Direct Sequencing Of Genomic DNA Research Articles

BIOM-35. IDENTIFICATION AND VALIDATION OF POTENTIAL BIOMARKERS FOR DECITABINE-MEDIATED SENSITIZATION TO TEMOZOLOMIDE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with limited treatment options and a poor prognosis. Our previous research demonstrated that the DNA hypomethylating agent decitabine (DAC) enhances the sensitivity to temozolomide (TMZ) of a subset of primary GBM cell lines derived from fresh surgical tissue specimens, and that this effect may be mediated by upregulation of DNA mismatch repair genes including MLH1. In this context, we aim to identify additional biomarkers for DAC-mediated sensitization to TMZ with potential clinical utility through in vitro and in vivo experiments. We performed single cell RNA sequencing on three DAC-sensitized and three DAC-desensitized cell lines and found 4 genes that exhibited significant differential expression: DCN, LUM, CRABP2 and NUPR1. We validated these findings using quantitative immunoblots, RT-qPCR and immunohistochemistry, finding that NUPR1 protein expression levels varied most significantly between DAC-sensitized and desensitized GBM tissues. Long-range direct methylation sequencing of non-amplified genomic DNA enriched using CRISPR/Cas9 demonstrated several CpG sites within the 4 genes that may be predictive of TMZ sensitization response after treatment with DAC. Finally, we performed mouse survival experiments using orthotopic patient-derived xenografts (PDXs) of the six GBM cell lines, with preliminary results showing that in selected PDX survival was significantly increased with combined DAC-TMZ versus TMZ monotherapy as predicted by in vitro assays. In conclusion, our study demonstrates that combining DAC with TMZ may be a potential treatment strategy for GBM in a subset of patients. There likely exist several biomarkers that can be used at the time of diagnosis, alone or in combination, to predict the response to epigenetic preconditioning using DAC for TMZ sensitization, which will be necessary for optimal patient selection in an early phase clinical trial. Further data and validation studies are necessary to strengthen these findings and to confirm their clinical relevance.

Frequency of the 'Asia type' DEL with weak D phenotype in Chinese.

DEL individuals account for 9-30% of serological RhD negative population in east Asia and majority of them carrying the RHD*DEL1 allele are referred to as 'Asia type' DEL individuals. There is a lack of data on the molecular basis for 'Asia type' DELs with weak RhD phenotype. Therefore, the aim of this study is to unveil 'Asia type' DELs by elucidating the genetic background and analyzing the serological results METHODS: With a microplate typing protocol, RhD characterization was performed in samples from one million blood donors collected at Chengdu blood center during the period from 2019 to 2022. RhD confirmatory test was performed by direct antiglobulin test and indirect antiglobulin test with five anti-D reagents to detect RhD variants. Molecular characterization of samples categorized as RhD variants was studied by using direct genomic DNA sequencing and RHD zygosity analysis, followed by adsorption and elution tests conduced for samples carrying RHD*DEL1 allele to confirm the presence of RhD antigens on the red cells RESULTS: We reported here 21 RhD variant samples were detected by micro-column gel agglutination assay with IgG anti-D antibodies. Moreover, the agglutination reaction was stronger with IgG anti-D reagents in micro-column gel card than with IgM/IgG blended anti-D antibodies. Each of the 21 samples carried the RHD*DEL1 allele, which indicated that they were 'Asia type' DEL. Of the 21 'Asia type' DEL samples, 9 samples were detected to be RHD+/ RHD+ homozygotes, whereas the other 12 samples were RHD+/RHD- hemizygotes. Among the samples phenotyped for RhCE, seven were CCee and four were Ccee. In this study, DEL samples carrying RHD*DEL1 showed weak RhD phenotype with some anti-D reagents in RhD confirmatory test, which suggest that a serology strategy using several anti-D reagents may be helpful to detect this 'Asia type' DEL. Further studies are needed to elucidate whether the 'Asia type' DELs with weak RhD phenotype have stronger antigenicity and could cause serious transfusion reaction.

Identification of the RPGR Gene Pathogenic Variants in a Cohort of Polish Male Patients with Retinitis Pigmentosa Phenotype.

