Whole Exome Sequencing in Patients With Ossification of the Spinal Ligaments

2021 ◽  
Author(s):  
Jiliang Zhai ◽  
Jiahao Li ◽  
Yu Zhao ◽  
Xing Wei
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Vital Role ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Clinical Presentations ◽  
Spinal Ligaments ◽  
Neurological Pathology

Abstract Background The clinical presentations of ossification of spinal ligament (OSL) are majorly myelopathy and/or radiculopathy, with serious neurological pathology resulting in paralysis of extremities and disturbances of motility (motor function) lowering the quality of life. Currently, studies find that missense single nucleotide polymorphisms (SNPs) play a vital role in the susceptibility for complex diseases. Methods In the present study, we used whole exome sequencing (WES) method to explore variants in exomes and found out novel potential responsible genes for OSL. Genomic DNA was extracted from ligamentum flavum collected from 5 OSL patients and 5 control patients. Whole-exome sequencing was then performed, while variation forecasts and conservation examination were subsequently assessed. Results 8 common SNP variants were exhibited in all 5 subjects of OSL, presented in the genes of GRHL2, CUL3, WHAMM, IL17RD, POM121L12, SLC26A8 and PTPN23. After screened with numbers of samples and additional screenings with deleterious Polyphen2_HDIV_score, Polyphen2_HVAR_score and SIFT, 4 common SNP variants were displayed in 4 subjects of OSL, presented in the genes of KRT84, KIF1B, NRAP and CETN1. 7 common SNP variants were existed in 3 subjects of OSL, presented in the genes of CCT3, ANLN, ESRRB, SRBD1, ODF3L1, BRAT1 and RBP3. Conclusion We found out novel potential variants in several genes, especially WHAMM, KIF1B and ESRRB, which represented potentially pathogenic mutations in patients with OSL.

scholarly journals The identification of novel gene mutations for degenerative lumbar spinal stenosis using whole-exome sequencing in a Chinese cohort

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xin Jiang ◽  
Dong Chen
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Spinal Stenosis ◽  
Lumbar Spinal Stenosis ◽  
Gene Mutations ◽  
Specific Gene ◽  
Single Nucleotide ◽  
Degenerative Lumbar Spinal Stenosis ◽  
Whole Exome ◽  
Lumbar Spinal

Abstract Background Degenerative lumbar spinal stenosis (DLSS) is a common lumbar disease that requires surgery. Previous studies have indicated that genetic mutations are implicated in DLSS. However, studies on specific gene mutations are scarce. Whole-exome sequencing (WES) is a valuable research tool that identifies disease-causing genes and could become an effective strategy to investigate DLSS pathogenesis. Methods From January 2016 to December 2017, we recruited 50 unrelated patients with symptoms consistent with DLSS and 25 unrelated healthy controls. We conducted WES and exome data analysis to identify susceptible genes. Allele mutations firstly identified potential DLSS variants in controls to the patients’ group. We conducted a site-based association analysis to identify pathogenic variants using PolyPhen2, SIFT, Mutation Taster, Combined Annotation Dependent Depletion, and Phenolyzer algorithms. Potential variants were further confirmed using manual curation and validated using Sanger sequencing. Results In this cohort, the major classification variant was missense_mutation, the major variant type was single nucleotide polymorphism (SNP), and the major single nucleotide variation was C > T. Multiple SNPs in 34 genes were identified when filtered allele mutations in controls to retain only patient mutations. Pathway enrichment analyses revealed that mutated genes were mainly enriched for immune response-related signaling pathways. Using the Novegene database, site-based associations revealed several novel variants, including HLA-DRB1, PARK2, ACTR8, AOAH, BCORL1, MKRN2, NRG4, NUP205 genes, etc., were DLSS related. Conclusions Our study revealed that deleterious mutations in several genes might contribute to DLSS etiology. By screening and confirming susceptibility genes using WES, we provided more information on disease pathogenesis. Further WES studies incorporating larger DLSS patient cohorts are required to comprehend the genetic landscape of DLSS pathophysiology fully.

