Exons sequencing identifies a novel mutation in GPR157 in a high myopia family

2020 ◽  
Author(s):  
Tao Tang ◽  
Mohan Liu ◽  
Ting Wei ◽  
Lin Deng ◽  
Yueyang Zhang ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
High Myopia ◽  
Sanger Sequencing ◽  
Novel Mutation ◽  
Rt Pcr ◽  
Genetic Characteristics ◽  
Reverse Transcription Pcr ◽  
Whole Exome ◽  
Reduced Penetrance ◽  
Next Generation Sequencing Ngs

Abstract Background Next-generation sequencing (NGS) and whole exome sequencing (WES) have identified many potential disease-causing loci and genetic mutations of high myopia(HM). However, these known genes can only explain the heritability of a small proportion of HM patients. A large proportion of variants have yet to be discovered. Herein we aimed to investigate the genetic characteristics of HM through a Chinese HM family(the inheritance pattern unknown) . Methods We performed WES on the parent-offspring trio and identified mutations by Sanger sequencing. All the members in this family were sequenced to validate phenotype co-segregated with candidate genes via Sanger sequencing as well. Besides, mutations detected were further evaluated in a cohort of 110 sporadic high myopia controls and 200 unrelated ethically-matched controls. And reverse transcription PCR(RT-PCR) was applied to measure the mRNA expression levels of GPR157 in the 4-week-old KM mice. Results A novel heterozygous nonsense mutation, c.859C>T (p.Arg287*) of GPR157 gene, was detected in the proband and her father by WES. And this disease-associated mutation was not found in 310 control individuals. For the family under study, HM was classified as autosomal dominant inheritance with reduced penetrance. And RT-PCR results showed GPR157 was abundantly expressed in the eye. Conclusion The hybrid nonsense mutation of the GPR157 gene identified in this study may constitute a novel genetic cause of HM. Keywords :high myopia, WES, GPR157

scholarly journals Novel manifestations of Warburg micro syndrome type 1 caused by a new splicing variant of RAB3GAP1: a case report

BMC Neurology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Raziyeh Khalesi ◽  
Ehsan Razmara ◽  
Golareh Asgaritarghi ◽  
Ali Reza Tavasoli ◽  
Yasser Riazalhosseini ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Cerebral White Matter ◽  
Global Developmental Delay ◽  
Spastic Diplegia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mutation System ◽  
Warburg Micro Syndrome

Abstract Background The present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients. Case presentation A 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes. Conclusions Due to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1568-1568
Author(s):  
Taehyung Simon Kim ◽  
Zhaolei Zhang ◽  
Marc Tyndel ◽  
Jae-Sook Ahn ◽  
Seunghyun Choi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Sanger Sequencing ◽  
Novel Mutation ◽  
Tyrosine Kinase Domain ◽  
Blastic Crisis ◽  
Whole Exome ◽  
Tki Resistance

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.

scholarly journals Diagnosis of Joubert Syndrome 10 in a Fetus with Suspected Dandy-Walker Variant by WES: A Novel Splicing Mutation in OFD1

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Siyuan Linpeng ◽  
Jing Liu ◽  
Jianyan Pan ◽  
Yingxi Cao ◽  
Yanling Teng ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Novel Mutation ◽  
Joubert Syndrome ◽  
Splicing Mutation ◽  
The Novel ◽  
Rt Pcr ◽  
Abnormal Karyotype ◽  
Whole Exome ◽  
The Family ◽  
Sequencing Result

Joubert syndrome (JBTS) is a clinically and genetically heterogeneous group of ciliary diseases. To date, 34 subtypes of JBTS have been classified due to different causative genes or extra clinical features. Most of them are autosomal recessive, while only the subtype 10 (JBTS10) is a quite rare X-linked recessive disorder caused by OFD1 mutations with few reports. In this study, by using whole exome sequencing (WES), a novel OFD1 splicing mutation (c.2488+2T>C) was identified in a male fetus with suspected Dandy-Walker variant (DWV) and syndactyly, for whom abnormal karyotype and pathogenic CNV have been excluded. This mutation was inherited from the mother who has experienced two similar pregnancies before. An abnormal skipping of exon 18 in OFD1 mRNA was confirmed by RT-PCR and sequencing. Result from quantitative RT-PCR also showed that total OFD1 mRNA in the index fetus was significantly lower than the control. After a combined analysis of genetic testing results and genotype-phenotype correlations, the novel mutation c.2488+2T>C in OFD1 was considered to be the genetic cause for the affected fetus. Thus the diagnosis should be JBTS10 rather than the primary clinical diagnosis of DWV. We report the first prenatal case of JBTS10 in Chinese population, which not only helps the family to predict recurrence risks for future pregnancies but also provides more information for understanding such a rare disease. The results also present evidence that WES is an effective method in prenatal diagnosis for those fetuses with Joubert syndrome.

