Parental balanced chromosomal rearrangement leading to major genomic imbalance and an autosomal trisomy resulting in consecutive pregnancy loss: a case report.

Chromosomal aberrations such as parental balanced translocation contribute to a significant proportion of recurrent pregnancy losses. These have extreme genetic implications on the foetus which can either cause physical and/or mental retardation or early death. In this study, we report a unique clinical case of a couple with three consecutive pregnancy losses and we aim to determine the genetic abnormalities causing the miscarriages. Conventional cytogenetic and molecular genetic analysis were performed on the products of conception as well as for the parents. Chromosomal analysis was performed based on the ISCN 2016 guidelines. This was followed by Chromosomal microarray analysis carried out using ISCA consortium probe set (8X60K). Genetic testing for the 1st product of conception was not performed. However, the 2nd and 3rd products of conception indicated an autosomal trisomy 22 and a 3.7 Mb deletion of 2p (cytoband p25.3) along with 13.6 Mb duplication of 16p (cytoband p13.3p13.12), respectively. The paternal karyotype was normal but mother showed a balanced translocation 46,XX,t(2;16)(p25.3;p13.3). This was parallel to the products of conception microarray findings, unbalanced chromosomal abnormality in the foetus. Balanced translocation carriers are susceptible for meiotic nondisjunction processes and early detection of genetic anomalies can be informative to parents trying to conceive. Genetic analysis of the abortus after the 1st loss can be helpful to understand the cause of miscarriage. A combined approach with microarray and karyotyping of the products of conception can be important to determine the specific aberrations in the foetal chromosomes leading to abortion.

Apert syndrome with S252W FGFR2 mutation and characterization using Phenomizer: An Indian case report.

Human genetic disease needs differential diagnosis to optimize clinical management, enable prenatal detection, and genetic counselling. The current methods of robust DNA sequencing also require next generation phenotyping to match with for better interpretation of genotypic and phenotypic heterogeneity commonly observed. We report use of human ontology based phenotypic characterization with Phenomizer that gives statistical score for possible diagnoses based on which, the gene mutation was studied. A case of craniosynostosis which refers to a group of syndromes characterized by a premature fusion of skull was studied. The phenotypic features viz, dental crowding and dental malocclusion, bulbous nose, downslanted palpebral fissures, radial deviation of thumb, syndactyly of fingers, macrocephaly, and oxycephaly were entered to query the web-based tool Phenomizer which indicated high probability of mutation in FGFR2 gene. The proband, a 13-year-old male born to non-consanguineous parents showed mutation on FGFR2 gene at c.755C>G indicative of Apert syndrome. Apert syndrome is one of the most severe craniosynostosis syndromes with two possible mutations in the exon IIIa of FGFR2 gene reported in majority of the cases. This case study shows the importance of Phenomizer and molecular genetic analysis in differential diagnosis of genetic diseases.

Titanium dioxide nanoparticles: an in vitro study of DNA binding, chromosome aberration assay, and comet assay.

Engineered titanium dioxide nanoparticles (TiO2 NPs) are extensively used in cosmetic, pharmaceutical and other industries globally due to their unique properties, which has raised concern for biosafety. Genotoxicity assessment is an important part of biosafety evaluation; we report in vitro cytogenetic assays for NPs considering their unique physicochemical characteristics to fill the gap of laboratory data regarding biological safety along with mechanistic study for mode of interaction of NP with genetic material. Comet and chromosome aberration assay (CA assay) using short-term human peripheral blood cultures following exposure to TiO2 NPs; along with physicochemical parameters for stability of nano form in cultures; and DNA binding activity were carried out. The dynamic light scattering and zeta potential measurements revealed mono dispersion in media. The fluorescence spectroscopy for binding affinity of TiO2 NPs and human genomic DNA showed binding constant (Kb), 4.158 × 106 M-1 indicating strong binding affinity and negative ΔG0 value suggesting spontaneous DNA binding supporting its genotoxic potential. Following in vitro exposure to TiO2 NPs for 24 h, the cultures were analyzed for comet and CA assays, which showed significant results (p < 0.05) for % DNA intensity in tail, Olive Tail Moment and frequency of Chromosomal aberrations (CA) at 75 and 125 µM but not at 25 µM.