The goal of the study was to explore the spectrum of pathogenic variants in the RPGR gene in a group of male Polish patients with a retinitis pigmentosa (RP) phenotype. A total of 45 male index patients, including twins, being members of 44 families, were screened for pathogenic variants in the RPGR gene via the direct sequencing of PCR-amplified genomic DNA and underwent a comprehensive ophthalmological examination in one center located in Poland. A total of two pathogenic and five likely pathogenic variants in eight patients (18%) were detected in the studied cohort. Of these, five variants were novel, and five disease-causing variants (71%) were identified within the ORF15 mutational hotspot of the RPGR gene. The median age of onset of the disease was 10 years (range 6-14 years), the median age during the examination was 30 years (range 20-47 years), and the median visual acuity was 0.4 (range 0.01-0.7). The majority of patients had middle constriction of the visual field and thinning of the central foveal thickness. Dizygotic twins bearing the same hemizygous mutation showed a different retinal phenotype in regard to the severity of the symptoms. This is the first RPGR mutation screening in Poland showing a prevalence of 18% of RPGR pathogenic mutations and likely pathogenic variants in the studied cohort of male patients with an RP phenotype.

A Study on the Candidate Gene Association and Interaction with Measures of UV Exposure in Pseudoexfoliation Patients from India

Purpose Environmental and genetic factors are associated with development of Pseudoexfoliation syndrome (XFS). Here we intended to elucidate the association of candidate genes in relevance to UV exposure in these patients. Methods This is a case-control study of 309 subjects (N = 219 controls and 90 XFS cases) from India. PCR based direct sequencing was performed for candidate genes (LOXL1, POMP and TMEM136) followed by genotype and haplotype analysis. The promoter methylation status was assessed by Methylation specific PCR based direct sequencing of genomic DNA for all samples. The methylation status was compared with that of primary fibroblasts cultures established from patient’s Tenon’s tissue samples in subset of these patients. Results SNPs rs3825942, rs41435250, rs8818 (LOXL1) and rs3737528 (POMP) showed significant association with XFS. LOXL1 gene haplotype GAGC (rs1048661- rs3825942- rs41435250–rs8818) was associated with lower risk for XFS with a p value 4.1961 × 10−6 (OR =0; 95%CI, 0.000–0.003). POMP gene haplotypes for intronic SNPs (rs1340815- rs3737528- rs913797) TCC and TTC were associated with increased risk for the disease (OR > 1.0). Significant correlation for SNPs rs3825942 of LOXL1 (ρ= −0.132) and rs3737528 of POMP (ρ = 0.12) was observed with measure of lifetime UV exposure (CUVAF value). Reduced LOXL1 gene expression was observed in cultured tenon fibroblasts from the patients that correlated with differential methylation of the Sp-1 binding sites at -253, -243bp upstream to the transcription start site of LOXL1 promoter region. Conclusion Our results suggest a possible interaction for LOXL1 gene haplotype (GAGC) with the measure of ocular UV exposure in pseudoexfoliation syndrome.

Cryptic splice mutation in the fumarate hydratase gene in patients with clinical manifestations of hereditary Leiomyomatosis and renal cell cancer (HLRCC).

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by qPCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.

Fibrinogen Bonn (p. Arg510Cys) in the Aα-Chain Is Associated with High Risk of Venous Thrombosis.

Inherited dysfibrinogenemia is a qualitative defect of fibrinogen caused by various mutations among three fibrinogen genes. Dysfibrinogenemia can be associated with an increased risk of thrombosis, bleeding, or both. Here, we report a 36-year-old female with dysfibrinogenemia who experienced two successful pregnancies under thromboprophylaxis after cerebral venous sinus thrombosis (CVST). In addition to plasmatic coagulation tests, fibrinogen genes FGA, FGB, and FGG were screened using direct genomic DNA sequencing. The structural-functional implications of the detected mutation were analyzed in silico. Inherited dysfibrinogenemia was diagnosed in an index patient after CVST in a risk situation. Anticoagulation with warfarin was stopped after 12 months when the first pregnancy was planned. Pregnancy and spontaneous delivery (2020) was uncomplicated. A second pregnancy was interrupted because of acute cytomegalovirus infection and the third pregnancy was successful in 2022. Pregnancies were accompanied by thromboprophylaxis with enoxaparin 40 mg once daily until 6 weeks postpartum. Substitution of fibrinogen has not become necessary in the index patient so far. Genetic analysis revealed a novel missense mutation (p. Arg510Cys) in the FGA gene ("fibrinogen Bonn") in the index patient, as well as an asymptomatic sister, and their father who experienced recurrent pulmonary embolism. Surface exposure of wild-type Arg510 suggested the mutated Cys510 to form nonnative disulfide bonds with surface-exposed reactive cysteines from other plasma proteins like albumin leading to formation of aggregates and impaired fibrinolysis. Fibrinogen Bonn might be associated with an increased risk of thrombosis, possibly due to impaired polymerization.

