Genomic DNA Samples Research Articles

MOLECULAR GENETIC ABNORMALITIES IN THE GENOME OF PATIENTS WITH Ph-NEGATIVE MYELOPROLIFERATIVE NEOPLASIA AFFECTED BY IONIZING RADIATION AS A RESULT OF THE CHORNOBYL NUCLEAR ACCIDENT.

to determine the frequency of major somatic mutations in the JAK2, MPL and CALR genes in the genomeof patients with Ph-negative myeloproliferative neoplasms that occur in individuals who have been exposed to ionizing radiation as a result of the Chornobyl accident. Molecular genetic analysis of genomic DNA samples isolated from blood was performed in90 patients with Ph-negative myeloproliferative neoplasia (MPN) with a history of radiation exposure and 191patients with spontaneous MPN utilizing allele-specific polymerase chain reaction (PCR). The presence of major mutations in the genes JAK2, CALR and MPL was revealed in patients with MPN witha history of radiation exposure with a frequency 58.9 % (53 of 90), 12.2 % (11 of 90), and 0 % respectively, and without exposure with frequency 75.4 % (144 of 191), 3.1 % (6 out of 191) and 1.6 % (3 out of 191) respectively.Mutations JAK2 V617F in patients with spontaneous MPN were observed in each clinical form: polycythemia vera (PV),essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations were detected exclusively inpatients with PMF and ET, significantly more often in groups with a radiation exposure history (18.9 % and 33.3 %,vs. 4.2 % and 6.5 %) than without one. At the same time, the occurence of MPL mutations was determined only inpatients with spontaneous MPN in 1.6 % of casees. Triple negative mutation status of genes JAK2, MPL and CALR prevailed in the group of patients with MPN with a history of radiation exposure and was 27.8 %, against 16.2 % inpatients without radiation exposure (p = 0.05). Genomic research of patients with Ph-negative MPN revealed features of molecular genetic damage inthose patients who were exposed to IR as a result of the Chornobyl accident and those with spontaneous MPN. Thedata obtained by determining of JAK2, MPL and CALR genes mutational status in the genome of patients with MPN isnecessary to expand the understanding of the mechanism of leukogenesis, especially caused by radiation.

573: A non-invasive prenatal test for Alpha Thalassemia Hb Barts and Hb Constant Spring

Hb Barts and Hb Constant Spring are the two most frequent severe forms of alpha thalassemia. The UNITYTM Carrier screen is a Lab Developed Test that performs reflex alpha thalassemia NIPT when the mother is determined to be an alpha-thalassemia carrier. This study analyzes the ability of the UNITYTM NIPT to exclude inheritance of Hb Barts and detect maternal inheritance of Hb Constant Spring. The UNITYTM Alpha Thalassemia NIPT was performed on simulated cell-free DNA samples. The assay is a multiplex PCR reaction that employs multiple technologies to evaluate the fetal genotype. Polymorphic alleles contained within the SEA deletion region are assayed to exclude inheritance of double deletions and therefore Hb Barts. Quantitative counting templates are used to accurately measure the number of DNA molecules present in the reaction. Relative mutation dosage is performed to determine the maternal and paternal inheritance of the Constant Spring allele. Gap-PCR is used to detect paternal inheritance of an SEA deletion. A likelihood ratio is used to combine these measurements and determine the Hb Constant Spring status of the fetus. Simulated cell-free DNA from pregnant mothers was prepared by shearing and combining genomic DNA samples. Hb Barts exclusion was tested using SEA carrier DNA mixed with healthy or Hb Barts DNA. Hb Barts was excluded from all healthy DNA samples and was not excluded from all affected samples (n=96). Maternal inheritance of the Constant Spring variant was tested on Constant Spring carrier DNA mixed with healthy SEA carrier DNA. All calls were correct at 5%, 10% and 20% fetal fractions, two 5% samples resulted in no-calls (n=12). These results analytically demonstrate the ability of the UNITYTM alpha thalassemia NIPT to identify healthy fetuses in carrier mothers. The UNITYTM alpha thalassemia carrier screen with reflex NIPT will enable detection of both maternal carrier status and fetal disease risk from a single blood tube. This removes the need for paternal DNA and decreases the turn around time to identify high-risk fetuses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Discovery of rare coding variants in OGDHL and BRCA2 in relation to breast cancer risk in Chinese women.

