Rouba Hoteit

JAK2 V617F Gene Mutation in the Laboratory Work-Up of Myeloproliferative Disorders: Experience of a Major Referral Center in Lebanon

AIMS JAK2 V617F mutation is gaining more acceptance in laboratory testing as part of the differential diagnosis work-up of myeloproliferative disorders (MPD). This report is the first of its kind from Lebanon that analyzes the distribution of this mutation among a series of referred cases to a major tertiary referral center. METHODS Real-time polymerase chain reaction using JAK2 V617F MutaScreen assay (IPSOGEN Cancer Profiler) was performed on 229 patients. RESULTS JAK2 V617F mutation was found to be positive in 100% of polycythemia vera cases, 68.29% of essential thrombocythemia cases, and 55.28% of all MPD cases whereas negative in idiopathic erythrocytosis, reactive thrombocytosis, and other non-MPD cases such as acute chronic myeloid leukemias. CONCLUSION Our unique study in this sample of Lebanese patients shows extensive similarities of positivity of JAK2 V617F as compared with the international literature and for the same categories of clinical entities. This will constitute a baseline for future clinical studies that would also help determine prognosis of cases based on the absence or presence of this mutation.

Study of KIR genes in Lebanese patients with tuberculosis

A total of 103 Lebanese tuberculosis (TB) cases and 38 controls without TB were studied for the killer cell immunoglobulin-like receptors (KIR) genotypic profile using polymerase chain reaction sequence-specific primers. Patients and controls were assigned to the AA, AB or BB genotypes based on their A or B haplotype genetic make-up, and KIR gene frequencies were compared. We found an increase in the KIR A haplotype in TB patients compared to controls, and only KIR 2DL3 was found to be significantly more prevalent among TB patients. This confirms the findings of another unique international study performed in the Mexican population showing a greater repertoire of inhibitory KIR genes among TB patients than controls.

Vitamin D receptor biochemical and genetic profiling and HLA-class II genotyping among Lebanese with multiple sclerosis — A pilot study

BACKGROUND Multiple sclerosis (MS) is an autoimmune demyelinating disease affecting mostly young adult females with multifactorial etiology. Recent studies suggested that adequate vitamin D levels may lower the risk of developing MS. OBJECTIVES Our aim was to explore the relationship between vitamin D receptor (VDR) polymorphism, HLA-DR locus genotype, and serum vitamins D and A levels in the Lebanese population. METHODS Fifty MS patients were recruited for this study. The control group consisted of 48 healthy and 51 patients with other neurological disorders (non-MS). Biochemical analysis included serum 25 hydroxyvitamin D (25OHD) and vitamin A. Molecular analysis targeted VDR genotypes (ApaI, TaqI and BsmI) and low resolution HLA typing for DRB1 locus. RESULTS Healthy and non-MS groups had comparable parameters and were combined into one control group. No significant differences were found between MS and control groups for VDR genotypes. The frequency of HLA-DRB1*15 was significantly higher in MS patients (22%) compared to controls (8%) (p=0.018). Odds ratio for MS in the presence of DRB1*15 allele was 3.21 (p=0.018). Cosegregation with A (ApaI) and b (BsmI) alleles did not influence the risk for MS. 25OHD levels were significantly higher in MS patients compared to controls (p=0.002), due to more frequent oral supplementation (p=0.005). Vitamin A levels were comparable between the two groups. When all parameters were included in a logistic regression model adjusted for supplementation, only HLA-DRB1*15 (OR=3.42; p=0.027) contributed significantly to MS risk. CONCLUSION There was no association between serum vitamin D or A or VDR genotypes and MS. HLA-DRB1*15 was the major factor imposing more than 3 folds greater risk for developing MS among Lebanese.

KIR genotype distribution among patients with multiple myeloma: Higher prevalence of KIR 2DS4 and KIR 2DS5 genes.

Introduction Natural killer (NK) cells possess an antitumor activity against multiple myeloma cells proven by the susceptibility of plasmocytes to NK lysis. In the early stage of MM, the killing of MM cells is mediated by natural cytotoxicity receptors (NRC) and NKG2D-dependent pathway, while in the late stage, NK cells lose their killing potential against MM cells due to the high expression of HLA class I molecules on MM cells. Aim The aim of this paper is to study KIR expression of NK cells in MM patients and in healthy controls, to check for any association between KIR genotypes and MM. Methods KIR genotype was analyzed in 120 healthy Lebanese individuals and 34 MM patients using the KIR Genotyping SSP kit. Results KIR 2DS4*001/002 and KIR 2DS5 were found to be significantly more prevalent among MM patients as compared to controls. For MM patients, the AA, AB, and BB genotype frequencies were, respectively, 38.23%, 47.06% and 14.71% with an A:B ratio of 1.62:1. As for the healthy controls, the AA, AB, and BB genotype frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.80:1. Conclusion The interesting observation of the significant presence of KIR2DS4 and KIR2DS5 genes more among multiple myeloma patients than controls is worth further clinical, translational as well as survival research studies in these cases.

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.

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.

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 Hodgkins 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 Hodgkins 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 Hodgkins 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.

Frequency of triple mutations involving factor V, prothrombin, and methylenetetrahydrofolate reductase genes among patients referred for molecular thrombophilia workup in a tertiary care center in Lebanon.

AIM Molecular diagnostics has markedly improved the diagnosis and workup of different clinical conditions including hypercoagulable state or thrombophilia where different genes are involved. In this report, which is the largest report in the medical literature and the first in Lebanon, we describe the prevalence of simultaneous mutations in the three major thrombophilia genes Factor V, Factor II, and methylenetetrahydrofolate reductase. MATERIALS AND METHODS Using a polymerase chain reaction and reverse hybridization assay for the corresponding mutations identification, 2248 referred cases were analyzed. RESULTS Only 25 cases were found to be simultaneously positive for the three mutations at a prevalence rate of 1.1%. CONCLUSION Compared with other populations, this prevalence rate is considered high, possibly the highest, and warrants future clinical studies and follow-up.

Proposed Algorithm for the Best Detection of Different bcr-abl Gene Fusion Transcripts in Molecular Diagnostics Laboratories: Experience of a Major Referral Center

AIMS Detection of bcr-abl transcripts is important both in diagnosis as well as in prognostication and treatment modalities of different types of leukemia, both chronic and acute. However, the techniques employed are variable and different among laboratories. Our aim was to share with other labs a strategy/algorithm that we find highly useful for implementation to best detect all bcr-abl fusion transcripts for proper patient management. METHODS We have used two techniques for the detection of bcr-abl transcripts, an in-house developed polymerase chain reaction and a real-time quantitative commercial polymerase chain reaction (PCR) kit and tested 849 patients referred for initial screening for bcr-abl. RESULTS Out of 849 cases, 146 (17.2%) were positive for bcr-abl. Around 92.11% of the total bcr-abl positive cases (N=76) detected by the real-time quantitative technique were also positive by the gel-based PCR assay; however, six cases (around 7.89%) were missed by the real-time assay and detected by the other technique in chronic myelogenous leukemia-proven cases. CONCLUSION We highly encourage other laboratories to perform testing using a simple and inexpensive gel-based PCR screening assay followed by a real-time quantitative assay for a baseline bcr-abl expression level. This combination will enable laboratories to detect all the reported fusion transcripts in accordance with the clinical presentation of the patient as well as other laboratory tests for the best use of this genetic test in patient management and care.