Sequence Variation In Exon Research Articles

Variability in 2-acetyl-1-pyrroline production and associated mutations in BADH2 gene in aromatic rice cultivars of Bangladesh

Aroma is a special trait and aspect of the end-use quality of rice and its principal component is 2-acetyl-1-pyrroline (2AP). Sequence variants of betaine aldehyde dehydrogenase (BADH2) gene on rice chromosome 8 are associated with fragrance, and the most prominent mutation is an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7. In this study, we analyzed sequence variations in exon 7 of BADH2 in four locally popular aromatic rice cultivars and six non-aromatic high-yielding rice varieties in Bangladesh. Sequencing and alignment analysis revealed that two aromatic rice cultivars, Kataribhog and Sakkorkhana, had an 8-bp deletion (discontinuous) and one SNP (A/T) in exon 7 of the BADH2 gene. However, deletions were absent in the other two aromatic rice cultivars (Chinigura and Ranisalut) and all high-yielding varieties (BRRI dhan28, BRRI dhan29, BRRI dhan86, BRRI dhan87, BRRI dhan92 and BINA dhan10). Gene expression analysis using specific markers also supports these findings. Our results suggest that aromaticity in local rice cultivars in Bangladesh may be regulated by differential genetic mechanisms. Further studies are needed to elucidate the genetic mechanisms and fragrant compound(s) involved in the fragrance traits of Chinigura, Ranisalut, and other locally cultivated aromatic rice varieties that are critical for the development of high-yield aromatic rice by knocking out the targeted gene(s) using CRISPR-Cas genome editing.

Association of Exon 14 of the SOX6 Gene Sequence Variations with Response to Hydroxyurea Therapy in Patients Carrying Non Transfusion-Dependent Thalassemia

Hydroxyurea (HU) activates the γ-globin gene, resulting in increased Hb F synthesis. The SOX6 gene is a member of the Sox (Sry-type HMG box) family of transcription factors, characterized by minor groove binding domain. The DNA binding domain of this gene is encoded by exon 14. We assessed the relationship between response to HU and exon 14 of the SOX6 gene sequence variations in patients with non transfusion-dependent thalassemia (NTDT). One hundred NTDT patients from southern Iran underwent HU therapy randomly participated in this cross-sectional study between February 2013 and October 2014. Based on response to HU therapy, the patients were divided into two groups: good and poor responder. Sequence variations of exon 14 of the SOX6 gene was assayed by the Sanger sequencing technique. From all evaluated single nucleotide polymorphisms (SNPs) as above, we found no significant association between sequence variations of exon 14 of the SOX6 gene and response to HU therapy (p > 0.05). It seems that no SNPs in exon 14 of the SOX6 gene is associated with response to HU in NTDT patients, but more studies are needed for further evaluation.

Association of genetic variants of Forebrain Embryonic Zinc Finger-like (FEZL) gene exon 3 with clinical mastitis in Murrah buffaloes (Bubalus bubalis)

Forebrain embryonic zinc finger-like (FEZL) gene is an important candidate gene, which affects the host disease resistance. The gene has been mapped on BTA22 and consists of 6 exons and 5 introns spanning over 4685 bp. Present investigation was carried out with an aim to identify genetic polymorphism within exon 3 of FEZL gene and its association with incidence of clinical mastitis in Murrah buffaloes. Genomic DNA extracted from 110 Murrah buffaloes were amplified using two sets of primers and respective PCR products of 554 bp and 474 bp were obtained. A total of 4 nucleotide sequence variations in exon 3 were observed. Two SNPs g.1556C>T and g.1707G>A were non synonymous, which resulted in amino acid substitution of isoleucine to threonine and methionine to valine at protein position 31 and 83, respectively. PCR-RFLP using MspI for primer 3.1 and DraI for primer 3.2 revealed monomorphic patterns, while TaqI for primer 3.1 exhibited polymorphism with CC, CT and TT genotypes with respective frequencies of 0.35, 0.21 and 0.44. Association of identified genotypes with clinical mastitis has been found to be significant (p<0.01).Odds Ratio (OR) analysis confirmed significant association (OR=6.8531; 95% CI) and TT genotype of SNP g.1556C>T of FEZL gene emerged as the most favoured and may be used as a potential marker for selecting Murrah buffaloes less susceptible to mastitis, after validation in a larger herd.