The missing heritability of breast cancer could be partially attributed to rare variants (MAF < 0.5%). To identify breast cancer-associated rare coding variants, we conducted whole-exome sequencing (~50×) in genomic DNA samples obtained from 831 breast cancer cases and 839 controls of Chinese females. Using burden tests for each gene that included rare missense or predicted deleterious variants, we identified 29 genes showing promising associations with breast cancer risk. We replicated the association for two genes, OGDHL and BRCA2, at a Bonferroni-corrected p < 0.05, by genotyping an independent set of samples from 1,628 breast cancer cases and 1,943 controls. The association for OGDHL was primarily driven by three predicted deleterious variants (p.Val827Met, p.Pro839Leu, p.Phe836Ser; p < 0.01 for all). For BRCA2, we characterized a total of 27 disruptive variants, including 18 nonsense, six frameshift and three splicing variants, whereas they were only detected in cases, but none of the controls. All of these variants were either very rare (AF < 0.1%) or not detected in >4,500 East Asian women from the genome Aggregation database (gnomAD), providing additional support to our findings. Our study revealed a potential novel gene and multiple disruptive variants of BRCA2 for breast cancer risk, which may identify high-risk women in Chinese populations.

BAG6 Variant rs805303 is Nominally Associated with ACEi-induced Cough among Filipinos

Cough is a common side effect of angiotensin converting enzyme inhibitor (ACEi) therapy. The incidence of ACEi-induced cough has been shown to correlate with genetic variation among different populations. This study aimed to determine the association of candidate genetic polymorphisms with ACEi-induced cough among Filipinos. Two hundred twenty (220) participants on ACEi therapy pressure-lowering in an unmatched case-control study (82 cases with ACEi-induced cough and 138 controls). Genomic DNA samples were extracted and genotyped for selected genetic variants. The association of genetic variants and clinical factors with ACEi-induced cough was determined using regression analyses. Univariate logistic regression showed that the BAG6 variant rs805303 is nominally associated with ACEi-induced cough among Filipinos, at a per-comparison error rate (PCER) of 0.05 (OR 2.10, p = 0.016). The association of the variant with ACEi cough was statistically significant after multiple regression analysis (adjusted OR 2.09, p = 0.022) while adjusting for confounding clinical factors (sex, alcohol intake, and diastolic blood pressure). Further studies are needed to validate these findings.

Phylogenetic Analysis of Chrysochamela (Fenzl) Boiss. (Brassicaceae) Taxa Growing in Turkey

In this study, we performed a genetic diversity analysis using RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter simple sequence repeat) markers for some Chrysochamela species growing in Turkey. PCR was performed using RAPD and ISSR primers and genomic DNA samples from each specimen, and the resulting bands were scored. Phylogenetic relationships and genetic distances between the studied taxa were calculated using the PAUP analysis program. According to the RAPD analysis, the closest genetic distance was found to be 0.20000 between C. noeana populations, while the most distant values were found as 0.60000 between C. noeana and C. elliptica. The ISSR analysis also revealed the closest genetic distance as 0.34043 between C. noeana populations, while the most distant values were found to be 0.55319 between C. noeana and C. elliptica. UPGMA (unweighted pair group method with arithmetic mean) dendrogram generated for both RAPD and ISSR analyses consisted of two groups. As a result, it was found that RAPD data were more appropriate for comparison with the previous anatomical and morphological data of the studied taxa.

Identification of novel variants in the FZD4 gene associated with familial exudative vitreoretinopathy in Chinese families.

