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.

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.

TPOX Triallelic Genotype: An Interesting Pattern to Be Noted in Bone Marrow Transplantation Monitoring.

AIMS: TPOX triallelic genotypic pattern has been described in the setting of forensic and paternity testing but not in bone marrow transplantation (BMT) monitoring for graft engraftment. MATERIALS AND METHODS: A total of 50 cases have been studied using the AmpFLSTR(®) Identifiler™ polymerase chain reaction amplification kit as part of the workup of patients and donors before and after BMT at the American University of Beirut Medical Center. RESULTS: Of the 50 studied cases, 49 showed typical allelic patterns of the variable short tandem repeats detected by the assay; however, one single patient showed a biallelic TPOX genotype in the pre-BMT specimen but a triallelic pattern in the post-BMT sample. CONCLUSION: Triallelic patterns of TPOX should also be considered in the context of BMT monitoring testing where misinterpretation of the allelic pattern can lead to wrong unwanted conclusions related to the graft condition and proper quantification of donor DNA.

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.

Should we screen for hereditary hemochromatosis in healthy Lebanese: a pilot study.

Hereditary hemochromatosis (HHC) is a genetic disorder of iron metabolism characterized by abnormal accumulation of iron that may lead to organ damage and death. Diagnosis is usually based on various genetic and phenotypic criteria. The study goals were to perform mutation analysis for 18 different mutations associated with HHC in healthy Lebanese, determine their allele frequency, and compare iron-overload status in identified carriers versus those found to be wild-type for mutations analyzed. 116 healthy adults (59 males and 57 females) underwent DNA testing for 18 different HHC mutations, and biochemical testing for percent transferrin saturation (%TS) and ferritin. C282Y mutation was not detected. Only H63D mutation (rs1799945) was found with an overall carrier frequency of 25.8% (24.1% heterozygous and 1.7% homozygous). %TS and ferritin differed significantly between genders. %TS and ferritin were significantly higher in males with H63D mutation when compared to males with wild-type (P=0.001, 0.019; respectively); but not in females. The proportion of subjects with increased %TS and serum ferritin was not statistically different between those with H63D mutation and the wild-type in either gender. In addition, none of the subjects had concurrent increase in %TS and ferritin. In conclusion, the H63D carrier frequency in healthy Lebanese is comparable to other populations in the region, and it does not result in significant biochemical iron overload. Moreover, in the absence of the C282Y mutation, genetic screening for HHC is not recommended according to this preliminary study in healthy Lebanese.

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.

Laboratory referral rates of genetic tests for thrombophilia workup in a major referral center.

AIMS: The rate of laboratory referrals for thrombophilia patients' genetic workup was assessed and compared among the medical and surgical specialties and subspecialties at a major tertiary care center in Lebanon. METHODS: DNA extraction was performed using the PEL-FREEZ extraction kit (PEL-FREEZ; DYNAL) and the Factor V, prothrombin, and methylenetetrahydrofolate reductase genotypic profiles were done using the FV-PTH-MTHFR StripAssay kit (ViennaLab) that employs a polymerase chain reaction-reverse hybridization method. A total of 2238 referred cases were analyzed. RESULTS: Around 42.23% of all referred cases turned out to have a thrombosis-associated mutation. Referrals from medical and surgical specialties were almost equal. In the surgical specialty, most referrals came from the department of Obstetrics and Gynecology, while in the medical speciality, most of the workup referrals originated from the Hematology/Oncology physicians. However, low referral rates were reported from the emergency department and family medicine practitioners. CONCLUSION: Genetic testing for thrombophilia workup is gaining more importance among the different medical and surgical specialties and is worth being introduced into the offered test lists of all established molecular diagnostics laboratories.

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.

The use of a reverse hybridization strip assay for the study of hemochromatosis-associated gene mutations in Lebanon.

AIMS: Hereditary hemochromatosis (HHC) is the most commonly identified autosomal recessive genetic disorder in the Caucasian population and HFE gene mutations are highly concentrated among European populations. This is the first study that screens for HHC-related gene mutations in a healthy Lebanese sample population. METHODS: Using the reverse hybridization Hemochromatosis StripAssay A from ViennaLab, the DNA extracted from a total of 116 healthy volunteers (59 males and 57 females) was analyzed, looking for 18 different mutations in the HFE, ferroportin, and transferrin genes. RESULTS: For the HFE gene, the C282Y mutation was not detected, but the H63D mutation was found with an overall carrier frequency of 25.8% (24.1% heterozygous and 1.7% homozygous). None of the mutations in the transferrin and ferroportin genes was identified. CONCLUSIONS: The Hemochromatosis StripAssay A from ViennaLab provides an easy and reliable technique for simultaneous screening of the different HFE gene mutations. This first study in Lebanon represents a baseline report for further future studies in the field using this easy technique with a reasonable turnaround time for diagnosis. We also note that ferroportin and transferrin gene mutations have not been detected in this population sample and larger clinical studies will be needed to better estimate their prevalence.

