A Case Study on PPM1D and 9 Other Shared Germline Alterations in a Family.

BACKGROUND: The use of high-throughput genotyping techniques has enabled us to identify the rare germline genetic variants with different pathogenicity and penetrance, and understand their role in cancer predisposition. We report here a familial cancer case, a study from Western Indian. METHODS: NGS-WES was carried out in a lung cancer patient who has a family history of multiple cancers across generations, including tongue, lung, brain, cervical, urothelial, and esophageal cancer. The results were validated by data mining from available data bases. I-TASSER, RasMol and PyMol were used for protein structure modelling. RESULTS: The sequencing by NGS-WES revealed PPM1D c.1654C>T (p.Arg552Ter) mutation in hotspot region exon 6 leading to sudden protein truncation and loss of the C-terminal, due to the substitution of C>T. This mutation was classified as a variant of uncertain significance (VUS), due to limited data on lung cancer, The three unaffected siblings of proband did not show any pathogenic variants and comparative analysis of the four siblings indicate 9 shared genetic variants, classified as benign as per ClinVar. CONCLUSION: PPM1D constitutional genetic alterations are rare and uncommon in different ethnic populations. This gene encodes a phosphatase playing role in regulating the P53 tumor suppressor pathway and DNA damage response. Genetic alterations in the PPM1D gene maybe linked to history of gliomas, breast cancer, and ovarian cancer onset in the proband's family.
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The Mystery of Cancer Resistance: A Revelation Within Nature.

Cancer, a disease due to uncontrolled cell proliferation is as ancient as multicellular organisms. A 255-million-years-old fossilized forerunner mammal gorgonopsian is probably the oldest evidence of cancer, to date. Cancer seems to have evolved by adapting to the microenvironment occupied by immune sentinel, modulating the cellular behavior from cytotoxic to regulatory, acquiring resistance to chemotherapy and surviving hypoxia. The interaction of genes with environmental carcinogens is central to cancer onset, seen as a spectrum of cancer susceptibility among human population. Cancer occurs in life forms other than human also, although their exposure to environmental carcinogens can be different. Role of genetic etiology in cancer in multiple species can be interesting with regard to not only cancer susceptibility, but also genetic conservation and adaptation in speciation. The widely used model organisms for cancer research are mouse and rat which are short-lived and reproduce rapidly. Research in these cancer prone animal models has been valuable as these have led to cancer therapy. However, another rewarding area of cancer research can be the cancer-resistant animal species. The Peto's paradox and G-value paradox are evident when natural cancer resistance is observed in large mammals, like elephant and whale, small rodents viz. Naked Mole Rat and Blind Mole Rat, and Bat. The cancer resistance remains to be explored in other small or large and long-living animals like giraffe, camel, rhinoceros, water buffalo, Indian bison, Shire horse, polar bear, manatee, elephant seal, walrus, hippopotamus, turtle and tortoise, sloth, and squirrel. Indeed, understanding the molecular mechanisms of avoiding neoplastic transformation across various life forms can be potentially having translational value for human cancer management. Adapted and Modified from (Hanahan and Weinberg 2011).

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.

Intrauterine growth restriction associated with paternal isodisomy of chromosome 5: a clinical report and literature survey.

Background: Uniparental disomy (UPD) is a condition where both the chromosomes are inherited from the same parent. The consequences of UPD can be ranging from normal to congenital anomaly depending on the parental origin and chromosome involved.Case characteristics: Here, we describe a case of 2-year-old male with central hypotonia, torticollis, and delayed motor skills born to a nonconsanguineous healthy parent. The proband was prenatally detected with paternal isodisomy 5 and birth was induced at 38 weeks of gestation due to intrauterine growth restriction. There was also confined placental mosaicism along with the isodisomy.Results: No major phenotypic correlation observed. This is the first case of paternal isodisomy 5 with phenotypically normal child.Conclusions: The present case supports the reports that genes on chromosome 5 are nonimprinted. The implications of abnormal genetic findings on genetic counseling are discussed.

Young mothers and higher incidence of maternal meiosis-I non- disjunction: Interplay of environmental exposure and genetic alterations during halt phase in trisomy 21.

