Whole Exome Sequencing Reveals That DNMT3A and ASXL1 Mutation Are Involved in the Development of Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia Patients without ABL1 Tyrosine Kinase Domain Mutation

Abstract BACKGROUND: Tyrosine kinase inhibitor (TKI) resistance is the most relevant event during the treatment of chronic myeloid leukemia (CML), which correlates with high risk of treatment failure, disease progression and death, explaining half of treatment failed CML patients. However, the remaining half with TKI resistance does not show any ABL1 tyrosine kinase domain (TKD) mutation indicating the presence of alternative pathogenic pathways behind TKI resistance. Thus we hypothesized that the novel mutation besides ABL1-TKD mutation occurs during the development of TKI resistance. Using whole exome sequencing, we screened 13 pairs of CML cases with TKI resistance, but without ABL1-TKD mutation. The present study attempts to: 1) explore novel mutation(s) developing TKI resistance to CML treatment and 2) validate the somatic variants in an independent cohort of CML patients (n=100). METHODS: Thirteen CML cases with TKI resistance but not having ABL1 TKD mutation were included prospectively. Reason for TKI resistance includes Loss of MCyR (n=7) with (n=2) or without additional cytogenetic abnormality (ACA; n=5), progression to blastic crisis (n=3), development of ACA (n=1), development of clonal evolution in Ph neg clone (n=1), primary cytogenetic resistance (n=1). TKI resistance were demonstrated to imatinib (n=12), dasatinib (n=5), nilotinib (n=4) or ponatinib (n=2). The latest treatment includes ponatinib (n=3), dasatinib (n=8) alone (n=4), with smoothen inhibitor (n=2), or with after systemic chemotherapy (n=2), omacetaxine (n=1), and nilotinib (n=1). Disease stage at the time of exome sequencing was chronic phase (n=10) or blastic crisis (n=3). Germline and tumor samples at the time of TKI resistance were compared using whole exome sequencing (Illumina TruSeq kit, HiSeq 2000). Targeted sequencing for selected variants was performed to validate the result. All patients were confirmed the absence of ABL1-TKD mutations using Sanger sequencing. RESULTS: 1) Exome sequencing (Illumina Truseq kit) was performed as per the manufacturer's protocol using an Illumina HiSeq 2000 sequencer. DNA from buccal mucosa was used as a control for variant calling. Exome sequencing reads processing includes mapping to human genome hg19, marking PCR duplicates, realignment of indels, fixing mate information, and discard the reads with more than 2 mismatches to increase the true positive rate. In the end, we have on-target-coverage of 57x. Lastly, 72% of target positions are mapped more than 30x. 2) One hundred nineteen somatic variants were identified in 13 patients in 108 genes. Among them 5 genes have variants in multiple patients including DNMT3A (n=3), ASXL1 (n=2), NPIPB5 (n=2), ATXN3 (n=2) and EFEMP1 (n=2) . We also found at least 1 mutation in well-known driver genes in 6 patients (6/13 = 46%). 3) Three out of 4 patients with ACA carry variants at least one of DNMT3A (n=2), ASXL1 (n=2), and SETBP1 (n=1). Also, 2 out of 3 cases progressed to blastic crisis demonstrate variants in DNMT3A (n=1) and IDH1 (n=1). 4) Interestingly, in one patient, exome sequencing reveals ABL1-TKD mutation (T315I), which was not detected at the initial screening by Sanger sequencing. 5) The result of targeted sequencing in an independent cohort of CML patients (n=100) will be presented in the annual meeting of American Society of Hematology in Dec 2015. CONCLUSION: Our study suggest that DNMT3A and ASXL1 mutations seem to be the driver mutations involved in the development of TKI resistance/progression, independent of ABL1-TKD mutation. Also, exome sequencing can detect ABL1-TKD mutations including T315I prior to be detected by initial Sanger sequencing. Disclosures Lipton: Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding. Kim:Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

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Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia

F1000Research ◽

10.12688/f1000research.9472.2 ◽

2017 ◽

Vol 5 ◽

pp. 2667

Author(s):

Amit Rawat ◽

Shamsudheen Karuthedath Vellarikkal ◽

Ankit Verma ◽

Rijith Jayarajan ◽

Anju Gupta ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Bacterial Infections ◽

Kinase Domain ◽

Clinical Findings ◽

Tyrosine Kinase Domain ◽

Affected Child ◽

Whole Exome

X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in theBTKgene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family.

