DNA repair functional analyses of NBN hypomorphic variants associated with NBN-related infertility.

Nijmegen breakage syndrome caused by biallelic pathogenic variants of the DNA-damage response gene NBN, is characterized by severe microcephaly, cancer proneness, infertility, and karyotype abnormalities. We previously reported NBN variants in siblings suffering from fertility defects. Here, we identify a new founder NBN variant (c.442A>G, p.(Thr148Ala)) in Lebanese patients associated with isolated infertility. Functional analyses explored preserved or altered functions correlated with their remarkably mild phenotype. Transcript and protein analyses supported the use of an alternative transcript with in-frame skipping of exons 4-5, leading to p84-NBN protein with a preserved forkhead-associated (FHA) domain. The level of NBN was dramatically reduced and the MRN complex delocalized to the cytoplasm. Interestingly, ataxia-elangiectasia mutated (ATM) also shifted from the nucleus to the cytoplasm, suggesting some interaction between ATM and the MRN complex at a steady state. The ATM pathway activation, attenuated in typical patients with NBS, appeared normal under camptothecin treatment in these new NBN-related infertile patients. Cell cycle checkpoint defect was present in these atypical patients, although to a lesser extent than in typical patients with NBS. In conclusion, we report three new NBN-related infertile patients and we suggest that preserved FHA domain could be responsible for the mild phenotype and intermediate DNA-damage response defects.

Pharmacogenetics of coumarin dosing: prevalence of CYP2C9 and VKORC1 polymorphisms in the Lebanese population.

BACKGROUND: Polymorphisms in the genes encoding the cytochrome P450 2C9 enzyme (CYP2C9) and the vitamin K epoxide reductase (VKORC1) are known to contribute to variability in sensitivity to coumarins. Patients with certain common genetic variants of CYP2C9 (*2 & *3) or a VKORC1 polymorphism (-1639A Allele) require a lower dose of coumarin and are also at higher risk for over-anticoagulation and serious bleeding. In August 2007, the FDA label for warfarin was updated to highlight the benefit of genetic testing to predict warfarin response. AIM: Since the prevalence of these variants in the Lebanese population has not yet been reported, our aim was to determine the genotypes of CYP2C9 and VKORC1 in our population and to compare allele frequencies with previous findings from other ethnic groups. MATERIALS AND METHODS: CYP2C9 (*1/*2/*3) and VKORC1 (*A/*G) allelic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism assays in a diversified sample of 161 unrelated healthy Lebanese volunteers. RESULTS: The allele frequencies of CYP2C9 *2 and *3 were 0.112 and 0.096 respectively, whereas VKORC1-1639A was 0.528. Carriers of the CYP2C9 *2 or *3 represented 34.2% of the subjects, whereas those of the VKORC1-1639A represented 73.9%. CONCLUSION: Our data show no significant difference in the frequency of CYP2C9 allelic variants when compared to the Caucasian population, whereas the allelic frequency of VKORC1-1639A was very high. Over 50% of the Lebanese population seem to be carrying more than two independent risk alleles, and is therefore potentially at high risk of over-anticoagulation.

Prevalence of CYP2C19 polymorphisms in the Lebanese population.

Clopidogrel is one of the most commonly prescribed drugs, as its combination with low-dose aspirin is the recommended oral anti-platelet therapy, to prevent ischaemic events following coronary syndromes or stent placement. Numerous recent studies have shown that polymorphisms in the gene encoding the cytochrome P450 (CYP450) 2C19 enzyme (CYP2C19) contribute to variability in response to clopidogrel; patients with certain common genetic variants of CYP2C19 (2, 3) have a reduced metabolism of clopidogrel and have a higher rate of cardiovascular events or stent thrombosis compared to patients with the CYP2C19 (1) allele. CYP2C19 2 is most common in Caucasians, Africans and Asians while CYP2C19 3 has been found mostly in Asians. Since the prevalence of these variants in the Lebanese population has not yet been reported, our aim was to determine the genotypes of CYP2C19 in our population. CYP2C19 (1/2/3) variants were assessed by Polymerase Chain Reaction-Restriction Length Polymorphism (PCR-RFLP) assays in a representative sample of 161 unrelated healthy Lebanese volunteers. The allele frequencies of CYP2C19 2 and 3 were 0.13 and 0.03. Carriers of the CYP2C19 2 or 3 represented 24.2% of the subjects. Our data show no significant difference in the frequency of CYP2C19 allelic variants when compared to Caucasian populations and demonstrate that the application of the recent FDA recommendations would also be beneficial in Lebanon, allowing physicians to identify patients at high risk for atherothrombotic events, and eventually advising them to consider other antiplatelet medications or alternative dosing strategies in poor metabolizers.