Predictive Value of the TP53 p.G245S Mutation Frequency for the Short-Term Recurrence of Hepatocellular Carcinoma as Detected by Pyrophosphate Sequencing.

Aims: We explored the relationship between the mutation at the p.G245S site in TP53 and the short-term recurrence of hepatocellular carcinoma (HCC). Materials and Methods: 101 HCC patients were included in this study. The TP53 p.G245S mutation frequency spectrum was examined by direct sequencing of genomic DNA from tissue specimens of HCC patients. Univariate and multivariate Cox regression analyses were performed to evaluate the independent prognostic factors of tumor recurrence. ROC curve analysis was applied to determine the cut-off value for the p.G245S mutation frequency and to verify the predictive ability of the Cox model compared with single risk factor indices. Results: A multivariate Cox regression analysis showed that TP53 p.G245S mutation frequency (HR = 1.231, 95% CI: 1.006-1.505, p = 0.043), AFP (HR = 2.432, 95% CI: 1.297-4.561, p = 0.006), MTM (HR = 2.656, 95% CI: 0.930-7.583, p = 0.068), and PVTT (HR = 14.297, 95% CI: 3.085-66.243, p = 0.001) were independent prognostic factors for short-term recurrence. The cut-off value for the TP53 p.G245S mutation frequency (18.5%) was determined by ROC analysis. A predictive model integrating the TP53 p.G245S mutation frequency with PVTT, MTM, and AFP values appears to an excellent predictive indicator of short-term recurrence in HCC patients (AUC = 0.849, 95% CI = 0.748-0.950, p = 0.000001). Survival analysis indicated that the probability of short-term recurrence-free survival was significantly different among different TP53 p.G245S mutation frequency, MTM, PVTT, and AFP risk groups (p < 0.05). Conclusion: The mutation frequency of the p.G245S site is a novel prognostic risk factor for the short-term recurrence of HCC.

A Genotype-Phenotype Study of Multiple Hereditary Exostoses in Forty-Three Patients.

Multiple hereditary exostoses (MHE) is a rare autosomal dominant skeletal disorder with a variety of clinical manifestations. We aimed to evaluate the general clinical phenotypic severity of MHE using our own scoring system and analyzed the risk factors associated with severe clinical phenotypes. In this study, 43 patients from 30 families were analyzed. The mutations were identified by direct sequencing of polymerase chain reaction-amplified genomic DNA or by multiplex ligation-dependent probe amplification. According to a new scoring system devised by the authors, the severity of the phenotype was assessed as mild, moderate, or severe based on the deformity of each segment, number of exostoses, leg length discrepancy, and functional limitations. Of 43 patients from 30 families, 39 patients (90.7%) and 24 families (80%) presented with EXT1 or EXT2 mutations. Patients with EXT1 mutations had a significantly worse phenotype than that of patients with EXT2 mutations or without any detectable mutation. The mean clinical score of patients with an EXT1 mutation (5.76; range, 2.0–8.0; SD = 1.60) was higher than that of patients with an EXT2 mutation (4.06; range, 2.0–7.0; SD = 1.47) or of those without any detectable mutation (4.63; range, 3.0–6.0; SD = 1.44; p = 0.005). According to our classification system, more patients with EXT1 mutations had ‘severe disease’ than those with EXT2 mutations. Deformity scores were also higher in patients with EXT1 mutations (p = 0.018). In the multivariate analysis, the deformity score was found to be associated with the ‘severe’ class (p = 0.031). In conclusion, 90.7% of patients with MHE showed EXT mutations. Our scoring system showed reliable results. We suggest that the extent of deformity is an important factor in determining the phenotype of MHE and close monitoring for the development of severe disease is recommended in patients with high deformity scores.

