Molecular basis for type 1 antithrombin deficiency: identification of two novel point mutations and evidence for a de novo splice site mutation

Inherited type 1 antithrombin (AT) deficiency is characterized by a reduction in both immunologically and functionally detectable protein. The disorder is associated with a high risk of thromboembolic disease. We have investigated the molecular basis of type 1 AT deficiency in three unrelated families. We have used the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and the intron-exon junctions of the AT gene. Two novel point mutations were identified. A T to C single-base substitution was found in codon 421 in exon 6 (nucleotide position 13380), leading to an AT 421 isoleucine to threonine substitution. In another kindred, one of three Cs at nucleotide (nt) positions 5448 to 5450 in exon 3A (codon 151 or 152) was deleted, resulting in a frameshift mutation and predicting premature termination of protein translation at codon 251. In a third family, a previously reported G to A substitution, at nt position 9788 in intron 4, 14 bp in front of exon 5, was found. We have demonstrated the creation of a de novo exon 5 splice site by ectopic transcript analysis of lymphocyte mRNA. In all cases, the affected individuals were heterozygous for the mutation and no variant AT protein was detected.

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The Molecular Basis of Antithrombin Deficiency in Belgian and Dutch Families

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615215 ◽

1998 ◽

Vol 80 (09) ◽

pp. 376-381 ◽

Cited By ~ 6

Author(s):

W. Lissens ◽

S. Seneca ◽

P. Capel ◽

B. Chatelain ◽

P. Meeus ◽

...

Keyword(s):

Molecular Basis ◽

Gene Deletion ◽

Direct Sequencing ◽

Genetic Defect ◽

Polymorphism Analysis ◽

Type I ◽

Type Ii ◽

Antithrombin Deficiency ◽

Venous Thromboembolic Events ◽

At Deficiency

SummaryThe molecular basis of hereditary antithrombin (AT) deficiency has been investigated in ten Belgian and three Dutch unrelated kindreds. Eleven of these families had a quantitative or type I AT deficiency, with a history of major venous thromboembolic events in different affected members. In the other two families a qualitative or type II AT deficiency was occasionally diagnosed.DNA studies of the AT gene were performed, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and intronexon junction regions. Six novel point mutations were identified: four missense, one nonsense mutation and a single nucleotide deletion near the reactive site, causing a frameshift with premature translation termination. In two kindreds the underlying genetic defect was caused by a whole gene deletion, known as a rare cause of AT deficiency. In these cases, Southern blot and polymorphism analysis of different parts of the AT gene proved useful for diagnosis. In another kindred a partial gene deletion spanning 698 basepairs could precisely be determined to a part of intron 3B and exon 4. In two type I and in both type II AT deficient families a previously reported mutation was identified. In all cases, the affected individuals were heterozygous for the genetic defect.

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A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

Biology ◽

10.3390/biology10121248 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1248

Author(s):

Dmytro Sirokha ◽

Olexandra Gorodna ◽

Yakov Vitrenko ◽

Nataliya Zelinska ◽

Rafal Ploski ◽

...

Keyword(s):

Splice Site ◽

Rna Binding ◽

De Novo ◽

Zinc Fingers ◽

Point Mutations ◽

Whole Exome ◽

Alternative Donor ◽

Wt1 Mutation ◽

Wt1 Protein ◽

Genetic Rearrangements

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

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Insertions/Deletions in the Antithrombin Gene: 3 Mutations Associated with Non-Expression

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648486 ◽

1992 ◽

Vol 67 (05) ◽

pp. 521-525 ◽

Cited By ~ 2

Author(s):

Martina Daly ◽

David J Perry ◽

Paul L Harper ◽

Helena M Daly ◽

Antoine W W Roques ◽

...

Keyword(s):

Molecular Basis ◽

Novel Mutation ◽

Direct Sequencing ◽

Protein Translation ◽

Thromboembolic Disease ◽

Cloning And Sequencing ◽

Antithrombin Deficiency ◽

The Third ◽

History Of ◽

Variant Alleles

SummaryWe have investigated the molecular basis of antithrombin deficiency in 3 individuals, 2 of whom had a proven family history of thromboembolic disease. An approximate 50% reduction in functional and immunologic levels of antithrombin was detected in plasma from the propositi indicating an allelic deficiency of antithrombin. In each case direct sequencing of amplified DNA revealed a novel mutation involving single bases: two being insertions, of a T in codon 48 and an A in codon 208, and the third being the deletion of an A in codon 370. The three mutations, which were confirmed by cloning and sequencing the normal and variant alleles, all caused frameshifts leading to premature termination of protein translation. In no case could a truncated antithrombin be detected in plasma from the propositus suggesting either that it fails to be secreted, or is rapidly degraded.