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Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia

F1000Research ◽

10.12688/f1000research.9472.1 ◽

2016 ◽

Vol 5 ◽

pp. 2667 ◽

Cited By ~ 1

Author(s):

Amit Rawat ◽

Shamsudheen Karuthedath Vellarikkal ◽

Ankit Verma ◽

Rijith Jayarajan ◽

Anju Gupta ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Bacterial Infections ◽

Kinase Domain ◽

Clinical Findings ◽

Tyrosine Kinase Domain ◽

Affected Child ◽

Whole Exome

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NAPG mutation in family members with hereditary hemorrhagic telangiectasia in China

BMC Pulmonary Medicine ◽

10.1186/s12890-021-01524-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yu Xu ◽

Yong-Biao Zhang ◽

Li-Jun Liang ◽

Jia-Li Tian ◽

Jin-Ming Lin ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Hereditary Hemorrhagic Telangiectasia ◽

Sanger Sequencing ◽

Conformational Stability ◽

Sequencing Analysis ◽

Large Pedigree ◽

Whole Exome ◽

Variant Filtering ◽

Genetic Contributions

Abstract Background Hereditary hemorrhagic telangiectasia (HHT) is a disease characterized by arteriovenous malformations in the skin and mucous membranes. We enrolled a large pedigree comprising 32 living members, and screened for mutations responsible for HHT. Methods We performed whole-exome sequencing to identify novel mutations in the pedigree after excluding three previously reported HHT-related genes using Sanger sequencing. We then performed in silico functional analysis of candidate mutations that were obtained using a variant filtering strategy to identify mutations responsible for HHT. Results After screening the HHT-related genes, activin A receptor-like type 1 (ACVRL1), endoglin (ENG), and SMAD family member 4 (SMAD4), we did not detect any co-segregated mutations in this pedigree. Whole-exome sequencing analysis of 7 members and Sanger sequencing analysis of 16 additional members identified a mutation (c.784A > G) in the NSF attachment protein gamma (NAPG) gene that co-segregated with the disease. Functional prediction showed that the mutation was deleterious and might change the conformational stability of the NAPG protein. Conclusions NAPG c.784A > G may potentially lead to HHT. These results expand the current understanding of the genetic contributions to HHT pathogenesis.

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Novel mutation in the TGFBI gene in a Moroccan family with atypical corneal dystrophy: a case report

BMC Medical Genomics ◽

10.1186/s12920-020-00861-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yahya Benbouchta ◽

Imane Cherkaoui Jaouad ◽

Habiba Tazi ◽

Hamza Elorch ◽

Mouna Ouhenach ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Clinical Symptoms ◽

Age Of Onset ◽

Novel Mutation ◽

Corneal Dystrophy ◽

Heterozygous Mutation ◽

Large Variability ◽

Whole Exome ◽

Moroccan Family

Abstract Background Corneal dystrophies (CDs) are a heterogeneous group of bilateral, genetically determined, noninflammatory bilateral corneal diseases that are usually limited to the cornea. CD is characterized by a large variability in the age of onset, evolution and visual impact and the accumulation of insoluble deposits at different depths in the cornea. Clinical symptoms revealed bilateral multiple superficial, epithelial, and stromal anterior granular opacities in different stages of severity among three patients of this family. A total of 99 genes are involved in CDs. The aim of this study was to identify pathogenic variants causing atypical corneal dystrophy in a large Moroccan family and to describe the clinical phenotype with severely different stages of evolution. Case presentation In this study, we report a large Moroccan family with CD. Whole-exome sequencing (WES) was performed in the three affected members who shared a phenotype of corneal dystrophy in different stages of severity. Variant validation and familial segregation were performed by Sanger sequencing in affected sisters and mothers and in two unaffected brothers. Whole-exome sequencing showed a novel heterozygous mutation (c.1772C > A; p.Ser591Tyr) in the TGFBI gene. Clinical examinations demonstrated bilaterally multiple superficial, epithelial and stromal anterior granular opacities in different stages of severity among three patients in this family. Conclusions This report describes a novel mutation in the TGFBI gene found in three family members affected by different phenotypic aspects. This mutation is associated with Thiel-Behnke corneal dystrophy; therefore, it could be considered a novel phenotype genotype correlation, which will help in genetic counselling for this family.

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Identification of a novel mutation of RUNX2 in a family with supernumerary teeth and craniofacial dysplasia by whole-exome sequencing

Medicine ◽

10.1097/md.0000000000011328 ◽

2018 ◽

Vol 97 (32) ◽

pp. e11328 ◽

Cited By ~ 7

Author(s):

Dan Ma ◽

Xuxia Wang ◽

Jun Guo ◽

Jun Zhang ◽

Tao Cai

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Novel Mutation ◽

Supernumerary Teeth ◽

Whole Exome

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Identification of Two Novel Mutations in PKHD1 Gene from Two Families with Polycystic Kidney Disease by Whole Exome Sequencing

Current Genomics ◽

10.2174/1389202922666210219111810 ◽

2021 ◽

Vol 22 ◽

Author(s):

Masoud Heidari ◽

Hamid Gharshasbi ◽

Alireza Isazadeh ◽

Morteza Soleyman-Nejad ◽

Mohammad Hossein Taskhiri ◽

...