Familial exudative vitreoretinopathy (FEVR, OMIM 133780) is a severe hereditary retinal disease characterized by incomplete retinal vascular development and pathological neovascularization. It has been reported that variants in nine genes are associated with FEVR, but they can only explain approximately 50% of FEVR patients, suggesting that other FEVR-associated variants or genes remain to be discovered. Whole-exome sequencing (WES) was carried out to analyse genomic DNA samples from the probands of 68 families with FEVR. Sanger sequencing was used to verify all identified variants. Western blot analysis was utilized to detect the expression of the variant mutant proteins. A luciferase assay was conducted to test the receptor activity of the mutant FZD4 proteins in Norrin-β-catenin signaling. Seven heterozygous FZD4 variants were found to cause FEVR in seven families, including six missense variants and one deletion variant: c.182C>T (p.T61I), c.205C>T (p.H69Y), c.217_234del (p.73T_78Qdel), c.264C>A (p.Y88X), c.344G>T (p.G115V), c.678G>A (p.W226X) and c.1310T>C (p.I437T). Among these variants, c.205C>T (p.H69Y) and c.678G>A (p.W226X) are known FEVR-causing variants, while the other five variants are novel pathogenic variants. Our study revealed the cause of FEVR in seven Chinese families and identified five novel pathogenic variants in FZD4, which expanded the mutation spectrum of FEVR in the Chinese population. These findings also provided further support for using WES in the clinical diagnosis of FEVR.

Genome-wide re-sequencing and transcriptome analysis reveal candidate genes associated with the pendulous comb phenotype in domestic chickens.

To determine the causative variations associated with two chicken comb phenotypes, pendulous comb (PC) or upright comb (UC), two pooled genomic DNA samples from PC and UC chickens were re-sequenced by Next-Generation Sequencer, and genome-wide Single nucleotide polymorphisms (SNPs) were detected. Using three selective sweep approaches, FST , θπ, and Tajima's D, with top 5% window values serving as the threshold, a total of 84 positively selective genes (PSGs) were identified. There were no SNPs in exons of the PSGs with significant differences in allele frequencies between the two comb phenotype groups. Then, 515 differentially expressed genes (DEGs) between the PC and UC were identified by RNA-seq. Three genes including CD36 (CD36 molecule), ADAMTSL3 (ADAMTS-like 3), and AOX1 (aldehyde oxidases 1) are overlapped between PSGs and DEGs. After genotyping seven candidate SNPs in the regulatory regions of the three overlapping genes in 120 chickens from two other breeds, two variants (rs14607046 and rs731818051) in the regulatory regions of AOX1 and ADAMTSL3 were found to have significant differences in allele frequency between the PC and UC, suggesting that the two variants may be causative mutations for PC. Overall, our study shed light on the genetic basis underlying the PC phenotype in chickens.

A laboratory practice that uses the polymerase chain reaction-sequence specific priming technique to rapidly screen for HLA-DR2 allotype from germline DNA in immunology course for undergraduate medical students.

In this article, we describe an in-house polymerase chain reaction-sequence specific priming (PCR-SSP) assay designed for undergraduate medical students as part of the experimental pathogen biology and immunology (EPBI) course. It screens human leukocyte antigen (HLA)-DR2 allotype from genomic DNA samples using a rapid and single-tube PCR technique, yielding definitive typing result without conventional post-amplification step like probing or Sanger sequencing. This laboratory exercise offers the undergraduate medical students an opportunity to learn about current molecular biology techniques in HLA genotyping with limited effort and cost, in addition to a better understanding of concepts presented in the classroom lectures. Upon completing this experiment module, the students show statistically significant improvement in several key indexes, such as the knowledge about the mainstream HLA DNA typing techniques, awareness of the relevance of this knowledge for their future scientific research, immunogenetics-related basic laboratory skills they acquire, and interest and desire for mastering this assay (all p < .05). This easy to implement set of experiments is composed of a two-session lab module occupying eight teaching hours, and has been run successfully in our laboratory.

Novel human sex-typing strategies based on the autism candidate gene NLGN4X and its male-specific gametologue NLGN4Y