Whole Exome Sequencing In Relapsed Pediatric T-ALL: Progression Into Relapse Is Characterized By An Increased Number Of Somatic Mutations and a Conservation Of Mutations In Leukemogenic Driver Genes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 228-228
Author(s):  
Joachim Kunz ◽  
Tobias Rausch ◽  
Obul R Bandapalli ◽  
Martina U. Muckenthaler ◽  
Adrian M Stuetz ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Initial Diagnosis ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Recurrent Mutations

Abstract Acute precursor T-lymphoblastic leukemia (T-ALL) remains a serious challenge in pediatric oncology, because relapses carry a particularly poor prognosis with high rates of induction failure and death despite generally excellent treatment responses of the initial disease. It is critical, therefore, to understand the molecular evolution of pediatric T-ALL and to elucidate the mechanisms leading to T-ALL relapse and to understand the differences in treatment response between the two phases of the disease. We have thus subjected DNA from bone marrow samples obtained at the time of initial diagnosis, remission and relapse of 14 patients to whole exome sequencing (WES). Eleven patients suffered from early relapse (duration of remission 6-19 months) and 3 patients from late relapse (duration of remission 29-46 months).The Agilent SureSelect Target Enrichment Kit was used to capture human exons for deep sequencing. The captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al., Nucl. Acids Res., 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, Genome Res. 2011), using default parameters. Following such preprocessing, the number of mapped reads was >95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al., Bioinformatics, 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible somatic driver mutations, candidate SNVs were filtered for non-synonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 50, and requiring absence of segmental duplications. Leukemia-specific mutations were identified by filtering against the corresponding remission sample and validated by Sanger sequencing of the genomic DNA following PCR amplification. We identified on average 9.3 somatic single nucleotide variants (SNV) and 0.6 insertions and deletions (indels) per patient sample at the time of initial diagnosis and 21.7 SNVs and 0.3 indels in relapse. On average, 6.3 SNVs were detected both at the time of initial diagnosis and in relapse. These SNVs were thus defined as leukemia specific. Further to SNVs, we have also estimated the frequency of copy number variations (CNV) at low resolution. Apart from the deletions resulting from T-cell receptor rearrangement, we identified on average for each patient 0.7 copy number gains and 2.2 copy number losses at the time of initial diagnosis and 0.5 copy number gains and 2.4 copy number losses in relapse. We detected 24/27 copy number alterations both in initial diagnosis and in relapse. The most common CNV detected was the CDKN2A/B deletion on chromosome 9p. Nine genes were recurrently mutated in 2 or more patients thus indicating the functional leukemogenic potential of these SNVs in T-ALL. These recurrent mutations included known oncogenes (Notch1), tumor suppressor genes (FBXW7, PHF6, WT1) and genes conferring drug resistance (NT5C2). In several patients one gene (such as Notch 1, PHF6, WT1) carried different mutations either at the time of initial diagnosis and or in relapse, indicating that the major leukemic clone had been eradicated by primary treatment, but that a minor clone had persisted and expanded during relapse. The types of mutations did not differ significantly between mutations that were either already present at diagnosis or those that were newly acquired in relapse, indicating that the treatment did not cause specific genomic damage. We will further characterize the clonal evolution of T-ALL into relapse by targeted re-sequencing at high depth of genes with either relapse specific or initial-disease specific mutations. In conclusion, T-ALL relapse differs from primary disease by a higher number of leukemogenic SNVs without gross genomic instability resulting in large CNVs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Analysis of f5 gene polymorphism in men with coronary atherosclerosis using whole exome sequencing

2021 ◽  
Vol 17 (1) ◽  
pp. 29-37
Author(s):  
E. S. Striukova ◽  
E. V. Shakhtshneider ◽  
D. E. Ivanoshchuk ◽  
Yu. I. Ragino ◽  
Ya. V. Polonskaya ◽  
...  
Keyword(s):  
Acute Coronary Syndrome ◽  
Angina Pectoris ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Coronary Atherosclerosis ◽  
Factor V ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Coronary Syndrome ◽  
Whole Exome