scholarly journals Detection and validation of six pathogenic variants in Chinese inherited heart disease patients using clinical whole-exome sequencing

2021 ◽  
Author(s):  
Lichao Cao ◽  
Fei Ye ◽  
Shuqi Xie ◽  
Ying Ba ◽  
Ying Zeng ◽  
...  
Keyword(s):  
Heart Disease ◽  
Next Generation Sequencing ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Sanger Sequencing ◽  
Targeted Next Generation Sequencing ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The targeted next-generation sequencing (NGS) was employed in detecting the pathogenic mutations in inherited heart disease patients in the present study. Two main methods, the NGS and the classic Sanger sequencing, were used in this study. And, the whole-exome sequencing (WES) was specifically used in this study.

Variable phenotype of Knobloch syndrome due to biallelic COL18A1 mutations in children

2020 ◽  
pp. 112067212097734
Author(s):  
Nadav Levinger ◽  
Karen Hendler ◽  
Eyal Banin ◽  
Mor Hanany ◽  
Adva Kimchi ◽  
...  
Keyword(s):  
Retinal Detachment ◽  
High Myopia ◽  
Sanger Sequencing ◽  
Autosomal Recessive Inheritance ◽  
Disease Awareness ◽  
Occipital Encephalocele ◽  
Knobloch Syndrome ◽  
Whole Exome ◽  
Neuroradiologic Imaging ◽  
Variable Phenotype

Purpose: Knobloch syndrome is a rare, recessively inherited disorder classically characterized by high myopia, retinal detachment, and occipital encephalocele. Our aim is to report the clinical and genetic findings of four Israeli children affected by Knobloch syndrome. Methods: Retrospective study of four patients diagnosed with Knobloch syndrome, who underwent full ophthalmic examination, electroretinography, and neuroradiologic imaging. Genetic analysis included whole exome sequencing (WES) and Sanger sequencing. Results: The four patients included in this study had high myopia and nystagmus at presentation. Ocular findings included vitreous syneresis, macular atrophy, macular coloboma, and retinal detachment. One child had iris transillumination defects and an albinotic fundus, initially leading to an erroneous clinical diagnosis of albinism. Electroretinography revealed a marked cone-rod pattern of dysfunction in all four children. Brain imaging demonstrated none to severe occipital pathology. Cutaneous scalp changes were present in three patients. WES analysis, confirmed by Sanger sequencing revealed COL18A1 biallelic null mutations in all affected individuals, consistent with autosomal recessive inheritance. Conclusions: This report describes variable features in patients with Knobloch syndrome, including marked lack of eye pigment similar to albinism in one child, macular coloboma in two children as well as advanced cone-rod dysfunction in all children. One patient had normal neuroradiologic findings, emphasizing that some affected individuals have isolated ocular disease. Awareness of this syndrome, with its variable phenotype may aid early diagnosis, monitoring for potential complications, and providing appropriate genetic counseling.

scholarly journals Analysis of the Genetic Characteristics of a Chinese Family With Otosclerosis

2020 ◽  
pp. 014556132091062
Author(s):  
Yongli Zhang ◽  
Qi Tang ◽  
Ruoyan Xue ◽  
Xiaohui Zhu ◽  
Hua Yang ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Sanger Sequencing ◽  
Autosomal Dominant ◽  
Family Members ◽  
Autosomal Dominant Inheritance ◽  
Dominant Inheritance ◽  
Genetic Characteristics ◽  
Whole Exome ◽  
The Family

Background: Otosclerosis is a focal lesion of the inner ear. The role of genetic factors in the pathogenesis of otosclerosis has received increasing attention. We analyzed the clinical manifestations, inheritance pattern, and pathogenic genes in a family with otosclerosis. Methods: We collected clinical data and generated a family pedigree. High-throughput second-generation sequencing technology was used to identify candidate genes by performing whole-exome sequencing of 7 members of the family, and Sanger sequencing was performed to validate candidate gene mutations in the 7 family members. Results: Otosclerosis was characterized by autosomal dominant inheritance in this family. Whole-exome sequencing did not reveal mutation sites in known deafness-related genes. However, a c.2209A > G (p.T737A) mutation was detected in exon 6 of the SP1 gene, which is associated with the COL1A1 gene. This mutation was a pathogenic mutation, and Sanger sequencing confirmed that this mutation cosegregated with the clinical phenotype among the family members. Conclusions: The pattern of otosclerosis in this family is consistent with autosomal dominant inheritance, and the SP1 gene, harboring the c.2209A > G (p.T737A) mutation in exon 6, may be the causative gene of otosclerosis in this family.

scholarly journals Whole exome sequencing reveals a homozygous nonsense mutation in HEXA gene leading to Tay-Sachs disease in Saudi Family