BackgroundSince the early days of PCR techniques, sex identification, “sex-typing,” of genomic DNA samples has been a fundamental part of human forensic analysis but also in animal genetics aiming at strategic livestock breeding. Most analyses are employing the AMELX/AMELY gene loci on the X and Y chromosomes present in most mammals. We hypothesize that sex-typing in humans is also possible based on the genes NLGN4X and NLGN4Y, which represent X and Y chromosome-specific copies of a common ancestral neuroligin-4 orthologue.MethodsGenomic DNA was isolated from human blood and buccal cell samples (total n = 111) and submitted to two different strategies: (a) a traditional two-primer PCR approach detecting an insertion/deletion (indel) polymorphism immediately upstream of the translational start on exon 1 and (b) detection of a single nucleotide polymorphism, SNP, on the translational stop carrying exon 7. The SNP detection was based on a quantitative PCR approach (rhAMP genotyping) employing DNA/RNA hybrid oligonucleotides that were blocked and which could only be activated upon perfect annealing to the target DNA sequence.ResultsAll indel PCR-tested human DNA samples showed two bands for males representing X- and Y-specific copies of NLGN4 and a single band for female samples, i.e., homozygosity of NLGN4X and absence of NLGN4Y, in accordance with the self-reported sex of the donors. These results were in perfect agreement with the results of the rhAMP-based SNP-detection method: all males were consequently positive for both alleles, representing either SNP variant, and females were interpreted as homozygous regarding the SNP variant found in NLGN4X. Both methods have shown reliable and consistent results that enabled us to infer the sex of donor DNA samples across different ethnicities.ConclusionsThese results indicate that the detection of human NLGN4X/Y is a suitable alternative to previously reported methods employing gene loci such as AMELX/Y. Furthermore, this is the first report applying successfully the rhAMP-genotyping strategy as a means for SNP-based sex-typing, which consequently will be applicable to other gene loci or different species as well.

Application of DNA repair for Streptococcus salivarius DNA-based identification of saliva from ultraviolet-exposed samples

Forensic samples are commonly influenced by various environmental factors, including ultraviolet (UV) irradiation; thus, forensic applications of DNA repair (e.g., PreCR™, Restorase®) have been investigated, focusing on short tandem repeat typing. However, current DNA-based examinations are used for both human and body fluid identification. This study thus aims to clarify the efficacy of a DNA repair approach for Streptococcus salivarius DNA-based identification of saliva from UV-damaged samples. Artificial UV-damaged genomic DNA of S. salivarius, drop saliva stains, and buccal swabs were used to evaluate the effects of DNA repair on S. salivarius DNA detection by using PreCR™ repair reagent. To evaluate forensic applications, we prepared mock forensic samples by exposing them to environmental conditions. Melting curve analysis following real-time PCR was applied for qualitatively detecting S. salivarius DNA with a specific melting peak of 80.5°C±0.4°C (n=10, mean ± 3SD). Single PCR was used for quantitative and qualitative analyses, whereas dual PCR was used for S. salivarius DNA qualitative detection. DNA repair experiments using artificial UV-damaged samples revealed a significant increase of only the quantitative value of genomic DNA samples by DNA repair. Moreover, significant quantitative DNA repair effects were not observed in all mock forensic samples, indicating the limitations of DNA repair for actual cell-derived DNA samples. Whereas, differences of qualitative results (with or without detection) were generated for mock forensic samples; thus, we consider the DNA repair strategy as an additional approach for S. salivarius DNA-based identification of saliva from environmentally damaged evidence.

Charcot-Marie-Tooth type 4B2 demyelinating neuropathy in miniature Schnauzer dogs caused by a novel splicing SBF2 (MTMR13) genetic variant: a new spontaneous clinical model.

BackgroundCharcot-Marie-Tooth (CMT) disease is the most common neuromuscular disorder in humans affecting 40 out of 100,000 individuals. In 2008, we described the clinical, electrophysiological and pathological findings of a demyelinating motor and sensory neuropathy in Miniature Schnauzer dogs, with a suspected autosomal recessive mode of inheritance based on pedigree analysis. The discovery of additional cases has followed this work and led to a genome-wide association mapping approach to search for the underlying genetic cause of the disease.MethodsFor genome wide association screening, genomic DNA samples from affected and unaffected dogs were genotyped using the Illumina CanineHD SNP genotyping array. SBF2 and its variant were sequenced using primers and PCRs. RNA was extracted from muscle of an unaffected and an affected dog and RT-PCR performed. Immunohistochemistry for myelin basic protein was performed on peripheral nerve section specimens.ResultsThe genome-wide association study gave an indicative signal on canine chromosome 21. Although the signal was not of genome-wide significance due to the small number of cases, the SBF2 (also known as MTMR13) gene within the region of shared case homozygosity was a strong positional candidate, as 22 genetic variants in the gene have been associated with demyelinating forms of Charcot-Marie-Tooth disease in humans. Sequencing of SBF2 in cases revealed a splice donor site genetic variant, resulting in cryptic splicing and predicted early termination of the protein based on RNA sequencing results.ConclusionsThis study reports the first genetic variant in Miniature Schnauzer dogs responsible for the occurrence of a demyelinating peripheral neuropathy with abnormally folded myelin. This discovery establishes a genotype/phenotype correlation in affected Miniature Schnauzers that can be used for the diagnosis of these dogs. It further supports the dog as a natural model of a human disease; in this instance, Charcot-Marie-Tooth disease. It opens avenues to search the biological mechanisms responsible for the disease and to test new therapies in a non-rodent large animal model. In particular, recent gene editing methods that led to the restoration of dystrophin expression in a canine model of muscular dystrophy could be applied to other canine models such as this before translation to humans.

Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR.

Genomic alterations in STK11 can predict clinical outcomes in cervical cancer patients

ObjectiveCervical cancer is the fourth most common cause of cancer-related deaths in Asian women, due to its poor prognosis. This study aimed to decipher genomic alteration profiles of a cohort of Japanese cervical cancer patients to understand why certain patients benefited from molecular targeted therapies and their prognostic significance. MethodsDuring 2008–2018, 154 cervical cancer patients underwent a potentially curative resection procedure at the National Cancer Center Hospital. Genomic DNA samples were analyzed using Ion AmpliSeq™ Cancer Hotspot Panel v2. Alterations in the copy number of PIK3CA, ERBB2, PTEN, and STK11 were detected using the TaqMan assay. HPV-positive results were confirmed by genomic testing and in situ hybridization assay. ResultsThe frequency of genomic alterations in PIK3CA (36%), STK11 (16%), PTEN (11%), TP53 (11%), and KRAS (8%) was >5%. KRAS mutations were preferentially detected in patients with adenocarcinomas, and the frequency of PIK3CA mutations in patients with squamous cell carcinomas was higher than that in patients with other histological cancer types. HPV-positive results were observed in 139/154 (90.3%) patients, and TP53 mutants were detected in HPV-negative specimens. In this study, the overall survival of patients with genomic alterations in STK11 was worse than in patients with wild–type STK11 (hazard ratio = 10.6, P = 0.0079) and TCGA dataset (hazard ratio = 2.46, P = 0.029). ConclusionsMore than one-third of Japanese cervical cancer patients exhibit mutations targeted by molecular targeted therapies. We have proposed the prognostic value of STK11 genomic alterations.

No Impact of Donor's Age-Related Clonal Hematopoiesis (ARCH) Observed on Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation: Result from Bar-Coded Error Corrected Sequencing in 33 Gene Mutations on 372 Pairs of Donor and Recipient