Factor V, encoded by the F5 gene, is a procoagulant blood clotting factor that increases the production of thrombin, the central enzyme that converts fibrinogen to fibrin, which leads to the formation of a blood clot. The F5 gene is localized to 1q24.2 chromosome and consists of 25 exons. There are various mutations in the F5 gene that lead to resistance of activated protein C (APC) (elimination of the APС cleavage site in factor V and factor Va), which can lead to arterial and venous thrombosis. The aim of the present study was to analyze variants of the F5 gene in patients diagnosed with coronary atherosclerosis without acute coronary syndrome with stable functional class II–IV angina pectoris, confirmed by coronary angiography data, using the method of whole exome sequencing.Material and methods. The study was conducted in the framework of the Program of joint research work IIPM — branch of the ICG SB RAS and the FSBI «Research Institute of Circulation Pathology named after E.N. Meshalkin» Ministry of Health of Russian Federation. The study included 30 men aged 40–70 years with coronary angiography-­verified coronary atherosclerosis, without ACS, with stable angina pectoris of the II–IV FC. Patients were admitted for coronary bypass surgery, and endarteriaectomy from the coronary artery (s) was performed during the operation according to intraoperative indications. Whole exome sequencing (SureSelectXT Human All Exon v.6+UTR) was carried out on an Illumina NextSeq 500 instrument (USA).Results. In 30 patients, 29 single-­nucleotide variants were found in the F5 gene. In patients with coronary atherosclerosis, rs9332701 of the F5 gene is 3.33 times more common, and rs6027 is 1.67 times more common than in the population. And rs184663825 was found in 3.33% of cases, while its occurrence in the population is 0.05%. For variants rs6034 and rs144979314, a possible damaging effect on the protein product is shown.Conclusion. The single-­nucleotide variants rs9332701, rs6027, rs184663825, rs6034, rs144979314 of the F5 gene are of interest for inclusion in the genetic panels for the analysis of risk factors for the development of acute coronary syndrome.

scholarly journals Whole Exome Sequencing of FLT3-ITD Sorafenib-Resistant Acute Myeloid Leukaemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1400-1400 ◽  
Author(s):  
Stephen Sze Yuen Lam ◽  
Chae Yin Cher ◽  
Nelson K. L. Ng ◽  
Cheuk Him Man ◽  
Anskar Y.H. Leung
Keyword(s):  
Drug Resistance ◽  
Acute Myeloid Leukaemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukaemia ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Drug Resistant ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Acute Myeloid

Abstract Acute myeloid leukaemia (AML) is a heterogeneous disease characterised by an abnormal increase in myeloblasts. Current treatment protocol employs the standard "7+3" chemotherapy regimen complemented with haematopoietic stem cell transplantation. However overall cure rate remains at 30-40%. Targeted therapy, especially the use of tyrosine kinase inhibitors, has been the main focus in the pursuit of an alternative solution. Internal tandem duplication at the juxatmembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) was found in around 30% of AML cases, conferring unfavourable treatment outcome and prognosis. Sorafenib, and other FLT3 inhibitors, successfully induced remission or partial remission in most of the relapsed/refractory FLT3-ITD AML patients (CR/CRi/nCR = 82.4%, n=34 in our sorafenib monotherapy cohort). Although consolidation with combination of sorafenib and chemotherapy or hypomethylating agents was reported to significantly extend the remission period, all patients relapsed with drug resistance. Mutations at the tyrosine kinase domain (TKD) were reported to alter the binding affinity of the drugs to the receptor and hence generate resistance. However, they were found only in around 25% of the patients while other non-mutational mechanisms of resistance have also been reported. Their relative importance in patients remains uncertain. We hypothesised that there is an emergence of drug-resistant clones acquiring new mutations or pathways to develop the resistance. We have performed whole exome sequencing (HiSeq pair-end sequencing of 101 bp) for sorafenib-sensitive and sorafenib-resistant samples obtained from 8 FLT3-ITD relapsed/refractory AML patients before sorafenib treatment and at relapse with drug resistance. Genomic content of the bulk population was observed to change drastically among the 8 patients, with disappearance of single nucleotide polymorphisms (SNPs) and emergence of new SNPs, suggesting the emergence of drug-resistant clones. Emergence of TKD D835Y mutation was observed in 3 patients with variant allele frequencies (VAF) at 11%, 44% and 50% that were undetectable in the sorafenib-sensitive samples. Mutations at other reported sites of TKD were not found. Filtering of the generated list of single nucleotide changes and insertion/deletions (indels) to sort out emerging non-synonymous mutations with high VAF at resistant samples but undetectable in the sensitive samples was done. They include SLC15A1, CDC27, NADKD1, DGAT2, UBXN11, TFR2 and TTBK1, potentially implicated in sorafenib resistance. They are involved in different pathways, including membrane transporter, cell cycle regulation and energy metabolism. Further functional validation of their role in sorafenib resistance is required to understand the mechanisms involved. Disclosures No relevant conflicts of interest to declare.

scholarly journals A study of new molecular genetic markers of sudden cardiac death identified in the analysis of the results of whole-exome sequencing