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Hereditary antithrombin deficiency: heterogeneity of the molecular basis and mortality in Dutch families

Blood ◽

10.1182/blood.v84.12.4209.bloodjournal84124209 ◽

1994 ◽

Vol 84 (12) ◽

pp. 4209-4213 ◽

Cited By ~ 23

Author(s):

HH van Boven ◽

RJ Olds ◽

SL Thein ◽

PH Reitsma ◽

DA Lane ◽

...

Keyword(s):

Genetic Heterogeneity ◽

Molecular Basis ◽

Excess Mortality ◽

Direct Sequencing ◽

Pcr Amplification ◽

Base Substitution ◽

Antithrombin Deficiency ◽

Single Base ◽

Molecular Defects ◽

Mild Defect

We studied the molecular basis and genetic heterogeneity of hereditary antithrombin (III) deficiency in nine Dutch families. Polymerase chain reaction (PCR) amplification and direct sequencing of all antithrombin gene exons and flanking intronic regions identified mutations in eight families. Given the opportunity to correlate the molecular basis with survival, we addressed the relevance of molecular defects to mortality in inherited antithrombin deficiency. The defects included single nucleotide deletions (7671 del G, 7768–69 del G) and insertions (5501 ins A, 2463 G-->TC) that lead to frameshifts, a single base substitution [5381 C-->T (129Arg-->stop)] leading to a premature termination codon, and single base substitutions resulting in amino acid substitutions [2652 A-->C (63Tyr-->Ser), 13380 T-->C (421Ile-->Thr), and 13407 G-->T (430Cys-->Phe)]. All affected individuals were heterozygous for the defects. Previously we found in Dutch families that antithrombin deficiency did not lead to higher mortality compared with the general population. In accordance with these findings, we observed no excess mortality in the nine families [Observed:Expected, 52:52.6; standardised mortality ratio (SMR) 1.0, 95% confidence interval (CI), 0.7–1.3]. Our findings confirmed a considerable genetic heterogeneity underlying antithrombin deficiency. We therefore concluded that the lack of excess mortality in these families is not caused by a Dutch mild defect. We suggest that the longevity is not affected by molecular defects in the antithrombin gene and hypothesize that differences in mortality or natural history between families most likely result from other (genetic) risk factors.

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P19 An extremely high rate of de novo base substitution mutations causes interruptions at the myotonic dystrophy type 1 locus

Neuromuscular Disorders ◽

10.1016/s0960-8966(10)70034-1 ◽

2010 ◽

Vol 20 ◽

pp. S10

Author(s):

J.M. Couto ◽

C. Braida ◽

F. Morales ◽

B. Adam ◽

P. Cuenca ◽

...

Keyword(s):

Myotonic Dystrophy ◽

De Novo ◽

High Rate ◽

Base Substitution ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Substitution Mutations

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RISK FACTORS, PREVALENCE AND DIAGNOSIS OF HUTCHISON GILFORD SYNDROME WITH SPECIAL REFERENCE TO CASE REPORTS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2017v9i5.16282 ◽

2017 ◽

Vol 9 (5) ◽

pp. 1

Author(s):

Bhawana Sharma ◽

Priyanka Sharma ◽

S. C. Joshi

Keyword(s):