Vitamin D receptor gene polymorphisms modulate the skeletal response to vitamin D supplementation in healthy girls.

OBJECTIVES: Vitamin D receptor (VDR) gene plays an important role in bone mass regulation. We have previously shown a beneficial effect of vitamin D supplementation on bone mass in girls. This study investigated whether the musculo-skeletal response to Vitamin D was modulated by polymorphisms in VDR gene. DESIGN: Randomized placebo-controlled trial. METHODS: 179 girls (10-17 years), were randomly assigned to placebo or Vitamin D3 for one year. VDR genotypes were determined in 167 girls using BsmI, TaqI and ApaI restriction enzymes. Bone mass at the spine, hip, forearm and total body, and lean mass were measured by DXA at baseline and at one year. RESULTS: After one year, VDR gene polymorphisms using Bsm1 and TaqI restriction enzymes were associated with percent changes in bone area, BMC and BMD at multiple skeletal sites in the Vitamin D3 group but not in the placebo group. The least increments were observed in the BB and tt genotypes. No similar effect was observed with ApaI enzyme. This relationship between VDR genotypes and changes in BMD and BMC remained significant after adjustment for puberty, changes in lean mass, height and bone area. CONCLUSION: VDR gene polymorphisms influence the skeletal response to vitamin D supplementation in healthy adolescent girls.

Molecular and clinical description of a girl with a 46,X,t(Y;4)(q11.2;p16)/45,X,der(4)t(Y;4)(q11.2;p16) karyotype and a small cryptic 4p subtelomeric deletion.

We report on a 13-year-old female with short stature, minimal axillary and pubic hair, no breast development, absence of uterus and ovaries, with the following karyotype on lymphocyte cultures: 46,X,t(Y;4)(q11.2;p16)[40]/45,X,der(4)t(Y;4)(q11.2;p16)[10]. Loss of the small derivative Y chromosome in 20% of the cells was also confirmed in skin fibroblast cultures. FISH analyses using Y centromere, SRY, subtelomere XpYp/XqYq, Y and 4 painting probes, confirmed the cytogenetic findings. High-resolution STS analyses using 40 markers covering the Y chromosome did not identify any deletion on the Y. However, de novo absence of the 4p subtelomeric region was noted by FISH, although this deletion was not revealed by Array-CGH at 1 Mb resolution, the last array clone being 0.35 or 1 Mb distal to the 4p FISH probe. The female phenotype of this patient must be due to the loss of the derivative Y chromosomes in some of her cells, especially the gonads, while the 4p subtelomeric deletion does not seem to contribute to her phenotype.

Outcome of chromosomally abnormal pregnancies in Lebanon: obstetricians' roles during and after prenatal diagnosis.

OBJECTIVES: To better understand obstetrician experiences in Lebanon when disclosing abnormal amniocentesis results. METHODS: Structured interviews with 38 obstetricians identified as caregivers from the American University of Beirut Medical Center Cytogenetics Laboratory database of patients with abnormal amniocentesis results between 1999 and 2005. RESULTS: Obstetricians were primarily male, Christian, and with an average of 14 years of experience. They reported doing most pre-amniocentesis counseling, including discussion of risk for common autosomal aneuplodies (95%), and procedure-related risk (95%). Obstetricians reported that 80% of patients at risk for aneuploidy underwent amniocentesis. The study population reported on 143 abnormal test results (124 autosomal abnormalities). When disclosing results, obstetricians reportedly discussed primarily physical and cognitive features of the diagnosis. They varied in levels of directiveness and comfort in providing information. Our records showed that 59% of pregnancies with sex chromosome abnormalities were terminated compared to 90% of those with autosomal aneuploidies; various reasons were proposed by obstetricians. CONCLUSIONS: This study is among the few to assess prenatal diagnosis practices in the Middle East, with a focus on the role of the obstetrician. Given the influence of culture and social norms on prenatal decision-making, it remains important to understand the various impacts on clinical practice in many nations.