Trisomy 21 is a genetic condition caused when chromosomes fail to separate during meiosis. We have studied conventional karyotype and QF-PCR using STR markers with high polymorphism and heterogeneity and the results were analyzed, to determine the paternal and meiotic origin of trisomy 21. This study was conducted using a detailed questionnaire to include: paternal, maternal, clinical and family history for various confounding factors such as age and regional environmental exposures where the parents resided. Out of 120 samples 95% (N = 114) were of maternal origin, including 92% (N = 105) of meiosis 1 errors and 8% (N = 9) meiosis 2 errors. Paternal origin accounted for 5% (N = 6) and were all due to meiosis-I errors. The higher incidence of maternal meiosis-I observed in the present study suggests that human trisomy 21 non-disjunction is a result of multiple factors contributing to the origin of the genetic condition.

MTHFR promoter hypermethylation may lead to congenital heart defects in Down syndrome.

Altered global methylation levels revealed LINE-1 methylation in young mothers of Down syndrome (DS) compared to controls suggesting the possibility of impaired DNA methylation causing abnormal segregation of chromosome 21. Methylene Tetrahydrofolate Reductase (MTHFR) is one of the major enzymes of the folate metabolism pathway. MTHFR gene polymorphism has been associated with maternal risk for DS. Studies have revealed that increased MTHFR promoter methylation results in the reduction of MTHFR protein activity further leading to increased risk of various diseases. The aim of this study is to compare the levels of MTHFR promoter methylation in all three study groups. A total of 120 subjects were recruited for the study and was divided into the following three groups: Group I (mothers of DS without Congenital Heart Defects (CHD), n = 40); Group II (mothers of DS with CHD, n = 40); and Group III (age matched control mothers, n = 40). Genomic DNA was isolated from 2 ml peripheral blood and bisulfite treatment was done to convert all unmethylated cytosines into uracil followed by PCR amplification for MTHFR promoter region and Sanger's sequencing. Results showed that there was a two fold increase in methylated promoter region of MTHFR gene in group II compared to other groups. None of the methylation pattern was observed in the control group. MTHFR promoter methylation affects folate metabolism which is known to play a role in chromosomal breakage, abnormal chromosomal segregation and genomic instability and therefore a developmental defect in the form of congenital cardiac anomaly.

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.

Effect of Phyllostachys parvifolia leaf extract on ionizing radiation-induced genetic damage: A preliminary in vitro cytogenetic study.

The ionizing radiation is a known carcinogen as well as cancer therapeutic agent however, the side effect on normal tissue is a limiting factor and inadequate doses necessitates search for an ideal radioprotective agent. Bamboo species are rich source of antioxidants hence have therapeutic value in many free radical mediated diseases. This is the first report regarding in vitro protective effect of bamboo leaf extract against radiation induced genetic damage in human peripheral blood lymphocytes by cytokinesis blocked micronuclei (CBMN) assay. Fresh whole blood was exposed to 5Gy of cobalt-6o gamma radiation with or without 30 min pre-treatment with 3 µl and 5 µl of hydro alcoholic leaf extract of Phyllostachys parvifolia. In addition to whole extract the effect of potential active compound orientin was also assessed. The frequency of radiation induced micronuclei decreased significantly in a dose dependent manner following treatment with whole extract as well as orientin. The extent of reduction in micronuclei frequency was higher with whole bamboo leaf extract as compared to orientin alone.

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.

Involvement of poly(ADP-ribose) polymerase in paraptotic cell death of D. discoideum.

Paraptosis is mediated by several proteins, poly(ADP-ribose) polymerase being one of them. D. discoideum lacks caspases thus providing a better system to dissect out the role of PARP in paraptosis. The cell death phenotype in unicellular eukaryote, D. discoideum is similar to the programmed cell death phenotype of multicellular animals. However, the events downstream to the death signal of PCD in D. discoideum are yet to be understood. Our results emphasize that oxidative stress in D. discoideum lacking caspases leads to PARP activation, mitochondrial membrane potential changes, followed by the release of apoptosis inducing factor from mitochondria. AIF causes large scale DNA fragmentation, a hallmark feature of paraptosis. The role of PARP in paraptosis is reiterated via PARP inhibition by benzamide, PARG inhibition by gallotannin and PARP down-regulation, which delays paraptosis. PARP, PARG and AIF interplay is quintessential in paraptosis of D. discoideum. This is the first report to establish the involvement of PARP in the absence of caspase activity in D. discoideum which could be of evolutionary significance and gives a lead to understand the caspase independent paraptotic mechanism in higher organisms.

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.