Single nucleotide polymorphisms in GBBSI and SSIIa genes in relation to starch physicochemical properties in selected rice (Oryza sativa L.) varieties

Single nucleotide polymorphisms in GBBSI and SSIIa genes in relation to starch physicochemical properties in selected rice (Oryza sativa L.) varieties

Variation in intronic microsatellites and exon 2 of the Plasmodium falciparum chloroquine resistance transporter gene during modification of artemisinin combination therapy in Thailand

The amino acid substitution at residue 76 of the food vacuolar transmembrane protein encoded by the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) is an important, albeit imperfect, determinant of chloroquine susceptibility status of the parasite. Other mutations in Pfcrt can modulate susceptibility of P. falciparum to other antimalarials capable of interfering with heme detoxification process, and may exert compensatory effect on parasite growth rate. To address whether nationwide implementation of artemisinin combination therapy (ACT) in Thailand could affect sequence variation in exon 2 and introns of Pfcrt, we analyzed 136 P. falciparum isolates collected during 1997 and 2016 from endemic areas bordering Myanmar, Cambodia and Malaysia. Results revealed 6 haplotypes in exon 2 of Pfcrt with 2 novel substitutions at c.243A > G (p.R81) and c.251A > T (p.N84I). Positive selection was observed at amino acid residues 75, 76 and 97. Four, 3, and 2 alleles of microsatellite (AT/TA) repeats occurred in introns 1, 2 and 4, respectively, resulting in 7 different 3-locus haplotypes. The number of haplotypes and haplotype diversity of exon 2, and introns 1, 2 and 4 were significantly greater among isolates collected during 2009 and 2016 than those collected during 1997 and 2008 when 3-day ACT and 2-day ACT regimens were implemented nationwide, respectively (p < 0.05). By contrast, the number of haplotypes and haplotype diversity of the merozoite surface proteins 1 and 2 of these parasite populations did not differ significantly between these periods. Therefore, the Pfcrt locus of P. falciparum in Thailand continues to evolve and could have been affected by selective pressure from modification of ACT regimen.

Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design

HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. We designed PCR primers to amplify HLA-B*57:01 along with closely related alleles, such as HLA-B*57:03, directly from genomic DNA. Most ABC-insensitive alleles, including HLA-B*58:01, would not produce any products in the PCR reaction. Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, alleles. In addition to using real-time PCR, we used restriction enzymes to generate differential digestion patterns that led to the development of an HLA-B*57:01 PCR-restriction fragment length polymorphism marker. When used to genotype a set of 75 selected clinical samples, our real-time PCR assay demonstrated 100% accuracy in distinguishing between the HLA-B*57:01-positive and -negative alleles when results were compared to those of sequence-specific oligonucleotide probe typing and reference laboratory testing. Our newly developed test will allow clinical laboratories with real-time PCR capabilities to perform HLA-B*57:01 genotyping in a timely and economical manner.

A novel mutation in exon 1 of GATA4 in Egyptian patients with congenital heart disease.

Congenital heart disease (CHD) is a common birth defect. Many studies have reported GATA4 mutations in patients with CHD, mainly septal defects. This study aimed to investigate the GATA4 exon 1 mutation in Egyptian patients with isolated congenital heart defects as a possible causative mutation. Screening for mutations or any sequence variations in exon 1 of the GATA4 gene was carried out by PCR amplification followed by direct sequencings in 165 Egyptian patients with different nonsyndromic congenital heart diseases and 93 controls who were matched in terms of age and sex. Thorough clinical assessments were done for all subjects, along with X-ray, 2D echocardiography, and Doppler examinations. The most common CHD among our cases was isolated ventricular septal defect (VSD) in 47.3% (78/165), followed by isolated atrial septal defect. A novel nonsynonymous sequence variation in fragment 2 (P193H) of exon 1 of GATA4 was detected in 15 (9.1%) of the subjects with septal defects. This mutation was not seen in any of the control group subjects. There is a high prevalence of exon 1 GATA4 mutation (9.1%) in our study compared to other studies in different populations, which may correlate with different ethnic populations.