A patient with duplication (7)(p22.1pter) characterized by array-CGH.

Approximately 40 patients with terminal duplication of the distal short arm of chromosome 7 have been reported, the smallest being dup(7)(p21). We report here on a patient with a smaller duplication, dup(7)(p22.1), detected on G-banding and characterized by array-CGH. We establish phenotype-karyotype correlations with the reported patients with other 7p duplications.

Peripheral neuropathy in thalassaemia.

BACKGROUND: Patients with thalassemia may complain of numbness and weakness of the lower extremities. The aim of the study was to document whether these patients suffer from a polyneuropathy and to determine any contributing factors for the development of neuropathy. PATIENTS AND METHODS: We examined 30 patients with thalassemia major and intermedia, clinically and electrophysiologically. We correlated these findings with demographics, blood status, and treatment and compared electrophysiologic data with 30 age- and sex-matched normal subjects or historical controls. RESULTS: We found that 78% of thalassemic patients suffered from a mild sensory polyneuropathy. The neuropathy seemed to be worse in the intermedia type. Thalassemic patients who received blood transfusions and deferoxamine had better nerve function than those who did not, irrespective of the dose of deferoxamine. The neuropathy was worse for older patients, irrespective of sex. The hemoglobin level, and the fact that some patients underwent splenectomy, did not affect the status of the patients' nerves. CONCLUSION: Patients with thalassemia may suffer from a sensory polyneuropathy especially as they grow older and if they are not optimally treated.

Prevalence of factor V Leiden, prothrombin and methylene tetrahydrofolate reductase mutations in women with adverse pregnancy outcomes in Lebanon.

OBJECTIVE: The purpose of this study was to determine the prevalence of factor V Leiden, prothrombin, and methylene tetrahydrofolate reductase gene mutations in women with adverse pregnancy outcome compared with women who had uneventful pregnancies. STUDY DESIGN: Between 2003 and 2005, pregnant women with > or = 1 unexplained second trimester abortion, > or = 1 intrauterine fetal death, severe preeclampsia, or severe intrauterine growth restriction (study subjects) were compared with control subjects (uneventful pregnancy) for the frequency of the mutations. RESULTS: The cases of 91 patients in each arm were analyzed. Obstetric complications were second trimester abortions (16.5%), intrauterine fetal death (53.8%), preeclampsia (8.8%), and severe intrauterine growth restriction (20.9%). Study subjects were more likely to be older and multiparous compared with control subjects. The 2 groups showed no difference in the incidence of smoking or family history of thrombosis, but study subjects were more likely to have a positive family history of obstetric complications. The prevalence of factor V Leiden (12.1% vs 18.7%; P = .304), prothrombin (7.7% vs 5.5%; P = .765), methylene tetrahydrofolate reductase gene mutations (53.8% vs 65.9%; P = .130), and > 1 mutation (11.0% vs 17.6%; P = .290) was not significantly different between study subjects and control subjects. CONCLUSION: Factor V Leiden, prothrombin, and methylene tetrahydrofolate reductase gene mutations did not seem to play a significant role in adverse pregnancy outcome in our population.

Apolipoprotein E gene polymorphism and allele frequencies in the Lebanese population.

Apolipoprotein E (ApoE) genotypes were studied in order to determine the prevalence in the Lebanese population and compare it with other populations. DNA from 160 unrelated healthy donors from our HLA-bank was used. ApoE genotype was determined using the CardioVascular Disease (CVD) StripAssay (this assay is based on a Polymerase Chain Reaction-Reverse Hybridization technique). The prevalence of genotypes E3/3, E3/4, and E2/3 was found to be 69%, 26%, and 22%, respectively, and 0.6% for each of E2/4 and E4/4 genotypes. The Lebanese population tested showed similarities to earlier reported ApoE genotypic distributions (high E3 allele frequency) but also peculiar differences especially to some Arabic countries (total absence of E2 allele among Saudis) and other populations. This is the first report from Lebanon that will serve as a template for future investigations of the prevalence of ApoE alleles in association with various clinical entities.

Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure.

Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.

Regression of skeletal manifestations of hyperparathyroidism with oral vitamin D.

