First report on HLA-DPA1 gene allelic distribution in the general Lebanese population.

AIMS: HLA-DPA1 is an important marker in bone marrow and organ transplantation and a highly emerging screening parameter in histocompatibility laboratories. Being highly polymorphic, it has another significant value in detecting population origins and migrations. This is the first study to assess DPA1 allele frequencies in an Arab population. METHODS: The HLA DPA1 alleles were identified using the One-Lambda assays on a Luminex reverse SSO DNA typing system. Our study included 101 individuals coming from different Lebanese geographical areas representing the different communities and religious sects of the country. RESULTS: We compared the results of this study to 16 different populations and found very interesting similarities and differences between Lebanese people and individuals of European ancestry. CONCLUSION: This study is the first to describe the different allelic frequencies of HLA-DPA1 in the Lebanese population and will serve as a template that can be later used for disease association studies both at the level of the country and internationally.

Use of the Human Coronavirus 2012 (MERS) GeneSig kit for MERS-CoV detection.

INTRODUCTION: Mortality due to MERS-CoV infection is common especially among immunocompromised patients. The pathogenesis and the transmission mode of this virus are still not well understood. The name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qRT-PCR assays for MERS-CoV detection was retrieved from the first detected case isolate. The employed assays target various regions including the area upstream of the envelope gene (upE) that is used for screening and the open reading frames (ORF) 1a and 1b used for confirmation. AIM: This study assesses the use of a MERS-CoV specific assay for screening of respiratory samples in anticipation of the possible spread of the virus in the region. METHODS: 46 respiratory specimens were tested using the qualitative one-step qRT-PCR GeneSig Human Coronavirus 2012 (MERS) kit (PrimerDesign™). RESULTS: Out of the 46 tested samples, 45 were negative for MERS-CoV and one sample was found MERS-CoV positive. CONCLUSION: The GeneSig Human Coronavirus 2012 (MERS) kit is very useful for the screening of suspected respiratory cases in the Middle East area as well as other regions.

KIR genotype distribution among Lebanese patients with Hodgkin's lymphoma.

INTRODUCTION: In addition to their important role in fighting infection, natural killer cells are cytotoxic to cancer cells. Studies demonstrated that some KIR genes were responsible for the reduction of the risk of Hodgkin's lymphoma (HL) while others were associated with an increased risk of HL. AIM: The aim of this study is to assess KIR genotypic distribution in Lebanese patients with Hodgkin's lymphoma. METHODS: KIR genotype was analyzed in 41 HL patients and 120 healthy Lebanese individuals using the KIR Genotyping SSP kit. RESULTS: No significant association between HL and any KIR gene was found. Among HL patients, the AA, AB, and BB genotype frequencies were, respectively, 41.46%, 43.9% and 14.63% with an A:B ratio of 1.73:1. As for the controls, the AA, AB, and BB genotype frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.79:1. CONCLUSION: In this first study from the Mediterranean region, KIR genotype does not seem to be associated with Hodgkin's lymphoma. Further clinical and translational research is needed to rule out the protective or predisposing role of KIR genes in this important clinical entity.

Invivoscribe BIOMED-2 primer mixes in B-cell immunoglobulin gene rearrangement studies: experience of a molecular diagnostics laboratory in a major tertiary care center.

AIMS: To determine the frequency of positive reactions obtained using the Invivoscribe BIOMED-2 kit for B-cell gene rearrangement studies in leukemias and lymphomas. MATERIALS AND METHODS: We reviewed the gel patterns for 192 samples tested, using the above-mentioned kit and matched the positive signal with the corresponding mix available in the assay kit. RESULTS: 92.2% had immunoglobulin heavy-chain clonality, of which 74% were detected by the IgH VH-FR1+JH primer set, 75.5% by IgH VH-FR2+JH primer set, 65.1% by IgH VH-FR3+JH primer set, 26% by IgH DH+JH primer set, and 2.1% by IgH DH7+JH primer set. In addition, 55.7% had clonality in the kappa light chain, where 33.3% were positive by the IgK Vκ +Jκ primer set and 39.6% by IgK Vκ and INTR+Kde primer sets. Clonality in the lambda light chain of immunoglobulins was detected in 17.7% of specimens tested using the IgL Vλ +Jλ primer set. CONCLUSION: All primer mixes provided by the assay were positive. Thus, the Invivoscribe BIOMED-2 B-cell gene rearrangement kit is very reliable in adequately covering all targets represented by the master mixes. This assay is an integral part of the differential diagnosis of clonal populations of cells. Our report is the first in the literature that describes the full range of coverage of the BIOMED-2 primer mixes provided in this assay.