Genetic association of sequence variation in exon 3 of the swine leukocyte antigen-DQA gene with piglet diarrhea in Large White, Landrace, and Duroc piglets.

Piglet diarrhea is one of the primary factors that affects the benefits of the swine industry. Recent studies have shown that exon 2 of the swine leukocyte antigen-DQA gene is associated with piglet resistance to diarrhea; however, the contributions of additional exon coding regions of this gene remain unclear. Here, we detected and sequenced variants in the exon 3 region and examined their associations with diarrhea infection in 425 suckling piglets using the polymerase chain reaction-single-strand conformational polymorphism and sequencing analysis. The results revealed that exon 3 of the swine leukocyte antigen-DQA gene is highly polymorphic and pivotal to both diarrhea susceptibility and resistance in piglets. We identified 14 genotypes (AA, AB, BB, BC, CC, EE, EF, BE, BF, CF, DD, DH, GG, and GF) and eight alleles (A-H) that were generated by 14 nucleotide variants, eight of which were novel, and three nucleotide deletions. Statistical analyses revealed that the genotypes AB and EF were associated with resistance to diarrheal disease (P < 0.05), and the genotype DD may contribute to diarrhea susceptibility but was unique to Large White pigs (P > 0.05). These results elucidate the genetic and immunological background to piglet diarrhea, and provide useful information for resistance breeding programs.

Absence of mutations in HCRT, HCRTR1 and HCRTR2 in patients with ROHHAD

Background and objectivesRapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) is a rare pediatric disease of unknown cause. Here, in response to a recent case report describing a ROHHAD patient who suffered from secondary narcolepsy confirmed by an absence of hypocretin-1 in the cerebrospinal fluid, we consider whether the ROHHAD phenotype is owing to one or more mutations in genes specific to hypocretin protein signalling. MethodsDNA samples from 16 ROHHAD patients were analyzed using a combination of next-generation and Sanger sequencing to identify exonic sequence variations in three genes: HCRT, HCRTR1, and HCRTR2. ResultsNo rare or novel mutations were identified in the exons of HCRT, HCRTR1, or HCRTR2 genes in a set of 16 ROHHAD patients. ConclusionsROHHAD is highly unlikely to be caused by mutations in the exons of the genes for hypocretin and its two receptors.

Sequence variation of Bemisia tabaci Chemosensory Protein 2 in cryptic species B and Q: New DNA markers for whitefly recognition

Bemisia tabaci Gennadius biotypes B and Q are two of the most important worldwide agricultural insect pests. Genomic sequences of Type-2 B. tabaci chemosensory protein (BtabCSP2) were cloned and sequenced in B and Q biotypes, revealing key biotype-specific variations in the intron sequence. A Q260 sequence was found specifically in Q-BtabCSP2 and Cucumis melo LN692399, suggesting ancestral horizontal transfer of gene between the insect and the plant through bacteria. A cleaved amplified polymorphic sequences (CAPS) method was then developed to differentiate B and Q based on the sequence variation in exon of BtabCSP2 gene. The performances of CSP2-based CAPS for whitefly recognition were assessed using B. tabaci field collections from Shandong Province (P.R. China). Our SacII based CAPS method led to the same result compared to mitochondrial cytochrome oxidase-based CAPS method in the field collections. We therefore propose an explanation for CSP origin and a new rapid simple molecular method based on genomic DNA and chemosensory gene to differentiate accurately the B and Q whiteflies of the Bemisia complex around the world.

The Anopheles stephensi odorant binding protein 1 (AsteObp1) gene: A new molecular marker for biological forms diagnosis

Anopheles (Cellia) stephensi Liston 1901 is known as an Asian malaria vector. Three biological forms, namely “mysorensis”, “intermediate”, and “type” have been earlier reported in this species. Nevertheless, the present morphological and molecular information is insufficient to diagnose these forms. During this investigation, An. stephensi biological forms were morphologically identified and sequenced for odorant-binding protein 1 (Obp1) gene. Also, intron I sequences were used to construct phylogenetic trees. Despite nucleotide sequence variation in exon of AsteObp1, nearly 100% identity was observed at the amino acid level among the three biological forms. In order to overcome difficulties in using egg morphology characters, intron I sequences of An. stephensi Obp1 opens new molecular way to the identification of the main Asian malaria vector biological forms. However, multidisciplinary studies are needed to establish the taxonomic status of An. stephensi.

