Walid Al-Achkar

Mutations of the Connexin 26 gene in families with non-syndromic hearing loss.

Autosomal recessive non-syndromic hearing impairment (ARNSHI) is caused by mutations in the gap junction gene GJB2 (Connexin 26; Cx26) in numerous human populations. The aim of this study was to determine the frequency of six GJB2 mutations in 50 Syrian families with congenital deafness and in 180 controls. PCR-RFLP was used to detect the 35delG, 167delT, M34T, W24X, W77R and E47X mutations, and direct sequencing was performed for the 35delG mutation. The data revealed a high prevalence of the 35delG mutation among deaf families. Homozygous 35delG was detected in fifteen of the Syrian families (30%). A compound heterozygous genotype was observed in two families: one with the 35delG/167delT mutation (2%) and one with the 35delG/M34T mutation (2%). Nine families were heterozygous with no second identified mutation in Cx26: four with 35delG+/unknown (8%), four with 167delT/unknown (8%) and one with M34T/unknown (2%). The W24X, W77R and E47X mutations were not detected in any of the study subjects. Three individuals with the heterozygous 35delG genotype (1.66%) and five with the heterozygous 167delT genotype (2.77%) were detected among the controls. No other mutations were found among the controls. These results have important implications for the diagnosis and counseling of families with Cx26 deafness.

A new t(9;11;20;22)(q34;p11.2;q11.21;q11) in a Philadelphia-positive chronic myeloid leukemia case

The so-called Philadelphia (Ph) chromosome is found in over 90% of cases of chronic myeloid leukemia (CML). Of these cases, 2–10% demonstrate complex translocations involving a third chromosome in addition to chromosomes 9 and 22. Since the majority of CML cases are currently treated with imatinib, variant rearrangements tend to have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. This study evaluated a CML case with complex chromosomal aberrations not previously observed. A four-chromosome translocation involving chromosomal regions including 11p11.2 and 20q11.21 in addition to 9q34 and 22q11 was characterized in detail using array-proven multicolor banding (aMCB), a technique which has proven to be of significance in characterizing breakpoint regions in detail. Underlying mechanisms and prognostic factors are discussed.

Molecular Update of β-Thalassemia Mutations in the Syrian Population: Identification of Rare β-Thalassemia Mutations

Abstract β-Thalassemia (β-thal) is an autosomal recessive disorder characterized by variable degrees of anemia, bone marrow hyperplasia, splenomegaly, and complications related to the severity of the anemic state. The β-thalassemias result from mutations in and around the β-globin gene (HBB) located as a cluster on the short arm of chromosome 11. In Syria, β-thal is highly prevalent. The main aim of this study was to identify the frequency of HBB mutations in 189 Syrian β-thal patients and carriers of β-thal. Out of the 189 patients and carriers recruited in this study, 181 patients had at least one HBB mutation and eight patients did not show any mutation. The 10 most frequent ones constituted 77.5% of all HBB mutations. These mutations in order of frequency were: IVS-I-110 (G > A) (17.0%), IVS-I-1 (G > A) (14.7%), codon 39 (C > T) (14.4%), IVS-II-1 (G > A) (9.8%), codon 8 (–AA) (6.2%), IVS-I-6 (T > C) (5.2%), IVS-I-5 (G > C) (4.9%), codon 5 (–C) (3.2%), IVS-I-5 (G > A) (3.2%) and codon 37 (G > A) (2.2%). Another 21 mutations were less frequent or sporadic. These results provide important tools for adapting a prenatal molecular diagnostic test for the Syrian population.

Mutation spectrum of phenylketonuria in Syrian population: genotype-phenotype correlation.

Characterization of the molecular basis of phenylketonuria (PKU) in Syria has been accomplished through the analysis of 78 unrelated chromosomes from 39 Syrian patients with PKU. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by using molecular detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR and direct DNA sequencing. 56.4% of the patients had cPKU. A mutation detection rate of 79.49% was achieved and sixteen different mutations were found: missense 56.25%, splice site 37.5%, and frameshift 6.25%. The predominant mutation in this population sample was p.R261Q G>A, p.F55>Lfs and p.R243Q G>A. No mutation in six PKU patients was observed. In 57.9% of patient genotypes, the metabolic phenotype could be predicted. The identification of the mutations in the PAH gene and the genotype-phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counseling for the patients relatives.

