Evaluation of a new flow cytometry based method for detection of BCR-ABL1 fusion protein in chronic myeloid leukemia.

BACKGROUND: Philadelphia chromosome, a hallmark of chronic myeloid leukemia (CML), plays a key role in disease pathogenesis. It reflects a balanced reciprocal translocation between long arms of chromosomes 9 and 22 involving BCR and ABL1 genes, respectively. An accurate and reliable detection of BCR-ABL fusion gene is necessary for the diagnosis and monitoring of CML. Previously, many technologies, most of which are laborious and time consuming, have been developed to detect BCR-ABL chimeric gene or chromosome. METHODS: A new flow cytometric immunobead assay was used for detection of BCR-ABL fusion proteins and applicability, sensitivity, reliability, efficacy and rapidity of this method was evaluated. RESULTS: From February 2009 to January 2014, a total 648 CML patients were investigated for the status of BCR-ABL1 protein. Among them, 83 patients were enrolled for comparative study of BCR-ABL1 positivity by three routinely used procedures like karyotyping, and quantitative real time PCR (RT-PCR) as well as immunobead flow cytometry assay. BCR-ABL protein analysis was found consistent, more sensitive (17% greater sensitivity) and reliable than the conventional cytogenetics, as flow cytometry showed 95% concordance rate to RT-PCR. CONCLUSION: BCR-ABL fusion protein assay using a new flow cytometric immunobead might be useful in the diagnosis and monitoring CML patients.

Fannin-Lubbock-I [α2ß2¹¹9(GLY>ASP)], a rare mutation in the beta-globin gene, has been detected for the first time in a Hindu Brahmin family in West Bengal, India.

This study aims to describe the hemoglobin Fannin-Lubbock-I, which has a rare mutation substituting the amino acid glycine with aspartic acid at codon 119 of the ß-globin chain. A Bengalee Hindu Brahmin family from Kolkata in West Bengal was the focus of this study. Molecular analysis using ARMS-PCR and direct DNA sequencing revealed the presence of a GGC > GAC mutation in codon 119 of the ß-globin gene in a heterozygote state in three women of the same family. This is the first report of the hemoglobin Fannin-Lubbock-I from India. Our results will help to identify this mutation, which is relatively infrequent in our population.

Descriptive profile of ß-thalassemia mutations in West Bengal population: a hospital-based study.

The present study was based in a hospital at which 660 individuals have been screened for thalassemia in the past 4 years. The main purposes of the study were to identify different types of beta mutations prevailing among these patients, and to establish a genotype-phenotypic correlation. Complete blood count, high-performance liquid chromatography, and amplification refractory mutation system-based polymerase chain reaction were performed on peripheral blood samples to detect beta mutations. Of the 660 subjects studied, 380 (57.6 %) were male and 280 (42.4 %) were female. These included 258 (39.09 %) normal individuals, 176 (26.67 %) ß-thalassemia carriers, 44 (6.67 %) ß-thalassemia major, 6 (0.91 %) cases of sickle ß-thalassemia, 6 (0.91 %) carriers of sickle cell anemia, 102 (15.45 %) Hb Eß-thalassemia, 42 (6.36 %) HbE carriers, 16 (2.42 %) HbE homozygous, and 10 (1.52 %) carriers of other mutations. Genotypic study of beta mutations revealed the prevalence of IVS1-5 mutation among the studied beta carriers to be 46.6 %, and codon 26 (G>A) mutation to be 31.54 %. Other prevailing mutations among the screened individuals include codon 30 (7.53 %), codon 15 (5.01 %), codon 41/42 (3.58 %), and codon 8/9 (1.07 %). Genotype-phenotype correlation revealed that the phenotype of the above-mentioned mutations is associated with mild, moderate, and severe forms of thalassemia.

Incidence of the Hb E [ß26(B8)Glu→Lys, GAG>AAG] variant in Totos, one of the smallest primitive tribes in the world.

Toto is one of the smallest tribes in the world. This primitive sub Himalayan, endogamous tribe lives in a small, isolated village called Totopara in the Jalpaiguri district of West Bengal in India. The tribal communities of West Bengal are vulnerable to various genetic disorders such as ß-thalassemia (ß-thal). We have studied 443 Totos to define their Hb E [ß26(B8)Glu→Lys, GAG>AAG] status. Awareness and screening camps have been organized in various parts of Totopara during the last 2 years. We collected 3 mL peripheral blood from each individual aseptically on which to use the naked eye single tube red cell osmotic fragility test (NESTROFT); complete hemogram and high performance liquid chromatography (HPLC) were done to detect their carrier status. The Hb E variant had been found to be prevalent among the Totos. To confirm the codon 26 (GAG>AAG) mutation in the ß-globin gene, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed. Restriction fragment length polymorphism (RFLP)-PCR was carried out with 44 Hb E alleles to construct the haplotype(s) of the Totos. Our extensive studies have revealed that 49.21% of Totos are Hb E heterozygotes and 19.19% Totos are Hb E homozygotes. The most prevalent haplotype linked with the codon 26 mutation in the Totos is [+ - - - - -] (HincII 5'ϵ, HindIII (G)γ, HindIII (A)γ, HincII 5'ψß, HincII 3'ψß and HinfI 3'ß). Consanguineous marriages have resulted in a significant increase of the percentages of heterozygotes and homozygotes of Hb E in the Totos. Genetic counseling is essential and important to prevent the spread of this mutation and hence to save them from having any kind of clinically significant hemoglobinopathy in the future.

Frequency of 5382insC mutation of BRCA1 gene among breast cancer patients: an experience from Eastern India.

The incidence of breast cancer in India is on the rise and is rapidly becoming the number one cancer in females pushing the cervical cancer to the second position. The mutations in two breast cancer susceptibility genes, BRCA1 and BRCA2, are frequently associated with familial breast cancer. The main objective of the study was to determine the frequency of the mutation 5382insC in BRCA1 of eastern Indian breast cancer patients and also study the hormonal receptor status and histopathology of the patients. Altogether 92 patients affected with breast cancer were included in this study. ARMS-PCR based amplification was used to detect the presence of mutation. The mutations were considered only after pedigree analysis. Out of 92 patients (age range: 20-77 years) with family history (57 individuals) and without family history (35 individuals) were screened. Fifty controls have been systematically investigated. Seven patients and two family members were found to be carriers of 5382insC mutation in BRCA1 gene. We have found 42.64 % ER(-)/PR(-) cancer and 20.58 % triple negative cancer. Invasive ductal carcinoma is the most common histology among the investigated individuals. The presented data confirm a noticeable contribution of BRCA1 5382insC mutation in BC development in Eastern India, which may justify an extended BRCA1 5382insC testing within this patient population. We found HER-2/neu negativity and BRCA1 positivity associated with familial breast cancer. From the hospital's patient history, it was revealed that the age of menarche plays an important role in development of breast cancer.