Natural Killer Cell Immunoglobulin-like Receptor (KIR) genotypes in Follicular Lymphoma patients: results of a pilot study.

AIMS: The Natural Killer Cell Immunoglobulin-like Receptor (KIR) genotype profiling in Follicular Lymphoma has not been reported before in the literature. MATERIALS AND METHODS: DNA extracted from 20 Follicular Lymphoma patients and 62 healthy controls was analyzed for KIR genotyping using a polymerase chain reaction/sequence specific primers technique (PCR/SSP) for the presence of 16 KIR gene and pseudogene loci. RESULTS: The AA, AB, and BB genotype frequencies were, respectively, 20%, 60% and 20% with an A:B ratio of 1:1. KIR 2DL4, KIR 3DL2, KIR 3DL3, and KIR 3DP1*003 were presented in all individuals. No significant difference between patients and controls was detected. CONCLUSION: KIR genotyping profile does not seem to be associated with Follicular Lymphoma. The results presented in this pilot research represent the first international report about this important clinical entity.

Significance of the use of the ViennaLab "Cardiovascular Disease panel" (CVD) Assay as a reflex test for the "Factor V/II/MTHFR Assay".

INTRODUCTION: Trends toward identifying risk factors of thrombotic complications had become essential as an attempt to prevent and decrease the incidence of the complications. Thrombosis has been associated with predisposing factors like mutations in FV, PTH, MTHFR and other genes. AIM: Evaluate whether the CVD StripAssay has an added value in the screening for more thrombophilia risk factors, which may predispose for the development of cardiovascular diseases and other thrombotic clinical conditions. METHODS: We compared the results for 94 patients who were previously tested for Factor V, Factor II and MTHFR gene mutations using the ViennaLab FV-PTH-MTHFR StripAssay, and for whom additional testing for the Cardiovascular Disease panel (CVD StripAssay, ViennaLab) was requested. RESULTS: Using the CVD StripAssay, 66% of patients who had no mutations when tested using the FV-PTH-MTHFR StripAssay or carried a mutation for MTHFR, were found to have additional genes' SNPs or mutations that are highly associated with a risk of thrombosis as per the available international literature. CONCLUSION: This observation is of extreme importance in clinical practice for the introduction of the extended CVD panel into routine molecular diagnostic test menus and highlights the importance of genetic analysis of the implicated genes in the management of patients with a thrombotic episode presentation.

HLA class I allele frequencies in the Lebanese population.

The highly polymorphic Human Leukocyte Antigen system encompasses different loci that have been studied in transplantation as well as diseases and population associated research. This study is the first and largest of its kind to describe the distribution of HLA-A, -B and -C alleles in Lebanon. Respectively, 1994, 1309 and 1163 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-A, HLA-B and HLA-C alleles using the polymerase chain reaction/Sequence specific priming (PCR-SSP) method. Our data were compared to that of several populations with interesting and common findings shared with the Moroccan, Jordanian, Tunisian, Omani, Korean, Chinese, Japanese, Peruan, Bulgarian, Irish, Polish, Spanish, Swiss, American, African and Brazilian populations. The following data concerning the Lebanese population will help future investigators to study the relation of HLA-A, -B and -C alleles with common diseases in Lebanon and will add to the available international literature. This new data will serve as a major reference report in the region.

HLA class II allele frequencies in the Lebanese population.

AIMS: Being one of the most polymorphic genetic systems , the Human Leukocyte Antigen system is divided into class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DP, -DQ and -DR). This study is the first and largest of its kind to describe the distribution of HLA-DQB1 and HLA-DRB1 alleles in Lebanon and the region. METHODS: Respectively, 560 and 563 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-DQB1 and HLA-DRB1 alleles using the polymerase chain reaction/sequence specific priming (PCR-SSP) method. RESULTS: Our data were compared to that of several populations with interesting common findings between the Lebanese, Jordanian, Bahraini, Saudi, Kuwaiti, Tunisian, Korean, Japanese, Thai, Irish, Bulgarian and Polish populations. CONCLUSION: These data about the Lebanese population are going to aid future researchers to study the relation of HLA-DQB1 and HLA-DRB1 alleles with major and common diseases in the Lebanese population and will add to the available international literature associated with these loci. In addition it will serve as a reference for the future national bone marrow registry program in our country. We also reviewed the literature for the described association between HLA-DRB1 and -DQB1 loci and different disease entities.

JAK2 V617F "indeterminate" results by MutaScreen can be easily resolved using MutaQuant kits.