Abstract 5250: Association between UGT1A1 gene polymorphism and safety and efficacy of irinotecan monotherapy as the second-line treatment for advanced gastric cancer

Abstract Objective: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) is a key enzyme in irinotecan metabolism. However, the relationship between UGT1A1 genotype and the safety and efficacy of irinotecan monotherapy in the treatment of Chinese advanced gastric cancer remains unclear. Methods: A total of 110 patients were enrolled. Intravenous irinotecan was administered every 3 weeks. Irinotecan dose was selected according to the polymorphism of UGT1A1 gene, which was divided into 3 groups: UGT1A1*6 wild-type (GG type): 125mg/m2, d1, d8; UGT1A1*6 mutant heterozygosity (GA type) 100mg/m2, d1, d8; UGT1A1*6 homozygosity mutation (AA type) 75mg/m2, d1, d8 or paclitaxel 125mg/m2, d1, d8. UGT1A1 genotypes were determined by direct sequencing or next-generation sequencing of genomic DNA extracted from peripheral blood. Results: Among 110 patients of the subjects, the genotypes of UGT1A1*28 were wild-type in 78 patients (70.91%), mutant heterozygosity in 28 (25.45%) and mutant homozygosity in 4 (3.64%). UGT1A1*6 were GG in 67 cases (60.91%), GA in 35 cases (31.82%), and AA in 8 cases (7.27%). There were two heterozygous mutations in 3 patients, and no homozygous mutation was detected. A total of 10 cycles of irinotecan were administered, with a median of five cycles per patient. There was no significant difference in the risk of delayed diarrhea and granulocytopenia in patients with different UGT1A1*28 genotypes (P&amp;gt;0.05), while there was significant difference in the risk of delayed diarrhea and granulocytopenia in patients with different UGT1A1*6 genotypes (P&amp;lt;0.05). The dose intensity of irinotecan was different in patients with different subtypes of UGT1A1*6 gene, and the dose intensity of heterozygous mutant patients was lower than that of wild-type patients. However, there were no significant differences in PFS and OS among patients with different subtypes after dose adjustment and program adjustment (P&amp;gt;0.05). Conclusion: UGT1A1*6 gene polymorphism was significantly associated with diarrhea and neutropenia induced by irinotecan. The safety and effectiveness of antitumor therapy might be significantly improved by individualized drug administration according to the results of UGT1A1*6 gene polymorphism. Citation Format: Huifang Lv, Yunduan He, Caiyun Nie, Beibei Chen, Jianzheng Wang, Weifeng Xu, Jing Zhao, Xiaobing Chen, Junling Zhang. Association between UGT1A1 gene polymorphism and safety and efficacy of irinotecan monotherapy as the second-line treatment for advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5250.

Lack of association between single nucleotide polymorphisms in TCF7L2 and T2DM in the Chinese Yao population: A case-control study.

Single-nucleotide polymorphisms (SNPs) in the transcription factor 7-like 2 (TCF7L2) gene have been identified to be associated with the susceptibility to type 2 diabetes mellitus (T2DM) in various populations worldwide, but the results in Chinese are conflicting, and no data are available about the Liannan Yao population. Therefore, this study aimed to investigate the association of the TCF7L2 gene polymorphisms (rs12255372, rs7903146, rs7901695, rs11196205, and rs7895340) with T2DM in the Yao population living in the rural areas in the Liannan Yao Autonomous County.This was a case-control study of 28 subjects with T2DM or prediabetes and 52 non-T2DM controls, all from the Chinese Yao population and recruited between January 2019 and June 2020. Patients with T2DM and prediabetes were grouped as the case group. The five SNPs (rs12255372, rs7903146, rs7901695, rs11196205, and rs7895340) were examined by polymerase chain reaction and direct genomic DNA sequencing in case and control groups.The subjects in case group were older than the controls (55±14 vs 48 ± 15 years, P = .047), had higher FBG levels (9.31 ± 5.43 vs 4.09 ± 0.81, P < .001), higher TC (5.79 ± 1.29 vs 5.13 ± 1.18 mmol/L, P = .025), and higher triglycerides (2.94 ± 2.04 vs 1.86 ± 1.39 mmol/L, P = .003). The genotypic distribution for each of the SNPs was in agreement with the Hardy-Weinberg equilibrium. There were no statistically significant differences in the distributions of genotypes or alleles at all five SNPs of the TCF7L2 gene between the case and control groups (all P > .05).TCF7L2 SNPs were not associated with T2DM in the Liannan Yao population.