2020 ◽  
pp. 33-35
Author(s):  
А.А. Иванова ◽  
Е.С. Мельникова ◽  
А.А. Гуражева ◽  
С.К. Малютина ◽  
В.П. Новоселов ◽  
...  
Keyword(s):  
Sudden Cardiac Death ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Genetic Markers ◽  
Cardiac Death ◽  
Molecular Genetic ◽  
Control Group ◽  
Nucleotide Polymorphisms ◽  
Molecular Genetic Markers ◽  
Whole Exome

Целью исследования является подтверждение ассоциации с внезапной сердечной смертью однонуклеотидных полиморфизмов rs77270326, rs34643859, выявленных в ходе собственного полноэкзомного секвенирования как возможных молекулярно-генетических маркеров внезапной сердечной смерти. В группе внезапной сердечной смерти (n=400, средний возраст умерших 53,2±8,7 года, доля мужчин - 70,9%, женщин - 29,1%) и контрольной группе (n=400, средний возраст 53,1±8,3 года, мужчины - 68,3 %, женщины - 31,7%) проведено генотипирование по выбранным полиморфизмам методом ПЦР-ПДРФ по авторским протоколам. По частотам генотипов и аллелей полиморфизма rs77270326 не найдено статистически значимых различий между группами (p>0,05). В группе женщин в возрасте до 50 лет выявлено статистически значимое уменьшение доли носительниц генотипа ТТ в группе внезапной сердечной смерти (32,3%) по сравнению с контрольной группой (60,0%) (ОШ=0,32, 95%ДИ 0,11-0,91, р=0,04). Таким образом, однонуклеотидный полиморфизм rs77270326 не ассоциирован с внезапной сердечной смертью. Для женщин младше 50 лет генотип ТТ полиморфизма rs34643859 ассоциирован с протективным эффектом в отношении внезапной сердечной смерти. The aim of the study is to confirm the association with sudden cardiac death of single-nucleotide polymorphisms rs77270326, rs34643859, identified during own whole-exome sequencing as possible molecular genetic markers of sudden cardiac death. In the group of sudden cardiac death (n = 400, the average age of the dead - 53.2 ± 8.7 years, the proportion of men - 70.9%, women - 29.1%) and the control group (n = 400, average age - 53 , 1 ± 8.3 years, men - 68.3%, women - 31.7%) genotyping of the selected polymorphisms was conducted by PCR-RFLP method according to the authors’ protocols. According to the frequencies of genotypes and alleles of rs77270326 polymorphism, no statistically significant differences were found between the groups (p>0.05). A group of women under the age of 50 revealed a statistically significant decrease in the proportion of carriers of the TT genotype in the group of sudden cardiac death (32.3%) compared with the control group (60.0%) (OR = 0.32, 95% CI 0, 11-0.91, p = 0.04). Thus, the rs77270326 is not associated with sudden cardiac death. For women under the age of 50, the TT genotype of rs34643859 polymorphism is associated with a protective effect against sudden cardiac death.

scholarly journals Dysregulation of IL12 Signaling As a Novel Cause of an Autoimmune Lymphoproliferative like Syndrome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1420-1420
Author(s):  
Schafiq Nabhani ◽  
Sebastian Ginzel ◽  
Hagit Miskin ◽  
Shoshana Revel-Vilk ◽  
Dan Harlev ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Candidate Genes ◽  
Whole Exome Sequencing ◽  
Death Receptor ◽  
Surface Expression ◽  
Complete Loss ◽  
Fasl Expression ◽  
Single Nucleotide ◽  
Death Receptor Pathway ◽  
Whole Exome