AIM: Janus kinase 2 (JAK2) V617F mutation testing has revolutionized the classification of myeloproliferative disorders, for which several tests have been introduced for qualitative and quantitative diagnostics including the MutaScreen and MutaQuant kits by IPSOGEN. One interesting technical observation are those values detected by MutaScreen kits, which have typically "indeterminate" meaning at a provided reference strand cutoff point and cannot be classified as either positive or negative for JAK2 V617F mutation. RESULTS: We ran 10 different patients with such a finding using the MutaQuant kit and got a better resolution and interpretation into clear-cut negative or positive cases, which were also clinically followed and confirmed as being nonmyeloproliferative or myeloproliferative entities, respectively. CONCLUSION: We propose that it is important to not consider the indeterminate or "at-the-reference strand" results obtained by MutaScreen as positive but rather perform additional testing using MutaQuant kits or other JAK2 quantitative assays. For laboratories that can afford it and utilize both assays, it may be a better strategy to directly initiate diagnostic testing using the MutaQuant rather than the MutaScreen kit.

Human leukocyte antigen-DQA1 gene allelic distribution: experience of a major tertiary care center in Lebanon.

AIMS: DQA1 is a human leukocyte antigen (HLA) class II molecule that is similar to the other class II molecules DR and DP. This study is the first of its kind to describe the distribution of HLA-DQA1 alleles in Lebanon. METHODS: HLA-DQA1 typing was detected using the polymerase chain reaction/sequence-specific priming method in 111 Lebanese individuals referred for HLA typing and possible bone marrow donation. RESULTS: Our data was compared to that of several populations. Some similarities were found between the Lebanese, Tunisian, Spanish, and Kuwaiti populations. CONCLUSION: This very first report from Lebanon will be of great help for later research to study the association of DQA1 alleles with major diseases in the Lebanese population and will add to the published international literature related to this important histocompatibility locus.

First report on HLA-DPB1 gene allelic distribution in the general Lebanese population.

AIMS: Interest in the importance of the HLA-DPB1 gene has increased lately, especially in transplantation outcomes in addition to other HLA CII molecules. This is the first study that assesses HLA-DPB1 typing among healthy Lebanese individuals. METHODS: The HLA-DPB1 alleles were detected using the polymerase chain reaction/sequence-specific priming method and our study included 130 individuals. HLA-DPB1 was determined in different ethnic groups and in relation to various clinical entities. RESULTS: We compared our data with the existing literature and found an interesting similarity between the Lebanese and Greek populations with highly specific alleles not reported in other communities. CONCLUSION: Our study will form the basis for further research to study the correlation between HLA-DPB1 and various clinical entities as well as a primer study for other transplantation centers.

JAK2 V617F mutation detection: laboratory comparison of two kits using RFLP and qPCR.

AIM: The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis with an increased demand for testing using molecular techniques. The latter are diversified and all aim to simplify the methods employed for detection. MATERIALS AND METHODS: In this study, two detection kits were compared: one using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) (JAK2 Activating Mutation assay; InVivoScribe Technologies, San Diego, CA) and the other using real-time quantitative PCR (JAK2 MutaScreen Kit assay; Ipsogen, Marseilles, France). RESULTS: A total of 80 reactions were compared using the two techniques and the results showed a perfect concordance between the two methods. CONCLUSION: We conclude that both PCR-RFLP and quantitative PCR are extremely useful and sensitive techniques for the detection of the JAK2 V617F mutation with quantitative PCR being more time effective and less expensive.

Distribution of cytokine gene polymorphisms in the general Lebanese population: the first report.

CONTEXT: Cytokines are polypeptide regulatory molecules that play a significant role in inflammatory and regulatory responses of the immune system. Several cytokine gene polymorphisms have been studied to date and have been found to be associated with distorted cytokine production or activity by affecting transcriptional regulation and with vulnerability to a variety of infectious and autoimmune diseases as well as to transplant rejection. RESULTS: We studied 106 healthy Lebanese individuals using polymerase chain reaction/sequence-specific priming technique to detect 22 single-nucleotide polymorphisms within 13 cytokine genes: IL1alpha 889-T/C, IL1beta 511-T/C, IL1beta +3962-T/C, IL1R pst1 1970-T/C, IL1RA mspa1 11100-T/C, IL4Ralpha 1902-G/A, IL12 1188-C/A, IFNgamma 874-A/T, TGFbeta codon 10-C/T, TGF-beta codon 25-G/C, TNFalpha 308-A/G, TNFalpha 238-A/G, IL2 166-G/T, IL2 330-T/G, IL4 1098-T/G, IL4 590-T/C, IL4 33-T/C, IL6 174-C/G, IL6 nt565-G/A, IL10 1082-G/A, IL10 819-C/T, and IL10 592-A/C. We compared our results to those reported in other populations with similarities observed between the Lebanese and the Italian populations. CONCLUSION: The study of different cytokine polymorphisms will aid in understanding the susceptibility of populations to various diseases, and this is the first report from the Lebanese community.