Molecular epidemiology of pachyonychia congenita in the Israeli population.

Pachyonychia congenita (PC) is a rare autosomal dominant disorder featuring palmoplantar keratoderma, nail dystrophy, oral leucokeratosis, pilosebaceous cysts and natal teeth. PC results from dominant mutations in one of five genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17) encoding keratin proteins. To delineate the clinical and genetic features of PC in a series of Israeli patients. We used direct sequencing of genomic DNA, and also used cDNA sequencing where applicable. We collected clinical information and molecular data in a cohort of Israeli families diagnosed with PC (n=16). Most of the patients were Ashkenazi Jews and had a family history of PC. The most common clinical findings were painful focal plantar keratoderma (94%) accompanied by nail dystrophy (81%), pilosebaceous cysts (31%) and prenatal/natal teeth (13%). In contrast to the high prevalence of KRT6A mutations in other populations, we found that KRT16 mutations were the most common type among Israeli patients with PC (56%). Most (77%) of the Israeli patients with PC with KRT16 mutation carried the same variant (c.380G>A; p.R127H) and shared the same haplotype around the KRT16 locus, suggestive of a founder effect. The data gleaned from this study emphasizes the importance of population-specific tailored diagnostic strategies.

TP53 R249S mutation detected in circulating tumour DNA is associated with Prognosis of hepatocellular carcinoma patients with or without hepatectomy.

Somatic mutation R249S in TP53 is highly common in hepatocellular carcinoma (HCC). We aim to investigate the effects of R249S in ctDNA on the prognosis of HCC. We analysed three cohorts including 895 HCC patients. TP53 mutation spectrum was examined by direct sequencing of genomic DNA from tissue specimens in HCC patients with hepatectomy (Cohort 1, N=260). R249S and other recurrent missense mutations were assessed for their biological functions and associations with overall survival (OS) and progression-free survival (PFS) of HCC patients in Cohort 1. R249S within circulating tumour DNA (ctDNA) was detected through droplet digital polymerase chain reaction (ddPCR) and its association with OS and PRS was analysed in HCC patients with (Cohort 2, N=275) or without (Cohort 3, N=360) hepatectomy. In Cohort 1, R249S occupied 60.28% of all TP53 mutations. Overexpression of R249S induced more serious malignant phenotypes than those of the other three identified TP53 recurrent missense mutations. Additionally, R249S, but not other missense mutations, was significantly associated with worse OS (P=.006) and PFS (P=.01) of HCC patients. Consistent with the results in Cohort 1, HCC patients in Cohorts 2 and 3 with R249S had worse OS (P=8.291×10-7 and 2.608×10-7 in Cohorts 2 and 3, respectively) and PFS (P=5.115×10-7 and 5.900×10-13 in Cohorts 2 and 3, respectively) compared to those without this mutation. TP53 R249S mutation in ctDNA may serve as a promising prognosis biomarker for HCC patients with or without hepatectomy.

Prevalence and Atypical Clinical Characteristics of NOTCH3 Mutations Among Patients Admitted for Acute Lacunar Infarctions.