Introduction: Recent advances in next-generation sequencing (NGS) enables us to reveal the clinical relevance of age-related clonal hematopoiesis (ARCH) in healthy individuals without having hematologic disorders. It is plausible that transfer of ARCH from a donor to the recipient following allogeneic hematopoietic stem cell transplantation (HCT) results in delayed engraftment, thus increasing the risk of non-relapse mortality (NRM). A recent study reported that the presence of ARCH in the donor is associated with increasing risk of chronic graft-versus-host disease (GVHD) and reduced risk of relapse after allogeneic HCT (JCO 2018). However, detailed analyses are lacking with respect to engraftment, the severity of GVHD, organ-specific GVHD. The present study aimed to assess the impact of donor ARCH on HCT outcomes regarding overall and each organ-specific acute/chronic GVHD as well as engraftment kinetics. Methods and patients: The present study includes 372 pairs of donors and/or recipients who received HCT from 2000 till 2007. Genomic DNA samples were archived from the samples taken prior to HCT. For barcoded error-corrected sequencing, a molecular inversion probe capture protocol was modified and applied (Gen. Research 2013). In short, we designed probes targeting 33 genes covering ARCH mutations along with other AML related mutations. Barcoded NGS library was generated and processed. Following sequencing (NovaSeq, Illumina) with 150 bp pair-end mode, the analysis was performed using a customized computational pipeline. Recipients and transplant characteristics are as following: male/female (n=223/149); median age, 48 yrs; conditioning regimen, reduced-intensity conditioning (RIC, n=105)/myeloablative conditioning (MAC; n=267). Median age of donors was 48 yrs. Transplant outcomes were analyzed according to the presence of ARCH in the donor in terms of overall survival (OS), NRM, relapse incidence (RI), overall acute and chronic GVHD as well as organ-specific incidences of GVHD. Engraftment parameters such as neutrophil and platelet recovery were also analyzed. Results: 30 mutations were detected in 25 donor samples (6.7%). Consistent with previous studies, the most frequently mutated gene was DNMT3A (n=10/25, 40%). The median variant allele frequency (VAF) for 30 mutations in donor was 1.18% (ranges, 0.57%-13.29%). With respect to the presence or absence of ARCH in donors, there were no significant differences in baseline and transplant characteristics such as age, sex, conditioning regimen (RIC vs MAC), GVHD prophylaxis. The presence of ARCH in donor also did not show any difference in OS (p=0.66, HR 0.86 [0.44-1.68]), RI (p=0.71, HR 0.83 [0.31-2.21]) or NRM (p=0.7, HR 1.16 [0.54-2.51]). With respect to the engraftment, the median time to neutrophil (defined as ≥0.5x109/L) and platelet engraftment (≥20x109/L with transfusion independence) was not associated with the presence of donor ARCH. Neutrophil engraftment rate was 88.0% in ARCH and 91.6% in the non-ARCH group at day 30 with a median time of 18 days and 19 days (p=0.45). The median time to platelet engraftment was 15 days both in ARCH and in the non-ARCH group (p=0.43). In terms of GVHD, donor ARCH status did not affect incidences of aGVHD/cGVHD. Incidence of grade II to IV acute GVHD at day 100 was 69.9% and 66.2% in the ARCH and non-ARCH group (p=0.94). There were no differences between the two groups in organ-specific aGVHD of the skin (p=0.23), liver (p=0.98) or gut involvement (p=0.48). The cumulative incidence of chronic GVHD (based on the NIH consensus criteria) was 58.3% in ARCH and 58.6% in the non-ARCH group (p=0.80). No differences were noted in the severity of cGVHD between the 2 groups (p=0.87). No differences in organ-specific cGVHD were also noted for the ocular, oral, skin, liver, GI, lung or musculoskeletal involvement. Multivariate analyses confirmed that the donor ARCH was not significantly associated with survival, a/cGVHD and, engraftment after allogeneic HCT. Conclusion: The presence of ARCH in the donor does not affect the mortality, relapse or engraftment following allogeneic HCT. Our data suggest that donor ARCH has no apparent association with HCT outcomes, although there was a concern on the presence of ARCH in the donor's HSCs and its transfer through allogeneic HCT. It also does not seem to delay engraftment or increase the risk of non-relapse mortality following allogeneic HCT. Figure Disclosures Michelis: CSL Behring: Other: Financial Support. Mattsson:Gilead: Honoraria; Therakos: Honoraria; Celgene: Honoraria.

Prenatal gene diagnosis of 200 fetuses at high risk of osteogenesis imperfect

Objective: The authors aim to provide genetic counselling and prenatal gene diagnosis to the families with osteogenesis imperfecta(OI), based on the identification of pathogenetic mutations in large cohort genetic testing. Methods: DNA was extracted from the peripheral blood of parents of the fetuses, and from the villi tissue, amniotic fluid or cord blood of the fetuses using a standard sodium dodecyl sulfate-proteinase K-phenol/chloroform extraction method. PCR combined with Sanger DNA sequencing was performed to validate the pathogenic mutations of 200 fetuses at risk of OI and their parents from 158 families. Allelic analysis of microsatellite markers was applied to exclude the false positive caused by maternal DNA contamination, when both the fetus and the mother harbored the same pathogenic genotype. Results: A total of 83 affected fetuses (83/200, 41.5%) and 12 (12/200, 6.0%) recessive carriers were identified among the 200 fetuses. The 83 affected fetuses included 78 heterozygotes (45 of COL1A1, 32 of COL1A2, one of IFITM5), and 5 compound heterozygotes or homozygotes of recessive OI (two of FKBP10, one of SEC24D, one of WNT1 and one of CRTAP); The 12 recessive carriers included 7 of WNT1, 4 of SERPINF1 and one of SERPINH1. Maternal DNA contamination was excluded from the genomic DNA samples of OI fetuses when their mother with the same affected genotypes. Conclusion: In this study, the authors used an optimized gene diagnosis system of OI to perform prenatal genetic diagnosis to 200 fetuses at high risk of OI, and provided precisely genetic counselling to the OI families.