Comparison of neutrophil volume distribution width to C-reactive protein and procalcitonin as a proposed new marker of acute infection.

BACKGROUND: The aim of this study was to assess the use of neutrophil distribution width (NDW) and to compare it to C-reactive protein (CRP) and procalcitonin (PCT), in the detection of early sepsis in the intensive care unit. METHODS: Subjects (N = 166) were divided into 4 groups: healthy, acute inflammatory non-infectious (AINI), localized infection, and systemic infection, according to clinical history and cultures. NDW, CRP, and PCT were compared among the different groups using multivariate analysis of variance (MANOVA). Diagnostic efficacy was assessed using receiver operating characteristic curves and areas under the curves (AUC). RESULTS: The lowest mean(NDW) was found in the healthy group (n = 41), followed by the AINI (n = 20), localized infection (n = 55), and systemic infection (n = 50) groups. AUC(NDW) was 0.877 for infected (localized + systemic) vs non-infected (healthy + AINI) groups, and 0.965 for systemic infection vs non-infected groups. A cut-off of 21.9 resulted in 90% sensitivity, 92% specificity, 90% positive predictive value, and 92% negative predictive value (AUC(NDW) = 0.965, 95% confidence interval 0.935-0.995). According to MANOVA, only NDW was able to differentiate an acute inflammatory process from early infection in postoperative patients, but not healthy from AINI subjects. CONCLUSIONS: NDW had the highest diagnostic accuracy and is available with the complete blood count with differential (CBC). It may be a promising parameter to aid in the diagnosis of acute infection in adults, provided the possibility of haematological disorders is first ruled out.

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.

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.

Successful pregnancy involving a man with chronic myeloid leukemia on dasatinib.

Dasatinib is a highly potent Bcr-Abl inhibitor that is approved for the treatment of imatinib-resistant or -intolerant chronic myeloid leukemia (CML). The potential effects of dasatinib on sperm counts, sperm function, and fertility have not been studied yet. There is only one report in the medical literature of successful pregnancies while patients were taking dasatinib, thus making our case the second report. Here, we present the case of a 38-year-old man who conceived a healthy baby while on dasatinib therapy.

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.

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.

Killer cell immunoglobulin-like receptor genotypes in Behçet's disease patients: any role for the 3DP1*001/002 pseudogene?

AIMS: Genotypic profiles of the natural killer cell immunoglobulin-like receptors (KIR) have been reported to vary among different ethnic groups and variable clinical entities. This study represents the second report on its distribution among patients with Behçet's disease (BD). We studied 43 unrelated Lebanese Behçet's patients, had their DNA typed using sequence-specific primer technique for the presence of 16 KIR genes and pseudogenes loci, and compared them to the general Lebanese population. RESULTS: In addition to sharing common features with the general population, the AA genotype was still the most frequent--however, with five new KIR profiles identified. There was no statistically significant distribution of the different KIR genes between the cases (BD patients) and controls (Lebanese population); however, KIR3DP1*001/002 was found to be significantly different between the BD patients and the Lebanese population, but this significance was lost after correction for all KIR loci. CONCLUSION: The results lead to an interesting future research question of whether or not KIR genotype is involved in the predisposition to or pathogenesis of BD especially that a pseudogene is controversially in question. This is the second report that describes the KIR genotypic profile in such an important clinical disease but the first to shed a light on the possible role of a pseudogene.

Distribution of killer cell immunoglobulin-like receptor (KIR) genotypes in patients with familial Mediterranean fever.

Genotypic profiles of the natural killer cell immunoglobulin-like receptors (KIR) have been reported to vary among different ethnic groups and variable clinical entities. This study represents the first report on its distribution among patients with familial Mediterranean fever (FMF). We studied 56 unrelated Lebanese FMF patients, had their DNA typed using sequence-specific primer (SSP) technique for the presence of 16 KIR gene and pseudogene loci, and compared them to the general Lebanese population. The AA1 genotype was the most frequent in both the FMF and control groups. Six new KIR profiles were identified. The FMF group showed a higher prevalence of KIR 3DP1*003 (p<0.05) and an increase in the BB genotype compared with controls. The results lead to an interesting future research question of whether or not KIR genotype is involved in the predisposition to or pathogenesis of FMF. This is the first report that describes the KIR genotypic profile in this important clinical disease.

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.

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.