Objectives: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary small vessel disease, with reported frequencies of 2-5/100,000 individuals. Recently, it has been reported that some patients with NOTCH3 gene mutations show atypical clinical symptoms of CADASIL. Assuming that CADASIL is underdiagnosed in some cases of lacunar infarction, this study was designed to examine the prevalence of NOTCH3 gene mutations in the patients at highest risk who were admitted for lacunar infarctions.Methods: From January 2011 to April 2018, 1,094 patients with lacunar infarctions were admitted to our hospital, of whom 31 patients without hypertension but with white matter disease (Fazekas scale 2 or 3) were selected and genetically analyzed for NOTCH3 gene mutations (Phase 1). Furthermore, 54 patients, who were 60 years or younger, were analyzed for NOTCH3 mutations (Phase 2). NOTCH3 exons 2–24, which encode the epidermal growth factor-like repeat domain of the NOTCH3 receptor, were analyzed for mutations by direct sequencing of genomic DNA.Results: Three patients presented NOTCH3 p.R75P mutations: two in the Phase 1 and one in the Phase 2 cohort. Among patients aged 60 years or younger and those without hypertension but with moderate-to-severe white matter lesions, the carrier frequency of p.R75P was 3.5% (3/85), which was significantly higher than that in the Japanese general population (4.7KJPN) (odds ratio [95% CI] = 58.2 [11.6–292.5]). All three patients with NOTCH3 mutations had family histories of stroke, and the average patient age was 51.3 years. All three patients also showed white matter lesions in the external capsule but not in the temporal pole. The CADASIL and CADASIL scale-J scores of the three patients were 6, 17, 7 (mean, 10.0) and 13, 20, 10 (mean, 14.3), respectively.Conclusion: Among patients hospitalized for lacunar infarctions, the p.R75P prevalence may be higher than previously estimated. The NOTCH3 p.R75P mutation may be underdiagnosed in patients with early-onset lacunar infarctions due to the atypical clinical and neuroimaging features of CADASIL. Early-onset, presence of family history of stroke, external capsule lesions, and absence of hypertension may help predict underlying NOTCH3 mutations despite no temporal white matter lesions.

ЭФФЕКТИВНОСТЬ ОПРЕДЕЛЕНИЯ 5-МЕТИЛЦИТОЗИНА В ДНК КЛЕТОК ESCHERICHIA COLI, НЕСУЩИХ ГЕНЫ БАКТЕРИАЛЬНЫХ ДНК- МЕТИЛТРАНСФЕРАЗ, С ПОМОЩЬЮ УСТАНОВКИ OXFORD NANOPORE

The MinION system (Oxford Nanopore Technologies Limited) was used for direct sequencing of genomic DNA of two recombinant E. coli strains. In one case, the cells contained a plasmid with the M.HpaII gene of DNA methyltransferase, which methylates the second cytosine in CCGG site; in the second case, the E. coli strain contained M.HspAI DNA methyltransferase, which modifies the central cytosine in the GCGC sequence. In both cases, DNA methyltransferases methylate cytosine to 5-methylcytosine. It has been shown that when DNA is sequenced at high coverage, the presence of 5-methylcytosine in DNA can be detected with high accuracy. In particular, in E. coli DNA containing the cloned gene of DNA methyltransferase M.HspAI, at 1300coverage, 98.9% of the GCGC sites are identified as methylated at the first cytosine. At the same time, only 0.09% of the remaining tetranucleotides, which have the CpG dinucleotide in the middle, give a false positive result being identified as methylated at the central cytosine...

Approaches to Whole Mitochondrial Genome Sequencing on the Oxford Nanopore MinION.

Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next-generation or massively parallel sequencing has made it possible to routinely obtain nucleotide-level data with relative ease. These short-read sequencing platforms offer deep coverage with unparalleled read accuracy in high-complexity genomic regions but encounter numerous difficulties in the low-complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy-number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long-read sequencing data on a pocket-sized device known as the MinION. Nanopore-based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor-intensive, time-consuming, and costly mainstay deep-sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR-free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean-up Support Protocol 3: Suggested data analysis pipeline.

The potential of VKORC1 polymorphisms in Mustelidae for evolving anticoagulant resistance through selection along the food chain

In response to strong selection, new mutations can arise quickly and sweep through populations, particularly, if survival and reproduction depend on certain allele copies for adaptation to rapidly changing environments, like resistance against deadly diseases or strong toxins. Since the 1950s, resistance to anticoagulant rodenticides in several rodents has emerged through single nucleotide mutations in the vitamin-K-epoxid-reductase-complex-subunit-1 (VKORC1) gene, often located in its exon 3. Detection of high prevalence and concentrations of anticoagulant rodenticides in non-target vertebrates, including carnivorous Mustelidae, let us assume that secondary exposure by feeding on poisoned prey may also cause selection along the food chain and we hypothesized that VKORC1-based resistance might also have evolved in rodents’ predators. Using newly-developed mustelid-specific primers for direct sequencing of genomic DNA, we studied VKORC1-DNA-polymorphisms in 115 mustelids of five species (Martes martes, M. foina, Mustela nivalis, M. erminea, M. putorius), obtained from northern Denmark, yielding six sites with nonsynonymous and several synonymous amino acid polymorphisms in exon 3. Comparison of these VKORC1-genotypes with hepatic rodenticide residues (obtained by HPLC combined with fluorescence or mass spectrometry) in 83 individuals (except M. martes), using generalized linear models, suggested that anticoagulant levels depended on species and specific polymorphisms. Although most VKORC-1 polymorphisms may present standing genetic variation, some are situated in resistance-mediating membrane parts of the VKORC1-encoded protein, and might be a result of selection due to exposure to anticoagulant poisons. Our new molecular markers might allow detecting indirect effects of anticoagulant rodenticides on rodent predator populations in the future.