Keyword(s):

Kidney Disease ◽

Exome Sequencing ◽

Polycystic Kidney Disease ◽

Whole Exome Sequencing ◽

Novel Mutation ◽

Genetic Diagnosis ◽

Genetic Alterations ◽

Polycystic Kidney ◽

Novel Mutations ◽

Whole Exome

Background:: Polycystic kidney disease (PKD) is an autosomal recessive disorder resulting from mutations in the PKHD1 gene on chromosome 6 (6p12), a large gene spanning 470 kb of genomic DNA. Objective: The aim of the present study was to report newly identified mutations in the PKHD1 gene in two Iranian families with PKD. Materials and Methods: Genetic alterations of a 3-month-old boy and a 27-year-old girl with PKD were evaluated using whole-exome sequencing. The PCR direct sequencing was performed to analyse the co-segregation of the variants with the disease in the family. Finally, the molecular function of the identified novel mutations was evaluated by in silico study. Results: In the 3 month-old boy, a novel homozygous frameshift mutation was detected in the PKHD1 gene, which can cause PKD. Moreover, we identified three novel heterozygous missense mutations in ATIC, VPS13B, and TP53RK genes. In the 27-year-old woman, with two recurrent abortions history and two infant mortalities at early weeks due to metabolic and/or renal disease, we detected a novel missense mutation on PKHD1 gene and a novel mutation in ETFDH gene. Conclusion: In general, we have identified two novel mutations in the PKHD1 gene. These molecular findings can help accurately correlate genotype and phenotype in families with such disease in order to reduce patient births through preoperative genetic diagnosis or better management of disorders.

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Screening of Candidate Pathogenic Genes for Spontaneous Abortion using Whole Exome Sequencing

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207324666210628115715 ◽

2021 ◽

Vol 24 ◽

Author(s):

Qingwen Zhu ◽

Yiwen Zhou ◽

Jiayi Ding ◽

Li Chen ◽

Jia Liu ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Spontaneous Abortion ◽

Gene Fusion ◽

High Throughput Sequencing ◽

Kegg Pathway ◽

Novel Mutation ◽

Common Disease ◽

Pathogenic Genes ◽

Whole Exome

Background: Spontaneous abortion is a common disease in obstetrics and reproduction. Objective: This study aimed to screen candidate pathogenic genes for spontaneous abortion using whole-exome sequencing. Methods: Genomic DNA was extracted from abortion tissues of spontaneous abortion patients and sequenced using the Illumina HiSeq2500 high-throughput sequencing platform. Whole exome sequencing was performed to select harmful mutations, including SNP and insertion and deletion sites, associated with spontaneous abortion. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and gene fusion analyses were performed. MUC3A and PDE4DIP were two novel mutation genes that were screened and verified by PCR in abortion tissues of patients. Results: A total of 83,633 SNPs and 13,635 Indel mutations were detected, of which 29172 SNPs and 3093 Indels were screened as harmful mutations. The 7 GO-BP, 4 GO-CC, 9 GO-MF progress, and 3 KEGG pathways were enriched in GO and KEGG pathway analyses. A total of 746 gene fusion mutations were obtained, involving 492 genes. MUC3A and PDE4DIP were used for PCR verification because of their high number of mutation sites in all samples. Conclusion: There are extensive SNPs and Indel mutations in the genome of spontaneous abortion tissues, and the effect of these gene mutations on spontaneous abortion needs further experimental verification.

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A homozygous mutation of GNRHR in a familial case diagnosed with polycystic ovary syndrome

Acta Endocrinologica ◽

10.1530/eje-16-0968 ◽

2017 ◽

Vol 176 (5) ◽

pp. K9-K14 ◽

Cited By ~ 5

Author(s):

Sandrine Caburet ◽

Ronit Beck Fruchter ◽

Bérangère Legois ◽

Marc Fellous ◽

Stavit Shalev ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sanger Sequencing ◽

Polycystic Ovary ◽

Missense Variant ◽

Genetic Alterations ◽

Homozygous Mutation ◽

Gonadotropin Secretion ◽

Whole Exome ◽

A Genome

Context PCOS is a heterogeneous condition characterized by hyperandrogenism and chronic anovulation and affects about 10% of women. Its etiology is poorly known, but a dysregulation of gonadotropin secretion is one of its hallmarks. Objective As the etiology of PCOS is unclear, we have performed a genome-wide analysis of a consanguineous family with three sisters diagnosed with PCOS. Methods Whole-exome sequencing and Sanger sequencing confirmation. Results Whole-exome sequencing allowed the detection of the missense variant rs104893836 located in the first coding exon of the GNRHR gene and leading to the p.Gln106Arg (p.Q106R) substitution. Sanger sequencing of all available individuals of the family confirmed that the variant was homozygous in the three affected sisters and heterozygous in both parents. Conclusions This is the first description of a GNRHR gene mutation in patients diagnosed with PCOS. Although we do not exclude a possible interaction of the identified variant with the genetic background and/or the environment, our result suggests that genetic alterations in the hypothalamo–pituitary axis may play role in the pathogenesis of PCOS.