First Report of an Association of ‘Candidatus Phytoplasma australasia’ with Cashew Nut Inflorescence Decline in Tripura, a Northeastern State of India

HomePlant DiseaseVol. 104, No. 3First Report of an Association of ‘Candidatus Phytoplasma australasia’ with Cashew Nut Inflorescence Decline in Tripura, a Northeastern State of India PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of an Association of ‘Candidatus Phytoplasma australasia’ with Cashew Nut Inflorescence Decline in Tripura, a Northeastern State of IndiaSurabhi Mitra, Prasenjit Debnath, Amar Bahadur, Suken Chandra Das, Kiran Kirdat, Amit Yadav, and G. P. RaoSurabhi MitraDivision of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, IndiaSearch for more papers by this author, Prasenjit DebnathCollege of Agriculture, Tripura, Lembucherra, West Tripura 799210, IndiaSearch for more papers by this author, Amar BahadurCollege of Agriculture, Tripura, Lembucherra, West Tripura 799210, IndiaSearch for more papers by this author, Suken Chandra DasCollege of Agriculture, Tripura, Lembucherra, West Tripura 799210, IndiaSearch for more papers by this author, Kiran KirdatNational Centre for Microbial Resource, National Centre for Cell Science, Sai Trinity Complex, Sus Road, Pashan, Pune 411021, IndiaSearch for more papers by this author, Amit Yadavhttp://orcid.org/0000-0002-4882-9075National Centre for Microbial Resource, National Centre for Cell Science, Sai Trinity Complex, Sus Road, Pashan, Pune 411021, IndiaSearch for more papers by this author, and G. P. Rao†Corresponding author: G. P. Rao; E-mail Address: gprao_gor@rediffmail.comhttp://orcid.org/0000-0003-1057-7826Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, IndiaSearch for more papers by this author AffiliationsAuthors and Affiliations Surabhi Mitra1 Prasenjit Debnath2 Amar Bahadur2 Suken Chandra Das2 Kiran Kirdat3 Amit Yadav3 G. P. Rao1 † 1Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India 2College of Agriculture, Tripura, Lembucherra, West Tripura 799210, India 3National Centre for Microbial Resource, National Centre for Cell Science, Sai Trinity Complex, Sus Road, Pashan, Pune 411021, India Published Online:30 Dec 2019https://doi.org/10.1094/PDIS-08-19-1715-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Cashew nut (Anacardium occidentale L.), an evergreen perennial tree, is an economically important cash crop extensively used in many Indian desserts and cuisines. Cashew nuts are used for their nutritional value and are a major source of protein, fats, vitamins, oleic acid, and palmitic acid. Cashew nut cultivation is undertaken in 17 states of India covering an area of 1.04 Mha. India is the second-largest cashew nut producer in the world, producing about 817,000 MT of raw cashew nuts per annum (https://cashew.icar.gov.in). During a survey in June 2018 of cashew nut orchards in Nagicherra (latitude N 23° 48′ 37.6″; longitude E 91° 19′ 29.7″), West Tripura, India, cashew nut plants were found exhibiting deformed nuts and inflorescence decline (CaID) with prevalence of 5.6% (in a 1-ha area), which were suggestive of phytoplasma infection. Three symptomatic and asymptomatic cashew nut samples were collected, and DNA was extracted from tender shoots using CTAB. To detect the association of phytoplasma with symptomatic cashew nut samples, the isolated genomic DNA samples were assayed for amplification of 16S rRNA region using phytoplasma-specific primer pairs, namely, P1/P7 followed by nested PCR primers R16F2n/R16R2. The 16S rRNA gene of phytoplasma was detected in all collected symptomatic CaID samples, but no amplification was observed in asymptomatic samples. The obtained nested PCR fragments of ∼1.25 kb were sequenced directly (MK076296, MK729150) which showed 99.8% sequence identity with ‘Candidatus Phytoplasma australasia’, strain ‘Carica papaya’ (Y10097). The virtual restriction fragment length polymorphism analysis of 16S rRNA gene sequences delineated the CaID strain to 16SrII-D subgroup with a similarity coefficient of 1.0 using iPhyClassifier. The association of CaID phytoplasma strain with symptomatic cashew nut samples was further established by amplifying phytoplasma-specific partial secA gene (Hodgetts et al. 2008) and tuf gene (Al-Subhi et al. 2018). The phylogenetic analysis of the obtained sequences of 16S rRNA, secA (MK726365, MK726366), and tuf (MK726367, MK726368) genes confirmed the close association of CaID phytoplasma isolates with ‘Ca. P. australasia’ 16SrII-D subgroup member strains. To the best of our knowledge, this is the first report of the association of ‘Ca. P. australasia’ (16SrII-D) with inflorescence decline symptoms of cashew nut in the world. This is significant because phytoplasma strains of 16SrII-C and D have widespread host range including several fruit trees all across India (Rao et al. 2017). These strains are also known to be associated with diseases in weeds, acting as alternate hosts, spreading phytoplasmas through polyphagous insect vectors (Thorat et al. 2016). Likewise, the cashew plants are intercropped in coconut plantations and with plants like maize, groundnut, cowpea, millets, pineapple, and cassava, which are known to be susceptible to phytoplasma-related diseases, posing a threat to cashew nut plantations (Rao et al. 2017; Visalakshi et al. 2015).The author(s) declare no conflict of interest.