CONTEXT: Parathyroidectomy is the only effective therapy for osteitis fibrosa cystica in hyperparathyroidism. OBJECTIVE: The objective of this study was to describe the changes of skeletal and nonskeletal manifestations in a patient with hyperparathyroidism and renal failure after oral vitamin D therapy. DESIGN: This was a descriptive case report. SETTING: The patient was followed up in a referral center. PATIENT: A 55-yr-old male patient with moderate renal failure was referred for expansile lytic lesions affecting several ribs and the spinous process of T12. His creatinine was 1.8 mg/dl; calcium, 8.9 mg/dl; PTH, 666 pg/ml; and 1,25 dihydroxy-vitamin D, 27 pg/ml. Bone mineral density (BMD) Z-scores by dual-energy x-ray absorptiometry were -4.1 at the spine, -1.7 at the hip, and -4.3 at the forearm. MAIN OUTCOME MEASURES: The main outcome measures were the skeletal manifestations of hyperparathyroidism. RESULTS: At 10 months of therapy, calcium level was 10 mg/d, PTH level declined to 71 pg/ml, and BMD increased by 12% at the spine and 18% at the hip. Computerized tomography (CT) cuts revealed marked regression in the lytic lesions. At 2 yr, BMD increased by an additional 6% at the spine, and there were no further changes in the lytic lesions by CT. The vitamin D receptor genotype using the restriction enzymes Bsm1, Taq1, and Apa1 was Bb, tt, and AA. CONCLUSIONS: We showed regression of severe skeletal abnormalities of hyperparathyroidism documented by serial CT images in response to oral vitamin D therapy. It is possible that the vitamin D receptor genotype of the patient modulated this response.

Molecular basis of oculocutaneous albinism type 1 in Lebanese patients.

Oculocutaneous albinism type 1 (OCA1) results from mutations in the tyrosinase gene, which lead to partial or complete loss of activity of the corresponding enzyme. A large number of mutations have been identified worldwide, providing insight into the pathogenesis of the disorder. We performed ophthalmic and dermatological exams on 30 Lebanese subjects with oculocutaneous albinism, then screened for mutations in the tyrosinase gene in an effort to establish the molecular basis of the disorder in our population and correlate it with phenotypic findings. The five exons of the gene together with the exon-intron boundaries and part of the promoter region were sequenced. Mutations were found in a total of 14 patients (47%) while no mutation was identified in the sequenced regions in 53% of patients. Fourteen different mutations were identified of which eight were novel while six had been previously reported. Mutations were mainly seen in patients with clinical findings, suggestive of OCA1A (64% of patients with OCA1A versus 25% of patients with OCA1B); therefore, the absence of mutations in some of the other patients may indicate the involvement of other genes.

Origin and history of the IVS-I-110 and codon 39 beta-thalassemia mutations in the Lebanese population.

Using restriction fragment length polymorphisms (RFLPs) and sequence haplotype analysis, we studied the chromosomal background of the beta-globin gene in 31 unrelated Lebanese IVS-I-110 or codon 39 (Cd39) subjects, and five normal betaAbeta/A individuals. Our results are compared with those from similar studies in other parts of the Mediterranean in an attempt to provide insights into historical patterns of selection and disease. The great majority of the Lebanese chromosomes with the IVS-I-110 mutation are associated with the RFLP haplotype I and sequence haplotype HT1, which is probably the ancestral structure on which the mutation first emerged. The remainder of the IVS-I-110 alleles are linked to the 5'-subhaplotype 12 RFLP haplotype and/or HTR sequence haplotype. In contrast, in Turkey, IVS-I-110 is associated with six distinct sequence haplotypes and four distinct RFLP haplotypes, suggesting that the mutation probably emerged there. The diversity of sequence haplotypes described in Turkey was probably generated through recombination or gene conversion events with the most frequent betaA autochthonous structures. Our data on Lebanese betaA chromosomes and Algerian betaA chromosomes, along with previously described Turkish betaA chromosomes, strengthen this hypothesis. Following its emergence in Turkey, the IVS-1-110 mutation was probably introduced to Lebanon later, by migration or settlements. Cd39 demonstrates a remarkable level of sequence and RFLP haplotype heterogeneity in Algeria, in contrast to its relative homogeneity in Turkish samples. However, its rarity in the Near East, and more specifically in Lebanon, does not allow us to draw any conclusions concerning its origin and gene flow.