Recurrent chromosomal translocations: Is proximity a rule?

The role of recurrent chromosomal translocations in pathogenesis is well characterized in many leukemia subtypes; however, the factors leading to such preferential gene fusions are yet to be understood. The proximity of the genetic regions is considered important for genetic exchange, and interphase molecular cytogenetic methods can be employed to measure the same. The interphase genomic location of gene pairs taking part in translocations which are non-randomly associated with leukemia subtypes was studied for the extent of proximity by measuring relative distance and radial location. The FISH (Fluorescence In Situ Hybridization) signals corresponding to gene pairs were scored for relative distance and percentage of possible translocation pairs showing proximity which was found higher for BCR-ABL, PML-RARA and AML-ETO. The radial position of the gene pairs was also recorded to see if there is any preferred location in terms of nuclear centre or periphery for translocation partners. The results suggested no preferential location of any of the gene pairs in periphery or centre of the interphase nucleus, rather random distribution was observed for all the three cases. We report here the use of simple interphase FISH method to assess the interphase proximity of gene fusion pairs which can be further employed for other translocations.

Constitutional Mosaic Trisomy 13 in Two Germ Cell Layers is Different from Patau Syndrome? A Case Report.

The heterogeneous phenotype of known syndromes is a clinical challenge, and harmonized description using globally accepted ontology is desirable. This report attempts phenotypic analysis in a patient of constitutional mosaic trisomy 13 in mesoderm and ectoderm to make globally comparable clinical description. Phenotypic features (minor/major abnormalities) were recorded and matched with the Human Phenotype Ontology terms that were used to query web-based tool Phenomizer. We report here a case of 24-year-old girl born to non consanguineous parents with history of one abortion. Her phenotypic evaluation included short columella, low-set ears, seizures, enlarged naris, bifid tongue, infra-orbital fold, smooth philtrum, microtia, microcephaly, carious teeth, downslanted palpebral fissures, proportionate short stature, high palate, thin upper lip vermilion, small for gestational age, broad fingertip, broad hallux, mandibular prognathia and dental malocclusion. Karyotype and interphase FISH (Fluorescence in situ hybridization) was done in blood cells. Interphase FISH was also performed on buccal epithelial cells. Cytogenetic analysis demonstrated trisomy 13 mosaicism in 25% cells i.e. 47, XX,+13(9)/46,XX(27). The interphase FISH in blood cells showed trisomy 13 in 15%, whereas in buccal mucosa cells showed nearly 6%. Mosaic aneuploidy in constitutional karyotype can be responsible for variation in clinical and morphological presentation of patient with genetic disorder.

Familial Constitutional Rearrangement of Chromosomes 4 & 8: Phenotypically Normal Mother and Abnormal Progeny.

Balanced chromosome translocations carriers mostly do not have recognizable phenotypic expression but may have more risk of recurrent spontaneous abortions &/or children with serious birth defects due to unbalanced chromosome complements. Unbalanced chromosomal rearrangements have variable clinical expression and are rare. We present here a case report of three siblings affected with intellectual disability and minor dysmorphic features of face and limbs, born to a non-consanguineous couple in which mother had 5 abortions. The constitutional chromosome analysis revealed balanced translocation t (4;8) in mother and all the three siblings were karyotypically normal. Chromosomal microarray in one of the probands revealed partial monosomy 8pter-p23 and a partial trisomy 4pter-p16. Phenotypic features were recorded in 3 probands using Human Phenotype Ontology terms to query web-based tool Phenomizer. The harmonized description using globally accepted ontology is very important especially in case of rare genetic conditions and the heterogeneous phenotypes which make it even more challenging. The prevalence of sub-microscopic unbalanced translocations may be under-reported due to lesser use of molecular genetic analysis. The familial expression of abnormal phenotypes including intellectual disability make the individuals candidate for molecular genetic analysis and phenotyping to help defer the status of idiopathic mental retardation and identify sub-entity of genetic condition.

Engineered nanoparticles: Revisiting safety concerns in light of ethno medicine.