Absence of JAK2 V617F mutation in thalassemia intermedia patients.

JAK2 is a cytoplasmic tyrosine kinase that has a vital role in signal transduction from several hemopoietic growth factor receptors. The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia Vera, essential thrombocythemia, and idiopathic Myelofibrosis but has not been previously described in Thalassemia patients. We studied 36 Lebanese patients diagnosed with thalassemia intermedia and assessed the presence or absence of the JAK2 V617F mutation using JAK2 activating mutation assay (In VivoScribe Technologies) and Polymerase Chain Reaction (PCR). None of the thalassemia intermedia patients were positive for this mutation. To our knowledge, this study is the first to determine the status of JAK2 V617F mutation in thalassemia intermedia patients and expands the international published literature on JAK2. The latter's V617F mutation does not seem to play a role in this hematologically important clinical entity.

Prevalence of the prothrombin G20210A polymorphism in the Lebanese population: use of a reverse hybridization strip assay approach.

The factor II (prothrombin) G20210A gene polymorphism is the second most common SNP reported in VTE where it is associated with elevated plasma prothrombin levels and with a 3-fold increased risk. We studied the distribution of the G/G, G/A, and A/A genotypes of the Prothrombin G20210A gene mutation in the general Lebanese population using a novel technique in order to assess their prevalence, compare the results to previously reported data and to describe an available method that will permit easy and fast identification of the mutation. Prothrombin different genotypes were determined using the Cardiovascular Disease (CVD) StripAssay which is based on a Polymerase Chain Reaction-Reverse hybridization technique and DNA from 205 unrelated healthy donors from our HLA-bank was used. The prevalence of G/G, G/A, and A/A genotypes was found to be 98.54, 1.46, and 0%, respectively, with G and A allelic frequency of 99 and 1%, respectively. The sampled Lebanese population showed prothrombin genotypes distribution similar to Caucasians, and our results are comparable to other reports on the Lebanese healthy individuals. However, this is the first report on the prevalence of prothrombin G20210A mutation using this technique. Our results suggest that this approach is reliable and can be used as an assessment for thrombophilia profile. In addition, future investigations should be conducted to assess the contribution of the prothrombin G20210A mutation, on its own and in collaboration with other factors, in various clinical entities notably VTE.

MEFV gene mutations spectrum among Lebanese patients referred for Familial Mediterranean Fever work-up: experience of a major tertiary care center.

Familial Mediterranean Fever (FMF) is an autosomal recessive inflammatory disorder predominantly affecting people living in or originating from areas around the Mediterranean Sea, mainly Jews, Armenians, Turks, and Arabs. It is characterized by recurrent attacks of inflammation of serosal membranes and fever resulting in acute abdominal, chest, or joint pain. Over 50 MEditerranean FeVer (MEFV) mutations and polymorphisms have been identified in FMF patients. The objective of this study was to analyze the distribution and frequencies of 12 MEFV mutations in 266 referred Lebanese patients using a reverse-hybridization assay. Of the 266 patients, 129 (48.5%) were positive for at least one mutation and 137 (51.5%) had no mutations detected. Of the 129 patients with mutations, 35 were homozygous, 41 were compound heterozygous and 53 were heterozygous. The five most common mutations M694V, E148Q, V726A, M694I and M680I (G/C) accounted for 26.1, 22.2, 21.3, 9.6 and 7.7%, respectively. The A744S, F479L, R761H and I692del were encountered in 2.9% of patients; P369S and M680I (G/A) were found in 1.2% of patients while K695R was absent. The spectrum of the MEFV mutations among our sampled Lebanese FMF patients shows the high heterogeneity at the allelic level when compared to Arab and non-Arab populations. The most important feature was the relatively high frequency of the E148Q in our study group that allows us to question it as a mutation rather than a polymorphism. Further studies should be conducted to evaluate the role of the E148Q allele.

Frequency distribution of the G/A alleles of the beta-fibrinogen gene in the Lebanese population.

