Assessment of Long-Term in vitro Multiplied Human Wharton's Jelly-Derived Mesenchymal Stem Cells prior to Their Use in Clinical Administration.

The quantity of mesenchymal stem/stromal cells (MSCs) required for a particular therapy demands their subsequent expansion through ex vivo culture. During in vitro multiplication, they undergo replicative senescence which may alter their genetic stability. Therefore, this study was aimed to analyze cellular, molecular, and chromosomal alterations in Wharton's jelly-derived MSCs (WJ-MSCs) during their in vitro sequential passages, where WJ-MSCs were sequentially passaged up to P14 and cells were evaluated at an interval of P2, P6, P10, and P14. They were examined for their morphology, tumorigenicity, surface markers, stemness markers, DNA damage, chromosomal aberration, and telomere length. We have processed five full-term delivered human umbilical cord samples to obtain WJ-MSCs. Morphological appearance observed at initial stages was small fine spindle-shaped WJ-MSCs which were transformed to flat, long, and broader cells in later passages. The cell proliferation rate was gradually decreased after the 10th passage. WJ-MSCs have expressed stemness markers OCT-4 and NANOG, while they showed high expression of positive surface markers CD90 and CD105 and lower expression of CD34 and CD45. They were non-tumorigenic with slow cellular aging during subsequent passages. There was no chromosomal abnormality up to the 14th passage, while increase in comet score and decrease in telomere length were observed in later passages. Hence, our study suggests that early and middle passaged (less than P10) WJ-MSCs are good candidates for clinical administration for treatment.

A Successful Case for Deselection of Albino Embryo and Live Birth of Albinism-Free Healthy Baby Followed by PGT-M.

In recent years, Preimplantation genetic testing for monogenic disorders (PGT-M) has gained a lot of focus in the field of assisted reproduction technology, various studies have been published in support of it and many are opposing its role. It has been criticized due to many ethical as well as scientific reasons, but there is no doubt that PGT-M has been one of the most important breakthroughs in in vitro fertilization. A critical aspect of this technology is the possibility that the biopsy itself can adversely affect the quality of embryo and compulsion of embryo freezing. Oculocutaneous albinism (OCA) is a condition which is related to skin, hair, eye color (pigments), where affected individuals typically have very fair skin and white- or light-colored hair. These patients are prone to skin cancers on prolonged sun exposure. It also reduces the pigmentation of the colored part of the eyes (the iris) and the light-sensitive tissue at the back of the eye (the retina). People with this condition usually have problem in vision such as reduced sharpness, involuntary eye movements, and photophobia. Here, we report the successful use of PGT-M and a novel protocol for the preimplantation genetic diagnosis of OCA following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant to a couple having previously affected child.

Clinicopathological profile of lung cancer at a tertiary care center.

BACKGROUND: Lung cancer is one of the most common malignancies with high morbidity and mortality. Nonsmall cell lung cancer (NSCLC) accounts for majority of cases. AIMS: This study aims to study the clinical and pathological features of lung cancer patients treated at our institute between January 2011 and December 2016. SUBJECTS AND METHODS: It is a retrospective study. 446 patients of lung cancer were retrospectively analyzed for demographic data, history of smoking, histological type, and presence of epidermal growth factor receptor (EGFR) mutation/anaplastic lymphoma kinase (ALK) mutations. RESULTS: Of the 446 patients analyzed, 304 (68%) were males and 142 (32%) were females, with the ratio being 2:1. Most of our patients had a lesion localizing to the right side (45.7%) than left (37.8%). NSCLC was reported in 81.1% of our patients. EGFR mutation was found in 60 (24%) patients, the most common mutation being the deletion of exon 19 (73%) followed by L858R mutation (21.6%). CONCLUSIONS: EGFR and ALK mutation testing of all the lung cancer patients is to be encouraged as these mutations form druggable targets.

Embryo genome profiling by single-cell sequencing for successful preimplantation genetic diagnosis in a family harboring COL4A1 c.1537G>A; p.G513S mutation.