The nanoparticles are a miracle invention of the century that has opened novel avenues of applications in various fields. The safety aspect of exposure to nanoparticles for humans, plants, animals, soil micro-flora, and ecosystem at large has been questioned. The safety concern can be addressed by laboratory studies to assess the actual risk and recommend exposure limits and related regulation. There is also a suggestion for considering the nanoparticle form of conventional compounds as a new chemical and subject it to safety assessment in line with the chemical regulatory agencies. In the light of the current scenario of popularity and safety concerns regarding nanoparticles, the use of ancient metal based forms like, Bhasma is revisited in the present article. The current approach of green synthesis of nanoparticles is compared with the Ayurveda Rasayana Shastra guidelines of Bhasma preparation and modern preparation of engineered nanoparticles. Since the benefits of nanotechnology are undeniable, and safety concerns are also not ungrounded, there is a pressing need to revisit the ways nanoparticles are manufactured, and to carry out safety assessment by the techniques specially adapted for this novel compound.

Phenotypic spectrum in uniparental disomy: Low incidence or lack of study?

CONTEXT: Alterations in the human chromosomal complement are expressed phenotypically ranging from (i) normal, via (ii) frequent fetal loss in otherwise normal person, to (iii) sub-clinical to severe mental retardation and dysmorphism in live births. A subtle and microscopically undetectable chromosomal alteration is uniparental disomy (UPD), which is known to be associated with distinct birth defects as per the chromosome involved and parental origin. UPD can be evident due to imprinted genes and/or activation of recessive mutations. AIMS: The present study comprises of data mining of published UPD cases with a focus on associated phenotypes. The goal was to identify non-random and recurrent associations between UPD and various genetic conditions, which can possibly indicate the presence of new imprinted genes. SETTINGS AND DESIGN: Data mining was carried out using the homepage "http://www.fish.uniklinikum-jena.de/UPD.html.", an online catalog of published cases with UPD. MATERIALS AND METHODS: The UPD cases having normal karyotype and with or without clinical findings were selected to analyze the associated phenotypes for each chromosome, maternal or paternal involved in UPD. RESULTS: Our results revealed many genetic conditions (other than the known UPD syndromes) to be associated with UPD. Even in cases of bad obstetric history as well as normal individuals chance detection of UPD has been reported. CONCLUSIONS: The role of UPD in human genetic disorders needs to be studied by involving larger cohorts of individuals with birth defects as well as normal population. The genetic conditions were scrutinized in terms of inheritance patterns; majority of these were autosomal recessive indicating the role of UPD as an underlying mechanism.

Trisomy 8 in leukemia: A GCRI experience.

Trisomy of chromosome 8 is frequently reported in myeloid lineage disorders and also detected in lymphoid neoplasms as well as solid tumors suggesting its role in neoplastic progression in general. It is likely to be a disease-modulating secondary event with underlying cryptic aberrations as it has been frequently reported in addition to known abnormalities contributing to clinical heterogeneity and modifying prognosis. Here, we share our findings of trisomy 8 in leukemia patients referred for diagnostic and prognostic cytogenetic assessment. Total 60 cases of trisomy 8, as a sole anomaly or in addition to other chromosomal aberrations, were reported (January 2005-September 2008). Unstimulated bone marrow or blood samples were cultured, followed by GTG banding and karyotyping as per the ISCN 2005. Patients with +8 were chronic myeloid leukemia (CML) (36), acute myeloid leukemia (AML) (17), and acute lymphoblastic leukemia (ALL) (7). In 7 patients, trisomy 8 was the sole anomaly, whereas in 6 patients +8 was in addition to normal clone, in 47 patients, the +8 was in addition to t(9;22), t(15;17), and others, including 3 with tetrasomy 8. Only one patient showed constitutional +8. The present study will form the basis of further cumulative studies to correlate potential differential effects of various karyotypic anomalies on disease progression and survival following a therapeutic regime. To unravel the role of extra 8 chromosome, constitutional chromosomal analysis and uniparental disomy will be considered.

Mutagen sensitivity in oral cancer patients, healthy tobacco chewers and controls.