Fibrinogen is a plasma protein that has been reported to be associated with an increased risk of atherothrombotic diseases and venous thrombosis. The most common polymorphism that has been studied so far in different populations is the G-455-->A polymorphism in the promoter region of the beta-fibrinogen gene. We studied 160 healthy unrelated Lebanese individuals for the prevalence of -455G/G, -455G/A and -455A/A genotypes of the beta-fibrinogen gene and the frequency of G and A alleles using a reverse hybridization PCR assay. The prevalence of the G/G, G/A, and A/A genotypes were found to be 60.6, 31.9 and 7.5%, respectively. The frequency of the G and A alleles were found to be 0.77 and 0.23, respectively. As compared to other ethnic groups, the Lebanese individuals were found to have a relatively high prevalence of the A allele which may predispose them to develop cardiovascular diseases as well as thrombotic events. This study provides additional unique genetic information pertaining to the Lebanese population.

Factor XIII gene V34L mutation in the Lebanese population: another unique feature in this community?

We studied the distribution of the Factor XIII gene V34L polymorphism in a sample of healthy Lebanese individuals to assess its prevalence and compare it with other populations. Factor XIII genotypes were determined using the Cardiovascular Disease (CVD) StripAssay (ViennaLab, Austria), which is based on a Polymerase Chain Reaction-Reverse hybridization technique. DNA from 205 unrelated healthy donors from our HLA database was used. The prevalence of Wild type, heterozygous, and homozygous genotypes was found to be 74.2%, 22.4%, and 3.4% respectively. The sampled Lebanese population showed that the prevalence of V34L carriers (25.8%) was lower than Caucasians in general (44.3%) and, interestingly, with a low allele frequency of 0.14 similar to that in Blacks and South Asians. This first report from Lebanon sheds light on an additional unique genetic feature of this population and will prospectively serve as a baseline statistical data for future investigations of the prevalence of Factor XIII V34L mutation in association with various clinical entities notably cardiovascular diseases.

Plasminogen Activator Inhibitor-1 (PAI-1) gene 4G/5G alleles frequency distribution in the Lebanese population.

Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. Increased plasma PAI-1 levels play an essential role in the pathogenesis of cardiovascular risk and other diseases associated with thrombosis. The 4G/5G polymorphism of the PAI-1 promoter region has been extensively studied in different populations. We studied 160 healthy unrelated Lebanese individuals using a reverse hybridization PCR assay to detect the 5G/5G, 4G/5G and, 4G/4G genotypes of the PAI-1 gene and the frequencies of the 4G and 5G alleles. We found that 4G/5G genotype was the most prevalent (45.6%) followed by 5G/5G (36.9%) and 4G/4G (17.5%). The frequencies of the 4G and 5G alleles were calculated to be 0.403 and 0.597, respectively. Compared to other ethnic communities, the Lebanese population was found to harbour a relatively high prevalence of the rare 4G allele. This, in turn, may predispose this population to develop cardiovascular diseases and other thrombotic clinical conditions. This study aids to enhance our understanding of the genetic features of the Lebanese population.

Molecular frequency of BCL2/JH t(14; 18) using PCR among Lebanese patients with follicular lymphoma: another piece of the geographical map revealed.

In follicular lymphoma the frequency of translocation t(14;18) varies considerably across different geographic regions ranging from up to 89% among the American follicular lymphoma to around 30% in the Japanese lymphoma. Neighboring and regional countries varied in their frequency reporting like in Israel (22 of 36 cases; 61%), Turkey (46 of 67 cases; 68.7%), and Jordan (4 of 5 cases; 80%). To our knowledge, this is the first study conducted in Lebanon to determine the frequency of this translocation in follicular lymphoma patients. Of 42 cases diagnosed with follicular lymphoma at the American University of Beirut Medical Center, amplifiable DNA was extracted from the corresponding paraffin embedded tissues and tested for t(14; 18) translocation using PCR amplification of the MBR and MCR breakpoints (INVIVOSCRIBE, CA, USA). We found that 19 patients were positive for t(14; 18) (45.2%) while 23 were negative (54.8%). Among the 19 positive cases, bcl2 was positive in 10 cases (52.6%). The majority of the cases were positive for MBR (40.47%), while only two cases were positive for MCR (4.76%). This study expands the geographical map of the distribution of bcl-2 gene rearrangement in follicular lymphoma patients in the Middle East region. The interesting low frequency of t(14;18) in Lebanese follicular lymphoma patients (45.2%) stands out among several other increased frequencies in surrounding and regional countries. In addition, in this patient population, there is a decreased frequency of the MBR breakpoint (40.47%) while that reported in the literature ranges from 50 to 60%.