Novel sequence variations in exon 25 of JHDM2A among overweight and obese sub/infertile males in South India

Novel sequence variations in exon 25 of JHDM2A among overweight and obese sub/infertile males in South India

RBFOX1 and RBFOX3 Mutations in Rolandic Epilepsy

Partial deletions of the gene encoding the neuronal splicing regulator RBFOX1 have been reported in a range of neurodevelopmental diseases, including idiopathic generalized epilepsy. The RBFOX1 protein and its homologues (RBFOX2 and RBFOX3) regulate alternative splicing of many neuronal transcripts involved in the homeostatic control of neuronal excitability. In this study, we explored if structural microdeletions and exonic sequence variations in RBFOX1, RBFOX2, RBFOX3 confer susceptibility to rolandic epilepsy (RE), a common idiopathic focal childhood epilepsy. By high-density SNP array screening of 289 unrelated RE patients, we identified two hemizygous deletions, a 365 kb deletion affecting two untranslated 5′-terminal exons of RBFOX1 and a 43 kb deletion spanning exon 3 of RBFOX3. Exome sequencing of 242 RE patients revealed two novel probably deleterious variants in RBFOX1, a frameshift mutation (p.A233Vfs*74) and a hexanucleotide deletion (p.A299_A300del), and a novel nonsense mutation in RBFOX3 (p.Y287*). Although the three variants were inherited from unaffected parents, they were present in all family members exhibiting the RE trait clinically or electroencephalographically with only one exception. In contrast, no deleterious mutations of RBFOX1 and RBFOX3 were found in the exomes of 6503 non-RE subjects deposited in the Exome Variant Server database. The observed RBFOX3 exon 3 deletion and nonsense mutation suggest that RBFOX3 represents a novel risk factor for RE, indicating that exon deletions and truncating mutations of RBFOX1 and RBFOX3 contribute to the genetic variance of partial and generalized idiopathic epilepsy syndromes.

Gene-based high-density mapping of the gene rym7 conferring resistance to Barley mild mosaic virus (BaMMV)

Barley yellow mosaic virus (BaYMV) disease seriously affects winter barley (Hordeum vulgare L.) production. Improvement of resistance to this disease can prevent yield losses. Based on previous reports, the gene rym7 (rmm7), conferring partial resistance to BaMMV, was assigned to chromosome 1H, closely linked to the centromere. In this study, newly developed barley genomic resources were applied to saturate the genetic map at the rym7 locus. Out of a set of 350 gene-based markers of chromosome 1H, we genetically assigned 23 to the rym7 region, delimiting the resistance locus to a 9.9-cM interval close to the centromere by genetic mapping in 53 doubled haploid progeny of a bi-parental mapping population. Nine gene-derived co-dominant markers co-segregated with the resistance locus. Among these, we identified the eukaryotic translation initiation factor Hv-eIF(iso)4E. Its homolog in Arabidopsisthaliana confers resistance to potyviruses. However, sequencing the entire open reading frame of resistant and susceptible genotypes could not reveal any sequence variation in exons of the gene. These results demonstrate how to combine newly developed barley genomic resources for rapid gene-based marker saturation. As a result, several easy-to-handle co-dominant markers have been identified for marker-assisted selection of rym7 in barley breeding.

Sequence Variant in the LPIN1 gene in Patients with Metabolic Syndrome

Objective: Metabolic syndrome (MetS) is a complex disease characterized by insulin resistance, abdominal obesity, hyperglycemia, hypertension, hypertriglyceridemia and low HDL-cholesterol level. The aim of the study was to evaluate the sequence variations in the LPIN1 gene in MetS. This gene codes lipin-1 protein which functions as Mg-dependent phosphatidic phosphatase enzyme and transcriptional coactivator. Material and Methods: The study groups included 73 MetS (19 M/54 F) and 56 non-MetS (16 M/ 40 F). Sequence variation in exons 2, 4, 5 and 14 of the LPIN1 gene were investigated by DNA sequencing method. Results: c.696 G>C variant (p.S232S) in exon 5 was observed in only one women with MetS. But this variation is not important because of coding same amino acid. Conclusion: Any important sequence variant was not detected in exons 2, 4, 5 and 14 in the LPIN1 gene in MetS.