2020 ◽  
Vol 36 (6) ◽  
Author(s):  
Muhammad Imran Naseer ◽  
Angham Abdulrahman Abdulkareem ◽  
Mohammed Mohammed Jan ◽  
Adeel G. Chaudhary ◽  
Mohammad H. Al-Qahtani
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Nonsense Mutation ◽  
Sanger Sequencing ◽  
Homozygous Mutation ◽  
Tay Sachs Disease ◽  
Whole Exome ◽  
Hexa Gene ◽  
Saudi Family ◽  
Homozygous Nonsense Mutation

Objective: To study the causative variants in affected member of a Saudi family with Tay-Sachs disorder. This disorder includes paralysis, decreasing in attentiveness, seizures, blindness, motor deterioration progresses rapidly leading to a completely unresponsive state and a cherry-red spot visible on the eye. Methods: Whole exome sequencing (WES) and Sanger sequencing was performed to study the variant leading to the disease. Results: WES data analysis and Sanger sequencing validation, identifies a homozygous nonsense mutation c.1177C>T, p.Arg393Ter as a result in protein change. This mutation was also studied in 100 unrelated healthy controls. Conclusions: We detected homozygous mutation in HEXA gene that may lead to cause Tay-Sachs disorder. Moreover, explain the possibility that HEXA gene may play important role for multiple aspects of normal human neurodevelopment. doi: https://doi.org/10.12669/pjms.36.6.2579 How to cite this:Naseer MI, Abdulkareem AA, Jan MM, Chaudhary AG, Al-Qahtani MH. Whole exome sequencing reveals a homozygous nonsense mutation in HEXA gene leading to Tay-Sachs disease in Saudi Family. Pak J Med Sci. 2020;36(6):---------.  doi: https://doi.org/10.12669/pjms.36.6.2579 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.

scholarly journals Identification of a Novel Astrovirus (Astrovirus VA1) Associated with an Outbreak of Acute Gastroenteritis

2009 ◽  
Vol 83 (20) ◽  
pp. 10836-10839 ◽  
Author(s):  
Stacy R. Finkbeiner ◽  
Yan Li ◽  
Susan Ruone ◽  
Christina Conrardy ◽  
Nicole Gregoricus ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Care Center ◽  
Gastrointestinal Illness ◽  
Unknown Etiology ◽  
Gastroenteritis Outbreak ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Rapid Amplification ◽  
Multiple Sequences ◽  
Multiple Patients

ABSTRACT The etiology of a large proportion of gastrointestinal illness is unknown. In this study, random Sanger sequencing and pyrosequencing approaches were used to analyze fecal specimens from a gastroenteritis outbreak of unknown etiology in a child care center. Multiple sequences with limited identity to known astroviruses were identified. Assembly of the sequences and subsequent reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends generated a complete genome of 6,586 nucleotides. Phylogenetic analysis demonstrated that this virus, named astrovirus VA1 (AstV-VA1), is highly divergent from all previously described astroviruses. Based on RT-PCR, specimens from multiple patients in this outbreak were unequivocally positive for Ast-VA1.

scholarly journals New mutations in KCNT2 gene causing early infantile epileptic encephalopathy type 57: Case study and literature review

2020 ◽  
Author(s):  
Meryem Alagoz ◽  
Nasim Kherad ◽  
Sureyya Bozkurt ◽  
Adnan Yuksel
Keyword(s):  
Novel Mutation ◽  
Bioinformatic Analysis ◽  
Epileptic Encephalopathy ◽  
Phenotype Correlation ◽  
Whole Exome ◽  
And Function ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
New Mutations

Purpose. Early infantile epileptic encephalopathy (EIEE) 57 belongs to a group of encephalopathies with early-onset and characterised by severe electroencephalogram abnormalities, seizures, developmental delay and intellectual disability. Method. We carried out Whole Exome analysis using Next Generation Sequencing (NGS) and bioinformatic analysis performed to find mutation associated with the patient phenotypes. The effect of the mutation on protein structure analysed by PolyPhen2 and Swissmodel ExPASy. Results. In this study, we evaluated two unrelated Turkish males diagnosed with EIEE type 57 to investigate the genetic cause of this disease. Whole exome sequencing revealed mutations in KCN2 gene, which is a member of Potassium channels (KCN) gene family associated with epileptic encephalopathies. Two mutations, c.545A>T (p.Asn182Ile and c.2638C>A (p.Leu880Met) were reported here as a novel mutation. Conclusions. Our findings implicate the genotype-phenotype correlation of these mutations. Furthermore, the computational analysis showed their effect on protein binding site and function suggesting their role in the development of early infantile epileptic encephalopathy 57.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

Sign in / Sign up

Export Citation Format

Share Document