When WAS Gene Diagnosis Is Needed: Seeking Clues Through Comparison Between Patients With Wiskott-Aldrich Syndrome and Idiopathic Thrombocytopenic Purpura.

Background: Wiskott-Aldrich syndrome (WAS) is a rare and severe X-linked disorder with variable clinical phenotypes correlating with the type of mutations in the WAS gene. The syndrome is difficult to differentiate from idiopathic thrombocytopenic purpura (ITP) before genetic diagnosis. We retrospectively reviewed patients suspected to have WAS who were referred to our hospital from 2004 to 2016 and compared the clinical features and laboratory examination of genetically confirmed WAS patients and of patients diagnosed with ITP in order to seek some clues to distinguish WAS and ITP before genetic diagnosis.Methods: Seventy-eight children suspected to have WAS from 78 unrelated families were enrolled in this study. The clinical data and laboratory examination of children were reviewed in the present study. The distribution of lymphocyte subsets from peripheral blood was examined by how cytometry. WASP mutations were identified by direct sequencing of PCR-amplified genomic DNA.Results: Forty-two patients were finally diagnosed with WAS genetically. The median onset age of these patients was 1 month (range: 1 day−10 months). The median diagnosis lag was 4.6 months (range: 0 months−9.42 years). Fifteen patients (35.71%) had positive family histories. More than half of the patients (n = 23, 54.76%) had diarrhea. Twenty-three (54.76%) had pneumonia, 7 with severe symptoms. Major bleeding events included skin spots or petechiae (n = 27, 64.29%), per-rectal bleeding (n = 21, 50.00%), epistaxis (n = 7, 16.67%) and intracranial bleeding (n = 2, 4.76%). Twenty-nine patients (69.05%) had eczema, and one patient had a drug allergy. Three patients had autoimmune diseases, among whom 2 had autoimmune hemolytic anemia and one had autoimmune hemolytic anemia and IgA nephropathy. A total of 42 mutations in WASP were identified, including 19 novel mutations. Eight patients received hematopoietic stem cell transplantation (HSCT) and all survived. Compared with the 30 patients diagnosed with ITP, the WAS patients had higher EOS counts and elevated IgE level, increased NK cell numbers but fewer CD8+T lymphocytes.Conclusion: The WAS gene diagnosis should be considered in all males with ITP-like features, especially for patients with a very early onset age, decreased MPV (<6.5 fl), higher EOS counts and elevated IgE level, increased NK cell number, diminished CD8+T lymphocyte count.

Phenotypic and molecular spectrum of Korean patients with Lesch-Nyhan syndrome and attenuated clinical variants.

Lesch-Nyhan syndrome (LNS) is an X-linked recessive disorder caused by mutations in the HPRT1 gene. The clinical features and mutation spectrum of 26 Korean LNS patients from 23 unrelated families were retrospectively reviewed. The HPRT1 gene was analyzed by direct sequencing of genomic DNA. The median age at diagnosis was 2.3years (range, 4months-22.6years) and the initial presenting features included developmental delay, orange colored urine, and self-injurious behaviors. Most patients were wheelchair-bound and suffered from urinary complications and neurologic problems such as self-mutilation and developmental delay. Twenty different mutations in HPRT1 were identified among 23 independent pedigrees, including six novel mutations. The most common mutation type was truncating mutations including nonsense and frameshift mutations (45%). Large deletions in the HPRT1 gene were identified in exon 1, exons 5-6, exons 1-9, and at chr X:134,459,540-134,467,241 (7702bp) including the 5'-untranslated region, exon 1, and a portion of intron 1. In conclusion, this study describes the phenotypic spectrum of LNS and has identified 20 mutations from 23 Korean families, including six novel mutations in Korean patients with LNS.