OBJECTIVE: To analyze chromosomal aberrations (CA) as an index of DNA damage, to measure DNA repair capability using mutagen sensitivity assay and to correlate tobacco exposure with CA. STUDY DESIGN: Oral cancer patients, healthy tobacco chewers and healthy tobacco nonusers were studied for spontaneous and mutagen-induced CA. An arbitrary unit obtained for lifetime tobacco exposure (LTE) was compared with CA. RESULTS: Mean levels of spontaneous and mitomycin-C-induced CA were higher in patients as compared to chewers and controls. DNA repair capability of patients was significantly deficient (p < or = 0.016) as compared to that of chewers. LTE was significantly higher (p = 0.004) in patients than chewers. Chewers having high LTE and spontaneous CA above cutoff levels might be at a greater risk of oral carcinogenesis. CONCLUSION: There is a probable risk of oral carcinogenesis in healthy tobacco consumers having higher CA and LTE. Whether the deficient DNA repair capacity of oral cancer patients is due to the disease process or the tobacco exposure needs to be confirmed with a larger population study.

Customized laboratory information management system for a clinical and research leukemia cytogenetics laboratory.

We developed a Microsoft Access-based laboratory management system to facilitate database management of leukemia patients referred for cytogenetic tests in regards to karyotyping and fluorescence in situ hybridization (FISH). The database is custom-made for entry of patient data, clinical details, sample details, cytogenetics test results, and data mining for various ongoing research areas. A number of clinical research laboratoryrelated tasks are carried out faster using specific "queries." The tasks include tracking clinical progression of a particular patient for multiple visits, treatment response, morphological and cytogenetics response, survival time, automatic grouping of patient inclusion criteria in a research project, tracking various processing steps of samples, turn-around time, and revenue generated. Since 2005 we have collected of over 5,000 samples. The database is easily updated and is being adapted for various data maintenance and mining needs.

A Case of ABL-BCR Negative, Philadelphia Positive CML with t(5;9)(q13;q34).

The Philadelphia chromosome is found in more than 90 percent of chronic myeloid leukemia (CML) patients. In most cases, it results from the reciprocal t(9;22)(q34;q11), with the ABL proto-oncogene from 9q34 fused to the breakpoint cluster region (BCR) locus on 22q11. In 5 to 10 percent of patients with CML, the Ph originates from variant translocations, involving various breakpoints in addition to 9q34 and 22q11. Here we report a rare case of a Philadelphia positive CML patient carrying t(5;9)(q13;q34) and deletion of ABL/BCR on der(9) as a separate event.

Atypical D-FISH patterns of BCR/ABL gene rearrangements in 169 chronic myeloid leukemia patients.

The Philadelphia (Ph) chromosome, a hallmark chromosomal anomaly observed in 95 percent of chronic myeloid leukemia (CML) cases, is known to involve the Abelson (ABL) proto-oncogene on chromosome 9 and the breakpoint cluster region (BCR) gene on chromosome 22, producing BCR/ABL mRNA encoding an abnormal tyrosine kinase protein. In the process of generating BCR-ABL fusion, the deletion of residual BCR or ABL occurs in 15-30 percent of CML patients. In addition, some rearrangements are complex, and do not yield the ABL/BCR fusion due to the involvement of a third chromosome in the rearrangement. The possible role of these deletions and complex rearrangements in disease outcome is an ongoing topic of research. We report our results of cytogenetic analysis with GTG banding and fluorescence in situ hybridization using dual color dual fusion probe (D-FISH) from Vysis Inc, USA in 169 (109 male and 60 female) CML patients registered at The Gujarat Cancer and Research Institute (GC and RI) from April 2004 to December 2005. GTG banding was carried out in 123 cases having analyzable metaphases. Of these 123 cases, D-FISH revealed atypical signal patterns in 57 patients (46%), and 12 cases revealed additional complex translocations indicative of disease progression. Out of 57 cases with atypical FISH patterns, 22 included metaphase FISH results, and the rest had only interphase FISH performed. In addition to the hallmark Philadelphia chromosome, other chromosomal aberrations in CML revealed heterogeneity of molecular events. Pooling of more data may lead to identification of new CML sub-groups and hence help in the analysis of clinical trials. Patients enrolled in our prospective study of prognostic significance will be followed up for disease free and overall survival in correlation with ABL-BCR deletion status.