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Molecular defects identified by whole exome sequencing in a child with atypical mucopolysaccharidosis IIIB

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2016-0333 ◽

2017 ◽

Vol 30 (4) ◽

Cited By ~ 6

Author(s):

Qingwen Zeng ◽

Yanjie Fan ◽

Lili Wang ◽

Zhuo Huang ◽

Xuefan Gu ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Novel Mutation ◽

Unknown Etiology ◽

Mendelian Disease ◽

Atypical Form ◽

Pathogenic Variants ◽

Diagnostic Potential ◽

Whole Exome ◽

Related Enzyme

AbstractBackground:Mucopolysaccharidosis IIIB (MPS IIIB) is a genetic disease characterized by mutations in theCase presentation:Whole exome sequencing (WES) was conducted and the putative pathogenic variants were validated by Sanger sequencing. The activity of MPS IIIB related enzyme in the patient’s blood serum was assayed. A heterozygous, non-synonymous mutation (c.1562C>T, p.P521L) as well as a novel mutation (c.1705C>A, p.Q569K) were found in theConclusions:Our results describe an atypical form of MPS IIIB and illustrate the diagnostic potential of targeted WES in Mendelian disease with unknown etiology. WES could become a powerful tool for molecular diagnosis of MPS IIIB in clinical setting.

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Whole exome sequencing identifies a novel mutation in ASPM and ultra-rare mutation in CDK5RAP2 causing Primary microcephaly in consanguineous Pakistani families

Pakistan Journal of Medical Sciences ◽

10.12669/pjms.38.1.4464 ◽

2021 ◽

Vol 38 (1) ◽

Author(s):

Ehtisham ul Haq Makhdoom ◽

Haseeb Anwar ◽

Shahid Mahmood Baig ◽

Ghulam Hussain

Keyword(s):

Intellectual Disability ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Novel Mutation ◽

Speech Impairment ◽

Primary Microcephaly ◽

Rare Mutation ◽

Homozygous Variant ◽

Whole Exome ◽

Pakistani Families

Background & Objectives: Primary Microcephaly (MCPH) is a rare neurogenetic disease, manifesting congenitally reduced head circumference and non-progressive intellectual disability (ID). To date, twenty-eight genes with biallelic mutations have been reported for this disorder. The study aimed for molecular genetic characterization of Pakistani families segregating MCPH. Methods: We studied two unrelated consanguineous families (family A and B) presenting >2 patients with diagnostic symptoms of MCPH, born to asymptomatic parents. We employed whole-exome sequencing (WES) of probands to find putative causal mutations. The candidate variants were further confirmed and analyzed for co-segregation by Sanger sequencing of all available members of each family. This study was conducted at Government College University, Faisalabad, Pakistan, and Cologne Center for Genomics (CCG), University of Cologne, Germany; during 2017-2020. Results: We identified a novel homozygous variant c.10097_10098delGA, p.(Gly3366Glufs*19) in exon 26 of ASPM gene in family A which presents with moderate intellectual disability, speech impairment, visual abnormalities, seizures, and ptyalism. Family B was found to segregate nonsense, homozygous variant c.448C>T p.(Arg150*) in CDK5RAP2. The patients also exhibited mild to severe seizures without ptyalism that has not been previously reported in patients with mutations in the CDK5RAP2 gene. Conclusion: We report a novel mutation in ASPM and ultra-rare mutation in the CDK5RAP2 gene, both causing primary microcephaly. The study expands the mutational spectrum of the ASPM gene to 212, and also adds to the clinical spectrum of CDK5RAP2 mutations. It also demonstrated the utility of WES in the investigation and genetic diagnosis of genetically heterogeneous disorders like MCPH. These findings would aid in diagnostic and preventive strategies including carrier screening, cascade testing, and genetic counselling. doi: https://doi.org/10.12669/pjms.38.1.4464 How to cite this:Makhdoom EH, Anwar H, Baig SM, Hussain G. Whole exome sequencing identifies a novel mutation in ASPM and ultra-rare mutation in CDK5RAP2 causing Primary microcephaly in consanguineous Pakistani families. Pak J Med Sci. 2022;38(1):---------. doi: https://doi.org/10.12669/pjms.38.1.4464 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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