Abstract Introduction Autoimmune lymphoproliferative syndrome (ALPS) is characterized by abnormal lymphocyte homeostasis caused by defective apoptosis. Mutations in genes involved in the Fas death receptor pathway (FAS, FASLG or CASP10 genes) are the cause for the pathogenesis of ALPS. However, in 20-30% of all ALPS cases, collectively classified as ALPS-U (undetermined), the genetic defect is still unknown. The objective of this study was to employ whole-exome sequencing to search for novel gene candidates underlying ALPS-U or ALPS-like disease. Resulting candidates should be validated and their impact on the Fas death receptor pathway studied. Methods Peripheral blood samples were collected from 26 patients diagnosed with ALPS (based on clinical phenotype and accumulation of DNT cells), relatives and healthy controls. PCR and Sanger sequencing were performed to check for germline and somatically acquired FAS, FASLG and CASP10 mutations. Whole-exome sequencing was carried out for all patients with unknown mutation and their parents. Candidate genes were identified by a KEGG-based protein interaction analysis interface of our in-house developed proprietary MySQL database driven workbench, termed SNuPy (Single Nucleotide Polymorphism Database). STRING 9.1 was used to identify high confidence (≥0.900) interaction partners of the Fas pathway. Single nucleotide variations (SNVs) were verified by PCR and Sanger sequencing. The impact of detected mutations on the candidate genes´ expression and functionality was analyzed by immunoblot. The candidate genes´ impact on Fas death receptor pathway and Fas/FasL expression was examined by flow cytometry, ELISA and qRT-PCR. Results Out of 26 analyzed ALPS cases 4 unrelated patients harbored heterozygous germline mutations in the death domain of the Fas receptor (p.Q282K, p.R249G, p.NVQ265-267KQT) or the extracellular domain (p.R191C), respectively. Two siblings with homozygous FASLGtruncating mutation (A69fs*138) and complete loss of FasL surface expression as a consequence were identified. Whole-exome sequencing also identified a homozygous R212* mutation in the IL12/IL23 receptor-component IL12RB1 (Interleukin 12 receptor, beta 1 subunit) in a patient who presented with classical ALPS symptoms (chronic non-malignant, noninfectious lymphadenopathy, splenomegaly, hepatomegaly, elevation of DNT cells, autoimmune cytopenias with polyclonal hypergammaglobulinemia, persistently increased vitamin B12 and IL10 levels). The p.R212* mutation in IL12RB1 leads to premature protein truncation by a stop codon gain, resulting in a complete loss of cell surface expression of IL12RB1 in the patient. IL12 is a factor known to regulate expression of both Fas and FasL. IL12 signaling was abrogated as demonstrated by deficient downstream STAT4 phosphorylation and IFNγ production. Low FasL expression on T-cells and low soluble FasL plasma levels were probably due to lack of IFNγ mediated transcriptional activation of FasL. In contrast to healthy controls, prolonged IL12 stimulation did not trigger upregulation of FasL nor apoptosis in the IL12RB1 deficient and FasL deficient (A69fs*138) patients, indicating that IL12 mediated apoptosis is FasL dependent. Heterozygous carriers of the IL12RB1 or the FASLG mutation showed an intermediate response but were asymptomatic and sub-clinically affected (showing e.g. moderate elevation of DNT cells). Conclusion Our data show that IL12 employs Fas signaling to achieve T-cell apoptosis and reveal IL12 signaling deficiency as a new cause of ALPS like disease via its impact on FasL expression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Whole Exome Sequencing Aids The Diagnosis of Fetal Skeletal Dysplasia

2020 ◽  
Author(s):  
Hui Tang ◽  
Qin Zhang ◽  
Linliang Yin ◽  
Jingjing Xiang ◽  
Jing Wang ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Skeletal Dysplasia ◽  
Clinical Symptoms ◽  
Single Nucleotide Polymorphism Array ◽  
Snp Array ◽  
Somatic Mosaicism ◽  
Single Nucleotide ◽  
Whole Exome

Abstract Background: Skeletal dysplasia is a complex group of bone and cartilage disorders with strong clinical and genetical heterogeneousity. Several types have prenatal phenotypes. And it is difficult to make a molecular diagnosis rapidly due to lacking family history and non-specific and limited clinical symptoms in utero. This study aims to diagnose 16 Chinese fetuses with skeletal dysplasia.Methods: Single nucleotide polymorphism-array (SNP-array) was performed in 12 of 16 samples. If no microdeletions or microreplications related to skeletal dysplasia were detected, whole-exome sequencing (WES) was adopted. And the last four cases only got whole-exome sequencing for analyzing copy number variants and single nucleotide variations at the same time.Results: Among the 16 cases, 12 patients received definitive diagnosis and we detected one deletion in DMD gene by SNP-array and 15 variants of 6 genes including FGFR3, COL1A1, COL1A2, ALPL, HSPG2 and DYNC2H1. 8 variants of COL1A1, COL1A2, ALPL and HSPG2 are novel. And somatic mosaicism in asymptomatic parent with mutations in COL1A1 or COL1A2 was observed.Conclusions: In general, our study expanded the prenatal phenotypes in Duchenne muscular dystrophy (DMD)/ Becker muscular dystrophy (BMD), found 8 novel variants and elucidated that the utilization of whole-exome sequencing improved the diagnosis yield of skeletal dysplasia and provided useful genetic counseling guidance for parents.

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