CONTEXT: Genetic profiling of embryos (also known as preimplantation genetic diagnosis) before implantation has dramatically enhanced the success quotient of in vitro fertilization (IVF) in recent times. The technology helps in avoiding selective pregnancy termination since the baby is likely to be free of the disease under consideration. AIM: Screening of embryos free from c.1537G>A; p.G513S mutation within the COL4A1 gene for which the father was known in before be in heterozygous condition. SUBJECTS AND METHODS: Processing of trophectoderm biopsies was done from twelve embryos for c.1537G>A; p.G513S mutation within the COL4A1 gene. DNA extracted from isolated cells were subjected to whole genome amplification using an isothermal amplification and strand displacement technology. Oligonucleotide primers bracketing the mutation were synthesized and used to amplify 162 base pairs (bp) polymerase chain reaction amplicons originating from each embryo which were subsequently sequenced to detect the presence or absence of the single base polymorphism. RESULTS: Three out of 12 embryos interrogated in this study were found to be normal while 9 were found to harbor the mutation in heterozygous condition. Implantation of one of the normal embryos following by chorionic villus sampling at 11th week of pregnancy indicated that the baby was free from c.1537G>A; p.G513S mutation within the COL4A1 gene. CONCLUSIONS: Single-cell sequencing is a helpful tool for preimplantation embryo profiling. This is the first report from India describing the birth of a normal child through IVF procedure where a potential pathogenic COL4A1 allele was avoided using this technology.

Pattern of variation in the mono- and dinucleotide repeat microsatellites associated with lynch syndrome in an Indian population.

BACKGROUND: Microsatellite instability (MSI) is associated with Lynch syndrome and hence is a surrogate marker for defective mismatch repair genes. The aim of this study was to investigate the degree of instability associated with each of the 5 microsatellite (MS) loci recommended by the National Cancer Institute (NCI), USA within an Indian population of mixed ethnicity and suffering from Lynch syndrome. METHODS: DNA from clinical samples originating from the study population (n = 130) were subjected to automated fragment analysis for all 5 MS loci, and data generated were analyzed to determine the frequency of variation of each of the MS in the resource population. RESULTS: Out of 130, 116 samples responded to polymerase chain reaction (PCR) for all 5 MS loci. 21 (16.15%) were MSI-high (MSI-H) while 27 (20.76%) and 68 (52.30%) were MSI-low (MSI-L) and microsatellite stable (MSS), respectively. D5S346 exhibited the highest instability (27 out of a total of 82 cases of instability recorded for all 5 MS in all 116 patients tested) followed by D2S123 (23/82), BAT26 (14/82), BAT25 (11/82), and D17S250 (7/82). CONCLUSION: MS D17S250 and BAT25 of the 5 MS panel recommended by the NCI are not informative enough and hence should be avoided for diagnosing Lynch syndrome in the Indian population.

ß-Thalassemia mutations in Western India: outcome of prenatal diagnosis in a hemoglobinopathies project.

Prenatal diagnosis (PND) is one of the most cost effective preventive methods, but it is available only in the large cities of India. Therefore, we initiated a program that offers PND and allows us to determine the prevalence of various mutations. Pregnant females (n = 111,426) were screened for hemoglobinopathies using complete blood count (CBC) and high performance liquid chromatography (HPLC). If the female had a hemoglobinopathy, her husband was then tested. If hemoglobinopathies were seen in both partners, a genetic mutation study was performed on the couple. Fetal samples were obtained by either chorionic villus sampling (CVS) in 70.6% or amniocentesis in 29.4%. The study included 282 couples. IVS-I-5 (G > C) was the most common mutation in all castes except in the Sindhis and Lohanas, where the 619 bp deletion was the most common. Prenatal testing was informative in 97.9% of the couples. A significant number of couples (41.0%) underwent PND during their first pregnancy. Seven patients with ß-thalassemia (ß-thal) trait had normal Hb A2 levels. The Hb A2 and Hb F values varied significantly (p < 0.0001 and 0.0082, respectively) among mutations associated with ß-thal. The IVS-I-5, 619 bp deletion, codons 41/42 (-CTTT), codons 8/9 (+G) and IVS-I-1 (G > T or G > A), were present in 81.0% of the couples tested. ß-Thalassemia mutation frequency varied among the different castes, underlining the need for evolving a testing strategy that considers the caste system. Targeting antenatal clinics could also prove to be a most cost effective way of preventing hemoglobinopathies.

Development, validation and clinical evaluation of a low cost in-house HIV-1 drug resistance genotyping assay for Indian patients.