A Novel Splice Site and Two Known Mutations of Androgen Receptor Gene in Sex-Reversed XY Phenotype

Molecular characterization of 27 cytogenetically confirmed Indian XY females was attempted by screening selected regions of candidate genes namely SRY (HMG box) and the ligand-binding domain of androgen receptor (AR) (Exons 5-8). Three of the index cases showed sequence variations in exons of the AR gene: a deletion mutation in exon 6 (c.2762 del C), a substitution mutation (c.2925 C>T), and a novel splice donor site mutation (IVS5+1 g>a; exon 5/intron 5). The proband's (case VA156) mother and one of the sisters were heterozygous for the novel splice donor site mutation while the father was normal. Review of literature suggested that an alternate spice donor site could be utilized leading to an aberrant splicing resulting in a truncated receptor. This could not be validated further through reverse transcriptase-polymerase chain reaction as the patient failed to cooperate for follow-up. Of the 16 spice site variations reported in various ethnic groups, this is a novel variation in the AR gene to be associated with Androgen Insensitivity Syndrome. The proband's sister, noted to be a heterozygous carrier, has high risk of having XY female progeny; hence prenatal screening of the mutation in case of an XY fetus is recommended.

Genetic characterization of growth hormone 1 gene in patients with isolated growth hormone deficiency

Introduction:Growth hormone (GH) secretion and release is a complex and highly regulated process. Any alteration disturbing synthesis, secretion or biological action of GH, results into growth hormone deficiency (GHD). GHD is of two types-isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD), of which IGHDis more common. The genes implicated in its etiology are growth hormone 1(GH1) and receptor of growth hormone-releasing hormone (GHRHR). Mutations within the coding region and/or either entire or partial deletions of the GH1gene lead to IGHD. In addition, GH1 possesses upstream regulatory elements and a promoter with binding sites for various transcription factors, which control its expression.Aim:The study was planned with an aim to identify entire GH1 locus deletion, mutations in the GH1 coding region and sequence variations (polymorphisms) in the promoter region of the gene in patients with IGHD.Materials and Methods:Thirty patients clinically diagnosed with IGHD and 30 healthy individuals who formed the controls were enrolled for the study. Genomic DNA was isolated from peripheral blood sample and processed for amplification of the desired regions followed by direct sequencing and/or restriction endonuclease digestion.Results:Out of the 30 IGHD patients screened, 20% of the cases showed consanguinity and 16% had a positive family history. Seven percentage of the patients showed homozygous deletion of the GH1gene while rest of them had heterozygous deletion. Screening of the coding region of GH1 showed sequence variations in exon 1 in 20% of the patients whereas the promoter region showed the presence of polymorphisms–rs2005171 in 20%, rs2005172 in 15% and rs11568828 in 18% of the cases. The haplotype comprising rs2005171 and rs2005172 was observed in four patients.Conclusion:The present study is an attempt to characterize the GH1 locus in IGHD patients. To the best of our knowledge this is the first study of its kind where entire GH1locus, upstream regulatory elements and promoter region have been studied. Such an analysis would provide valuable information on the etiology of IGHD.