Multiple copies of BCR-ABL fusion gene on two isodicentric Philadelphia chromosomes in an imatinib mesylate-resistant chronic myeloid leukemia patient

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. Amplification or duplication of the BCR-ABL gene has been found to be one of the key factors leading to drug resistance to imatinib mesylate (IM). In the present study, we identified the presence of isodicentric Ph chromosomes [idic(Ph)] in an IM-resistant patient. Fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes confirmed the heterogeneity and amplification of the fused BCR-ABL gene. FISH analysis superimposed on G-banding confirmed the presence of idic(Ph) chromosomes. Reverse transcription-polymerase chain reaction (RT-PCR) products revealed the presence of the BCR-ABL fusion transcript b3a2. The idic(Ph) chromosomes in CML were shown to be fused at the satellite regions of the short arms. The patient did not respond to IM chemotherapy and did not achieve remission. In this study, the impact of the idic(Ph) chromosomes on genomic instability, heterogeneity and amplification of the BCR-ABL gene in IM-resistant patients is discussed.

Three-way Philadelphia translocation t(9;10;22)(q34;p11.2;q11.2) as a secondary abnormality in an imatinib mesylate-resistant chronic myeloid leukemia patient

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome created by the reciprocal translocation t(9:22)(q34;q11), resulting in the chimeric gene breakpoint cluster region (BCR)-Abelson (ABL). Variant Ph chromosome translocations involving chromosomes other than 9 and 22 occur in 5–10% of CML cases. In the present study, a novel case of a Ph chromosome-positive CML in the chronic phase (CP) is reported, with a three-way Ph translocation involving three chromosomal regions, 9q34, 10p11.2 and 22q11.2, in addition to the loss of the Y chromosome, where the latter was a secondary abnormality. Since the majority of CML cases are currently treated with imatinib, variant rearrangements generally have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. The underlying mechanisms and prognostic implications of these cytogenetic abnormalities are discussed in the present study.

Detailed analysis of an idic(Y)(q11.21) in a mosaic karyotype

Abnormalities involving sex chromosomes account for approximately 0.5% of live births. The phenotypes of individuals with mosaic cell lines that exhibit structural aberrations of the X and Y chromosomes are variable and difficult to predict. Phenotypes associated with sex chromosome mosaicism vary from females with Turner syndrome to males with infertility, and include individuals with ambiguous genitalia. In this study, we report a 17-year-old male with phenotypic features of Klinefelter syndrome with an isodicentric Y chromosome and a final karyotype of 45,X[4]/46,X,idic(Y)(q11.21)[95]/47,XX,+idic(Y)(q11.21)[1]. Application of high resolution molecular cytogenetic techniques as well as molecular studies revealed two copies of the sex-determining region of Y chromosome (SRY) gene and two centromers. Additionally, the breakpoint in Yq11.21 was narrowed down between positions 13.4 and 14.3 MB (hg18). We present a patient with partial disomy of Ypter to Yq11.21 in the majority of the patient cells, showing phenotypic features of Klinefelter syndrome. The syndrome may have occurred due to a more prominent presence of the cell line 47,XX,+idic(Y)(q11.21) detected only once in 1% of the peripheral blood cells. This finding may prove helpful in similar cases with symptoms of Klinefelter syndrome, but which exhibit an absence of the cell line 47,XXY in peripheral blood.

