutations [odds ratio (OR), 4.000; 95% confidence interval (CI), 1.431–11.180].
Statistical analysis of UNC13D c.2588G>A mutation
UNC13D c.2588G>A/p.G863D was the most frequent mutation identified in the current study, which was identified in 9 patients (10.00%), including 1 homozygous and 8 heterozygous. This genetic variation was annotated as rs140184929 in dbSNP without frequency data. Data in the 1000 Genomes Project demonstrated that the c.2588A allele existed predominantly in the Chinese (0.83%), and rarely in the Japanese (0.48%) and Bengali (0.58%) populations. Other populations did not carry this variant (Table III). Data in ExAC also demonstrated that the allelic frequency of c.2588A was increased in East Asian populations (37/8,638; 0.43%) compared with that in South Asian populations (5/16,504; 0.03%). Only one individual out of 32,962 Europeans was heterozygous for c.2588G>A variant. This variation was not observed among 14,554 individuals analyzed from other populations. Considering the high allele frequency of this mutation in the present patient cohort and the distinctly different allele frequencies among diverse populations, genotyping of the c.2588 allele was performed in 210 unrelated healthy donors of Chinese Han nationality (Table III). Heterozygous c.2588G>A was observed in 4 of them. Combined with data in the 1000 Genomes Project (a total of 301 Chinese), a control cohort of 511 individuals, 9 of whom harbored c.2588A allele in a heterozygous state was obtained. The Chi-square test revealed that the allele frequency of c.2588A in patients was significantly increased compared with that in the control group (P<0.001; OR, 6.621; 95% CI, 2.652–16.532), suggesting an association between the c.2588G>A mutation, and the risk of developing lymphoma.
In silico analysis of UNC13D c.2588G>A mutation
The UNC13D c.2588G>A/p.G863D mutation resulted in a substitution of the nonpolar and hydrophobic glycine (often involved in the formation of the turn structure) in the Munc13 homology domain 2 of protein UNC13D by the polar, and neutral aspartic acid (often involved in the formation of the coil structure). Multiple sequence alignment demonstrated that the amino acid at this position was highly conserved in available vertebrate species (Fig. 2A) and the alteration is predicted to be possibly damaging using PolyPhen-2 (Fig. 2B), and deleterious with SIFT in silico analysis. I-TASSER also demonstrated significant differences in the 3D structures of the wild-type and mutant-type proteins (Fig. 2).
Discussion
In 2005, Clementi et al (10) first reported that 8/29 (27.6%) unrelated Italian patients with lymphoma carried PRF1 mutations and 5 of them carried PRF1 c.272C>T/p.A91V heterozygous mutation. In 2014, Ciambotti et al (11) observed mutations in 23/84 (27.4%) Italian patients with anaplastic large cell lymphoma following genotype analysis of PRF1, UNC13D and SH2D1A. Twenty-one patients (25%) carried PRF1 mutations and the other 2 patients had mutations of UNC13D. PRF1 c.272C>T/p.A91V mutation was also the most common mutant genotype (11/84).
In the present study 6 genes, which are all involved in cytotoxic function of natural killer cells and cytotoxic T lymphocytes, were identified in 90 Chinese patients with lymphoma. The results demonstrated the association of germline defective mutations and development of lymphoma. The majority of mutations detected in the current study were heterozygous missense mutations, which were consistent with previous reports (10,11). This may explain why these patients developed lymphoma later in life rather than outbreak fatal FHL during infancy. Such monoallelic mutations may contribute to the pathogenesis of the disease, but are not sufficient to initiate the disease phenotype alone. Additional unidentified genetic defects, or possibly even environmental factors, may contribute to the development of lymphoma (10). What was different from reports in Europe was that the most common mutant gene in the present study was UNC13D while PRF1 was less frequently involved, indicating a distinct mutation spectrum in Chinese patients with lymphoma.
Notably, no hot spot region or predominant pathogenic mutation in UNC13D had been previously identified (18). In the current study; however, 9/16 UNC13D mutation carriers exhibited c.2588G>A/p.G863D mutation, including 1 homozygous and 8 heterozygous. This single amino acid substitution occurred in an evolutionary conserved position and was predicted to be pathogenic using PolyPhen-2, SIFT, and I-TASSER. Furthermore, statistical analysis revealed that this mutation was significantly associated with the risk of developing lymphoma. In addition, none of our patient harbored the PRF1 c.272C>T/p.A91V mutation, which was most frequently reported in European populations (10,11). In the present consecutive cohort of >500 patients with diagnosed or suspected FHL, the PRF1 c.272C>T mutation was not identified (data not shown).
Data in the 1000 Genomes Project demonstrated that the allele frequency of PRF1 c272T was significantly higher in European population compared with that in Chinese and Japanese, supporting the concept of a Mediterranean origin of the mutation (11). However, the UNC13D c.2588A allele existed predominantly in Chinese, less in Japanese and Bengali, and was not identified in any other populations listed in this database (Table III). In regards to Korea, where UNC13D is the predominant causative gene in Korean patients with FHL, c.2588G>A was not reported (19). Collectively, the data obtained from the present study and the databases suggest that UNC13D c.2588G>A/p.G863D is a founder mutation of Chinese patients.
In conclusion, the current study provides a relatively comprehensive mutation spectrum of defective cytotoxicity associated genes in Chinese patients with lymphoma. Monoallelic germline mutations were identified to be most frequent in the present cohort, suggesting that partially impaired cytotoxic machinery may represent a predisposing factor for the development of lymphoma. In addition, UNC13D was identified as the predominant causative gene, while PRF1 was less frequently involved. Furthermore, UNC13D c.2588G>A/p.G863D, which is not reported in other populations, is a founder mutation in Chinese patients.
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