Human Immunodeficiency Virus-1 (HIV-1) drug resistance genotyping assay is a part of clinical management of HIV-1 positive individuals under treatment with highly active antiretroviral therapy (HAART). Routine monitoring of drug resistance mutations in resource limited settings like India is not possible due to high cost of commercial drug resistance assays. In this study we developed an in-house, cost effective HIV-1 drug resistance genotyping assay for Indian patients and validated it against the US-FDA-approved ViroSeq HIV-1 drug resistance testing system. A reference panel of 20 clinical samples was used to develop and validate the assay against ViroSeq HIV-1 drug resistance testing system which was subsequently used to genotype a clinical panel of 225 samples. The Stanford HIV database was used to identify drug resistant mutations. The analytical sensitivity of the assay was 1000 HIV-1 RNA copies/ml of plasma sample while precision and reproducibility was 99.68 ± 0.16% and 99.76 ± 0.18% respectively. One hundred and one drug resistant mutations were detected by the in-house assay compared to 104 by ViroSeq system in the reference panel. The assay had 91.55% success rate in genotyping the clinical panel samples and was able to detect drug resistant mutations related to nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse-transcriptase inhibitor (NNRTI) as well as protease inhibitor (PI) classes of antiretroviral drugs. It was found to be around 71.9% more cost effective compared to ViroSeq genotyping system. This evaluation of the assay on the clinical panel demonstrates its potential for monitoring clinical HIV-1 drug resistance mutations and population-based surveillance in resource limited settings like India.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting.

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000-25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Rare e14a3 (b3a3) BCR-ABL fusion in chronic myeloid leukemia in India: the threats and challenges in monitoring minimal residual disease (MRD).

OBJECTIVE: The primary objective of this work was to confirm the occurrence of rare BCR ABL fusion variant involving the a3 region of the ABL gene in a patient positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions and the challenges and threats that it poses in a routine laboratory setting which use commercial kits for monitoring the minimal residual disease. METHODS: A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. PCR amplicon generated from in the process was sequenced and analyzed. RESULTS: The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus of affected cells and presented a '1O1G2F' signal pattern. RT-PCR with probes from commercial kit designed to detect common breakpoints within the M- and m-BCR regions involving e13a2, e14a2 and e1a2 fusion variants respectively failed to generate any signal. Further investigation revealed presence of the rare e14a3 (b3a3) fusion. DISCUSSION: This is the first report of rare e14a3 fusion in the BCR ABL gene in a CML patient from India. The observation indicates the need for interrogating rare BCR ABL fusions when common breakpoint cluster regions are absent such that minimal residual disease (MRD), critical for disease monitoring, can be performed and false positive remission cases can be avoided. It also emphasizes the utility and significance of cytogenetics and FISH techniques in primary diagnosis of CML and use of RT-PCR based assays only for generating secondary information within special reference to MRD. CONCLUSION: The rare e14a3 (b3a3) fusion of the BCR ABL gene is present in Indian population as demonstrated from this first report and clinical laboratories using commercial kit that do not cover such rare fusions are likely to generate false result thereby declaring complete molecular remission in CML patients under therapy while conducting MRD assay using RT-PCR technology.

Distinct geographical clustering of HIV-1 and a signature amino acid at position 41 of the p24 unveiled by gag variability in India.

A portion of the gag gene cDNA for p24 protein from 30 Indian HIV-1 proviral DNA was amplified by PCR and sequenced. Phylogenetic analysis with reference samples of A1, A2, B, C, D, F1, F2, G, H, J, K, N and O subtypes revealed that 29 test samples aligned with subtype C reference strain while 1 matched with HIV-1 subtype A. Multiple alignment of predicted amino acid sequence of the Indian test samples and reference C subtype of HIV-1 samples from other countries indicated a molecular signature by way of rigid conservation of the amino acid 'S' at position 41 of the gag p24 protein in all Indian HIV-1 samples analyzed in this study as opposed to 'T' in the same position in C subtype sequences from other parts of the world. A phylogenetic analysis and visualization of the resulting tree in radial position showed distinct clubbing of all Indian C subtypes and formation of a cluster when compared to C subtype sequences from other countries with a single Chinese sample as an exception which was found in the Indian cluster. The use of a portion of p24 gene sequence as tool for subtyping as well as phylogenetic grouping with special reference to its geographical location is discussed.

Primary plasma cell leukemia with light chain secretion and multiple chromosomal abnormalities: How successfully treated? - A case report with review of literature.

Primary plasma cell leukemia is a rare form of plasma cell dyscrasia. We present a case which had leukocytosis with numerous circulating plasma cells in the peripheral blood. Flow cytometry revealed an unusual CD117 expression. Free light chain analysis in the serum showed a markedly elevated level of free lambda light chains. Radiography did not reveal any lytic lesions. Fluorescent in-situ hybridization analysis revealed deletion of 13q14.3 and t(4;14)/t(11;14), while the cytogenetic analysis was normal. The patient was given chemotherapy and was subjected to autologous stem cell transplant, after which she is in complete remission till date.