Splice Site ◽

De Novo ◽

Case Reports ◽

Genetic Disorder ◽

Subcutaneous Fat ◽

Point Mutations ◽

Lamin A ◽

Laboratory Findings ◽

Progeria Syndrome ◽

Hutchinson Gilford Progeria Syndrome

Progeria also known Hutchinson–Gilford progeria syndrome (HGPS), is an extremely rare genetic disorder. The prevalence of HGPS is 1 in 4-8 million newborns. Progeria causes premature, rapid aging shortly after birth present within the first year of life. Recently, de novo point mutations in the Lmna gene at position 1824 of the coding sequence have been found in persons with HGPS. Lmna encodes lamin A and C, the A-type lamins, which are an important structural component of the nuclear envelope and play a role in protein processing. The most common HGPS mutation is located at codon 608 (G608G). This mutation responsible for creating a cryptic splice site within exon 11, which deletes a proteolytic cleavage site within the expressed mutant lamin A. In Progeria, gene mutation results in the deletion of a Zmpste24/FACE1 splice site in prelamin A, preventing end terminal cleavage. The result of this point mutation can be observed by the main clinical and radiological features include alopecia, thin skin hypoplasia of nails, loss of subcutaneous fat, and osteolysis. The common symptoms of HGPS is a loss of eyebrows and eyelashes which can observed in early childhood and due to receding hairline and blading can also observed. Generally, this patient has facial character include microganthia (small jaw), craniofacial disproportion, prominent eyes, scalp veins and alopecia (loss of hair), restricted joint mobility and severe premature atherosclerosis. Laboratory findings are unremarkable, with the exception of an increased urinary excretion of hyaluronic acid. There is presently no effective therapy is available for Hutchinson-Gilford progeria syndrome (HGPS) but, it is essential to monitor carefully cardiovascular and cerebrovascular disease So, Treatment usually includes low dose aspirin which helps prevent the atherothrombotic events, stroke and heart attacks by hindering platelet aggregation

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Using Oxford Nanopore Technology direct RNA sequencing to identify depurination events induced by ricin and other ribosome inactivating proteins

10.1101/2021.08.13.456275 ◽

2021 ◽

Author(s):

Yan Ryan ◽

Abbie Jasmine Harrison ◽

Hannah Trivett ◽

Catherine Hartley ◽

Jonathan David ◽

...

Keyword(s):

Nucleic Acids ◽

Rna Sequencing ◽

Direct Sequencing ◽

Point Mutations ◽

Ribosomal Subunit ◽

Protein Translation ◽

28S Rrna ◽

Sequencing Data ◽

Ribosome Inactivating Proteins ◽

Oxford Nanopore

Depurination is a frequent modification to both DNA and RNA, in DNA causing point mutations through misincorporation, in RNA, disabling ribosomes and halting protein synthesis. Some modifications of nucleic acids can be determined by direct sequencing using Oxford Nanopore Technologies (ONT). However, the identification of modifications is often limited by noise and their variety and number. Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop of the large ribosomal subunit. This leaves only a ribose backbone, thus inhibiting protein translation. In humans, biological threat agents and ribosome inactivating proteins, such as ricin and saporin, depurinate base 4605 on the 28S rRNA providing a single defined target to try and identify. We postulated that the depurination event could be detected using ONT direct RNA sequencing through a change in charge in the ricin loop. A software tool was developed, RIPpore, that quantified the adenine modification from direct RNA sequencing data of ribosomal RNA purified from respiratory epithelial cells exposed to ricin. This provided a novel method of directly identifying ricin exposure and a basis for the utility of ONT in detecting lesions in nucleic acids caused by depurination events.

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Incidence and prognostic value of TET2 alterations in de novo acute myeloid leukemia achieving complete remission

Blood ◽

10.1182/blood-2009-07-234484 ◽

2010 ◽

Vol 116 (7) ◽

pp. 1132-1135 ◽

Cited By ~ 85

Author(s):

Olivier Nibourel ◽

Olivier Kosmider ◽

Meyling Cheok ◽

Nicolas Boissel ◽

Aline Renneville ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

Prognostic Value ◽

De Novo ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Point Mutations ◽

Disease Free Survival ◽

Acute Myeloid

Abstract Mutations of the ten eleven translocation 2 gene (TET2) have recently been reported in myelodysplastic syndrome and myeloproliferative neoplasms. We analyzed the incidence and prognostic value of TET2 point mutations and other genomic alterations by direct sequencing and single nucleotide polymorphism microarray analysis in 111 de novo acute myeloid leukemia, who had all achieved complete remission (CR). Mutations were observed in 19 (17%) of the 111 patients compared with 10 (27%) of 36 patients who had failed to achieve CR (P = .2). In the 111 patients who had achieved CR, TET2 alterations were only significantly associated with NPM1 mutations but not with other pretreatment characteristics. TET2 gene status was not significantly correlated with disease-free survival and overall survival, both in the entire cohort and in patients with normal karyotype.

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