\u2018Genome skimming\u2019 with the MinION hand-held sequencer identifies CITES-listed shark species in India\u2019s exports market

Chondrichthyes - sharks, rays, skates, and chimeras, are among the most threatened and data deficient vertebrate species. Global demand for shark and ray derived products, drives unregulated and exploitative fishing practices, which are in turn facilitated by the lack of ecological data required for effective conservation of these species. Here, we describe a Next Generation Sequencing method (using the MinION, a hand-held portable sequencing device from Oxford Nanopore Technologies), and analyses pipeline for molecular ecological studies in Chondrichthyes. Using this method, the complete mitochondrial genome and nuclear intergenic and protein-coding sequences were obtained by direct sequencing of genomic DNA obtained from shark fin tissue. Recovered loci include mitochondrial barcode sequences- Cytochrome oxidase I, NADH2, 16S rRNA and 12S rRNA- and nuclear genetic loci such as 5.8S rRNA, Internal Transcribed Spacer 2, and 28S rRNA regions, which are commonly used for taxonomic identification. Other loci recovered were the nuclear protein-coding genes for antithrombin or SerpinC, Immunoglobulin lambda light chain, Preprogehrelin, selenium binding protein 1(SBP1), Interleukin-1 beta (IL-1β) and Recombination-Activating Gene 1 (RAG1). The median coverage across all genetic loci was 20x and sequence accuracy was ≥99.8% compared to reference sequences. Analyses of the nuclear ITS2 region and the mitochondrial protein-encoding loci allowed accurate taxonomic identification of the shark specimen as Carcharhinus falciformis, a CITES Appendix II species. MinION sequencing provided 1,152,211 bp of new shark genome, increasing the number of sequenced shark genomes to five. Phylogenetic analyses using both mitochondrial and nuclear loci provided evidence that Prionace glauca is nested within Carcharhinus, suggesting the need for taxonomic reassignment of P. glauca. We increased genomic information about a shark species for ecological and population genetic studies, enabled accurate identification of the shark tissue for biodiversity indexing and resolved phylogenetic relationships among multiple taxa. The method was independent of amplification bias, and adaptable for field assessments of other Chondrichthyes and wildlife species in the future.

A Heterozygous Splice-Site Mutation in PTHLH Causes Autosomal Dominant Shortening of Metacarpals and Metatarsals.

Short metacarpals and/or metatarsals are typically observed in pseudohypoparathyroidism (PHP) type Ia (PHP1A) or pseudo-PHP (PPHP), disorders caused by inactivating GNAS mutations involving exons encoding the alpha-subunit of the stimulatory G protein (Gsα). Skeletal abnormalities similar to those in PHP1A/PPHP were present in several members of an extended Belgian family without evidence for abnormal calcium and phosphate regulation. Direct nucleotide sequencing of genomic DNA from an affected individual (190/III-1) excluded GNAS mutations. Instead, whole exome analysis revealed a novel heterozygous A>G change at nucleotide -3 upstream of PTHLH exon 3 that encodes the last two amino acids of the prosequence and the mature PTHrP. The same nucleotide change was also found in her affected mother and maternal aunt (190/II-2, 190/II-1), and her affected twin sons (190/IV-1, 190/IV-2), but not in her unaffected daughter (190/IV-3) and sister (190/III-2). Complementary DNA derived from immortalized lymphoblastoid cells from 190/IV-2 (affected) and 190/IV-3 (unaffected) was PCR-amplified using forward primers located either in PTHLH exon 1 (noncoding) or exon 2 (presequence and most of the prosequence), and reverse primers located in the 3'-noncoding regions of exons 3 or 4. Nucleotide sequence analysis of these amplicons revealed for the affected son 190/IV-2, but not for the unaffected daughter 190/IV-3, a heterozygous insertion of genomic nucleotides -2 and -1 causing a frameshift after residue 34 of the pre/prosequence and thus 29 novel residues without homology to PTHrP or any other protein. Our findings extend previous reports indicating that PTHrP haploinsufficiency causes skeletal abnormalities similar to those observed with heterozygous GNAS mutations. © 2018 American Society for Bone and Mineral Research.