Biclonal myelodysplastic syndrome involving six chromosomes and monoallelic loss of RB1 - A rare case

BackgroundMyelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia, and reflects to defects in erythroid, myeloid and megakaryocytic maturation. MDS is more frequently observed in older aged patients with cytogenetic abnormalities like monosomy of chromosome(s) 5 and/or 7. In 50% of de novo MDS cases, chromosomal aberrations are found and rearrangements involving the retinoblastoma (RB1) gene in 13q14 are found.ResultsHere, we are presenting a case report of a rare biclonal MDS with a karyotype of 45, XY,-4, der(6)t(4;6)(p15.1;p21.3), der(8)t(4;8)(q31.2;q22), t(13;16)(q21.3;p11.2)[11]/45, XY, der(7)t(7;13)(p22.2~22.3;q21.3),-13 [9]. The patient was diagnosed according to WHO classification as refractory anemia with excess of blasts (RAEB-II).Immunophenotyping was positive for CD11b, CD11c, CD10, CD13, CD15, CD16 and CD33.ConclusionWe report, a novel and cytogenetically rare case of a biclonal MDS with complex chromosomal aberrations and deletion of RB1-gene in both clones. These findings are associated with a poor prognosis as the patient died 3 months after diagnosis.

First report of prevalence c.IVS1+1G>A and del (GJB6-13S1854) mutations in Syrian families with non-syndromic sensorineural hearing loss.

OBJECTIVE Mutations in GJB2 and GJB6 genes are a frequent cause of congenital non-syndromic hearing loss (NSHL). Mutational screening has usually focused on coding region of GJB2 gene. A few studies have been conducted on the non-coding region and exon 1. c.IVS1+1G>A (a splice site mutation in GJB2 gene have been detected as disruptive mutation. Del (GJB6 D13S1830) is found in many populations, but del (GJB6 D13S1854) is reported from a few restricted countries. This study was carried out to investigate the prevalence of splice site mutation c.IVS1+1G>A and two common deletions in GJB6 gene as the genetic etiology of hearing impairment in 70 Syrian families. METHODS The frequency of the c.IVS1+1G>A mutation and two deletions were determined by PCR-RFLP and A multiplex PCR assay. RESULT Our results showed a high prevalence of IVS1+1G>A mutation (20%) and del(GJB6-D13S1854) (15.7%) in deaf families. The homozygous genotype (c.IVS1+1G>A/c.IVS1+1G>A) was observed in one family and the compound heterozygous genotypes (c.35delG/c.IVS1+1G>A) and (c.IVS1+1G>A/V153I) were observed in 7 families and one family respectively. Also, the heterozygous state (c.IVS1+1G>A/unknown) was detected in 5 families. The study of del((GJB6-D13S1854) was showed a compound heterozygous genotype del((GJB6-D13S1854)/c.IVS1+1G>A) in the same families (5 families) having heterozygous genotype of c.IVS1+1G>A mutation. Also, del(GJB6-D13S1854) is combined with c.35delG mutation in 2 families and it was observed in the heterozygous state del(GJB6-D13S1854)/unknown) in 4 families. In contrast, the del(GJB6-D13S1830) described in many population was absent in our patients. CONCLUSION Our findings indicate to significant contribution of the splice site mutation and del(GJB6-D13S1854) in our deaf families and these mutations were important causes of hearing impairment.

Prenatal Molecular Diagnosis of β-Thalassemia and Sickle Cell Anemia in the Syrian Population

Abstract Our objective was to evaluate the prenatal diagnosis (PND) of β-thalassemia (β-thal) and sickle cell anemia in Syria. Mutations detected from blood of at-risk couples and 55 amniotic fluid samples collected at the second trimester of pregnancy (14–22 weeks’ gestation) were characterized. Molecular screening and direct DNA sequencing of the HBB gene was carried out. DNA analyses showed 14 affected fetuses (25.45%), 32 (58.18%) carriers and eight (14.54%) normal fetuses. It appears that 20.0% of individuals carried the sickle cell anemia mutation and 80.0% carried the β-thal mutation. Thirteen different known mutations were detected in the fetuses. The most common mutations were: IVS-II-1 (G > A), codon 39 (C > T)], IVS-I-110 (G > A), IVS-I-1 (G > A) and IVS-I-5 (G > C). The Hb S [β6(A3)Glu → Val; HBB: c.20A > T] mutation was the only abnormal hemoglobin (Hb) that was found. The results point to a successful future for PND of β-thal and sickle cell anemia in Syria, using a rapid and accurate molecular method. We hope that this method will be used as a common application approach to decrease the incidence of β-thal major (β-TM).