Preimplantation genetic testing for a family with usher syndrome through targeted sequencing and haplotype analysis

BackgroundPreimplantation genetic testing for monogenic defects (PGT-M) has been available in clinical practice. This study aimed to validate the applicability of targeted capture sequencing in developing personalized PGT-M assay.MethodsOne couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. Customized capture probe targeted at USH2A gene and 350 kb flanking region were designed for PGT-M. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A hidden Markov model (HMM) assisted haplotype analysis was performed to deduce embryo’s genotype by using single nucleotide polymorphisms (SNPs) identified in each sample. The embryo without paternal rare variant was implanted and validated by conventional prenatal or postnatal diagnostic means.ResultsFour embryos were diagnosed as free of father’s rare variant, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGT-M and prenatal diagnosis results were further confirmed by the molecular diagnosis of the baby’s genomic DNA sample. The auditory test showed that the hearing was normal.ConclusionsTargeted capture sequencing is an effective and convenient strategy to develop customized PGT-M assay.

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study.

Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations. We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC). Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples. We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205days (median 95days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression. We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC. Clinical Trials.gov identifier: NCT02804100.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Screening of intronic mutation IVS9+141A>G in an Indonesian patient with Gaucher disease

Objective: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by the accumulation of the glycolipid glucosylceramide encoded by the GBA gene in certain organs. At present, more than 460 GBA intronic mutations have been reported in several subpopulations worldwide, but many have never been reported in Indonesia. Here, we aimed to screen for intronic mutations of GBA that might be present in patients with GD in Indonesia. Materials and Methods: Blood samples from patients with and without GD were obtained from the National Dr. Cipto Mangunkusumo Referral Hospital, Jakarta, Indonesia. Genomic DNA samples from peripheral leukocytes were extracted, purified, and amplified using the polymerase chain reaction (PCR) with specific primers. Products of PCR were visualized by gel electrophoresis and were further sequenced to analyze the presence of mutations in intron (intervening sequence [IVS]) 9 of GBA. Results: A mutant allele was identified at IVS9+141A>G, discovered at nucleotide 9335 in IVS 9. This mutation had been reported in India before and was categorized as nonpathogenic. Conclusion: Our study may be used as supplemental information for the GD database in Indonesia and will also open new research opportunities for predicting splicing processes in other intronic variants among patients with GD in Indonesia.