TP53 Mutation Is Rare In Primary Myelofibrosis

Abstract 5241 IntroductionTP53 is the most frequently mutated tumor suppressor gene in human cancers and is usually associated with an aggressive disease course. TP53 mutation has been described in a variety of hematopoietic neoplasms, and has been suggested to play a role in leukemic transformation of myeloproliferative neoplasms (MPN). However, the incidence as well as the clinical and pathogenetic implications of TP53 mutation in each sub-category of MPN, including primary myelofibrosis, have not been described. In this study, we investigated the presence and potential clinical significance of TP53 mutations in a large series of primary myelofibrosis cases. Patients and MethodsWe retrieved archival bone marrow DNA from 51 consecutive patients diagnosed with primary myelofibrosis at The University of Texas MD Anderson Cancer Center between the years 2005 and 2007. Diagnosis was based on morphologic, immunophenotypic, cytogenetic and molecular evaluation of bone marrow in conjunction with clinical data. Only 2 patients had blast counts >10% (11 and 14%). Twenty nine of 50 (58%) patients showed JAK2 p.V617F mutation and all patients were negative for BCR-ABL1 translocation on routine clinical testing. DNA samples were assessed for sequence variation in exons 4 through 9 of TP53 by both high resolution melting curve (HRM) analysis using LightCycler® 480 System (Roche, Indiana IN) and bidirectional Sanger sequencing using 3730XL DNA Analyzer (Life technologies, Carlsbad CA). ResultsThe mean overall survival was 5.7 years. Five patients developed acute leukemia, all of whom died of disease. By Sanger sequencing, only one (1.9%) case showed an amino acid-altering mutation in TP53: c.707A>G (TAC to TGC) in codon 236 (p.Y236C) of exon 7. In addition, 8 cases showed silent mutations/single nucleotide polymorphisms of unknown significance - c.36G>A (CCG to CCA) in exon 4 (n=3) and c.213A>G (CGA to CGG) in exon 6 (n=6). The p.R72P polymorphism in exon 4 which has been described in other hematopoietic neoplasms was present in 1 patient. All cases with a mutant sequence by Sanger sequencing also showed a variant melting curve pattern by HRM analysis. The patient with TP53 mutation died 2 years after presentation from progressive myelofibrosis without developing acute leukemia. ConclusionTP53 mutation is very rare in primary myelofibrosis. Disclosures:No relevant conflicts of interest to declare.

A single nucleotide polymorphism in the Waxy gene explains a significant component of gel consistency

Gel consistency (GC) is a standard assay used in rice improvement programmes to determine whether rice cultivars/breeding lines of high amylose content are soft or firm textured when cooked. In this study, we show that sequence variation in exon 10 of the Waxy (Wx) gene associates with GC using RILs derived from parents with high amylose content that differ in GC. The association was validated using a diverse set of traditional varieties, selected on the basis of amylose content, from the generation challenge programme. Structural investigations to explain how the mutation leads to differences in GC showed a strong association between GC and the proportion of amylose that leaches. It was shown that cooked rices of hard GC do not change in hardness over 24h, whereas rices of soft GC retrograde significantly over 24h. This leads to the conclusion that the mutation on exon 10 of the Wx gene affects the proportion of amylose bound to amylopectin and the proportion able to leach, and these structural differences alter the composition of the gel, which affects the amount of time the gel takes to reach a final hardness. The SNP described here completes the set of markers required to genotype for the current traits of cooking quality, but selecting the allele for soft texture has the negative result of also selecting for retrogradation potential.

Widespread sequence variation in the Epstein-Barr virus latent membrane protein 2A gene among northern Chinese isolates

Latent membrane protein 2A (LMP2A) is expressed in most Epstein-Barr virus (EBV)-associated malignancies. Besides its roles in the maintenance of latent infection and epithelial-cell transformation, LMP2A could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence polymorphisms in LMP2A from northern Chinese EBV-associated gastric carcinoma patients, nasopharyngeal carcinoma patients and healthy donors were identified and compared with the prototype B95-8 strain. Four consistent mutations were detected in all isolates. Frequent mutations in the analysed sequences distinguished two and seven types of sequence variation in exon 1 and exons 2-8, respectively, with no consistent association shown between the genotyping of the two gene fragments. The immunoreceptor tyrosine-based activation motif and PY motif in the amino terminus were strictly conserved. Nine of the 16 identified CTL epitopes were affected by at least one point mutation, which may confer complexity to proposed immunotherapeutic approaches for EBV-associated malignancies. Most changed epitopes showed higher mutation rates in tumour isolates than in throat-washing samples from healthy donors, in accordance with the idea that virus strains can evade immune surveillance by altering amino acids within LMP epitopes. This first detailed investigation of sequence variations in the LMP2A gene reveals classifiable sequence polymorphisms in exon 1 and exons 2-8, and encourages further work on the impact of viral gene variations on tumour persistence and CTL-based immunotherapy.