Molecular Basis of Antithrombin Type I Deficiency: The First Large In-frame Deletion and Two Novel Mutations in Exon 6

1994 ◽  
Vol 72 (04) ◽  
pp. 534-539 ◽  
Author(s):  
J Emmerich ◽  
G Chadeuf ◽  
M Alhenc-Gelas ◽  
M Gouault-Heilman ◽  
P Toulon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Stop Codon ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Repeat Sequence ◽  
Type I ◽  
Novel Mutations ◽  
Flanking Sequences ◽  
At Deficiency ◽  
Exon 4

SummaryWe report three novel mutations accounting for cases of inherited type I antithrombin (AT) deficiency. Using the polymerase chain reaction (PCR) and direct sequencing of the coding sequences of the AT gene, we found one mutation in exon 4 and two in exon 6. A deletion of 105 bp causing an in-frame deletion of 35 amino acids between Tyr 240 and Gly 276 was found in exon 4. In a second kindred, deletion of two adenines in codon 412-413 introduced a frameshift and a stop codon at position 431. The last mutation was an insertion of ACCG in codon 387, generating a frameshift with a stop codon located at the normal position.The finding of a sequence repeat of nine residues located at the 5’and 3’ ends of the deleted fragment might explain the 105 bp deletion by slippage and mispairing at the replication fork during DNA synthesis. The second mutation is the fourth described within a region of six amino acids (between Phe 408 and Arg 413), which seems to be a cluster of mutations. In this case, the presence of a double repeat sequence - TTCCT and AACA - flanking this region could be particularly favorable for slipped mispairing.These results confirm that human gene mutations are not random events but are strongly influenced by DNA flanking sequences.

The Molecular Basis of Antithrombin Deficiency in Belgian and Dutch Families

1998 ◽  
Vol 80 (09) ◽  
pp. 376-381 ◽  
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.

scholarly journals Identification of molecular defects in a subject with type I CD36 deficiency

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3545-3552 ◽  
Author(s):  
H Kashiwagi ◽  
Y Tomiyama ◽  
S Kosugi ◽  
M Shiraga ◽  
RH Lipsky ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Stop Codon ◽  
Type I ◽  
Molecular Defects ◽  
Cdna Sequences ◽  
Cd36 Deficiency ◽  
Translation Stop Codon ◽  
Polymerase Chain ◽  
Exon 4 ◽  
Splice Junctions

Abstract We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5′ half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191–1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3′ region of CD36 cDNA (nt 962–1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription- polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR- based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.

Novel ABCD1 gene mutations in Iranian pedigrees with X-linked adrenoleukodystrophy

2019 ◽  
Vol 32 (11) ◽  
pp. 1207-1215
Author(s):  
Babak Emamalizadeh ◽  
Yousef Daneshmandpour ◽  
Abbas Tafakhori ◽  
Sakineh Ranji-Burachaloo ◽  
Sajad Shafiee ◽  
...  
Keyword(s):  
Protein Structure ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Protein Product ◽  
Structure And Function ◽  
Novel Mutations ◽  
Chain Reaction ◽  
Abcd1 Gene ◽  
Polymerase Chain ◽  
And Function

Abstract Background X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the ABCD1 gene located on Xq28. X-ALD is characterized by a spectrum of different manifestations varying in patients and families. Methods Four pedigrees with X-ALD consisting of patients and healthy members were selected for investigation of ABCD1 gene mutations. The mutation analysis was performed by polymerase chain reaction (PCR) followed by direct sequencing of all exons. The identified mutations were investigated using bioinformatics tools to predict their effects on the protein product and also to compare the mutated sequence with close species. Results One previously known missense mutation (c.1978 C > T) and three novel mutations (c.1797dupT, c.879delC, c.1218 C > G) were identified in the ABCD1 gene, each in one family. Predicting the effects of the mutations on protein structure and function indicated the probable damaging effect for them with significant alterations in the protein structure. We found three novel mutations in the ABCD1 gene with damaging effects on its protein product and responsible for X-ALD.

Molecular analysis and genotype-phenotype correlation in patients with antithrombin deficiency from Southern Italy

2012 ◽  
Vol 107 (04) ◽  
pp. 673-680 ◽  
Author(s):  
Giuseppe Castaldo ◽  
Anna Cerbone ◽  
Anna Guida ◽  
Igor Tandurella ◽  
Rosaria Ingino ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Southern Italy ◽  
Stop Codon ◽  
Type I ◽  
Missense Mutations ◽  
Type Ii ◽  
Clinical Expression ◽  
Antithrombin Deficiency ◽  
At Deficiency ◽  
Molecular Bases

SummaryWe sequenced the SERPINC1 gene in 26 patients (11 males) with antithrombin (AT) deficiency (22 type I, 4 type II), belonging to 18 unrelated families from Southern Italy. Heterozygous mutations were identified in 15/18 (83.3%) families. Of them, eight were novel mutations, each being identified in one family. Seven clearly cause impaired protein synthesis (four frameshift, one non-stop, one splicing and one 21bp deletion). One, present in a single patient, is a missense mutation thought to be causative because: a) it is absent in 100 chromosomes from controls; b) it involves a highly conserved amino acid, whose change is predicted to impair AT activity; c) no other mutation is present in the propositus. Severe mutations (i.e. nonsense, frameshift, deletions) were invariably identified in type I patients. In type II patients, 3/4 were missense mutations; the fourth leads to a 19 nucleotides shift in the stop codon. In addition to the type of mutation, the co-existence of other predisposing factors in most patients helps explain the severity of the present type I cases (age at first event, recurrence during prophylaxis). In the five families in which there was more than one member affected, the same genotype and a concordant clinical expression of the disease were found. We conclude that the molecular bases of AT deficiency in Southern Italy are different as compared to other geographic areas, and that molecular analysis and the study of the effect of the mutation may help predict the clinical expression of the disease.

scholarly journals Five novel mutations located in exons III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 159-168 ◽  
Author(s):  
S Gandrille ◽  
M Alhenc-Gelas ◽  
P Gaussem ◽  
MF Aillaud ◽  
E Dupuy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein C ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Anticoagulant Activity ◽  
Direct Sequencing ◽  
Catalytic Triad ◽  
Activity Levels ◽  
Novel Mutations ◽  
Protein C Deficiency

We describe five families presenting with type II hereditary protein C deficiency characterized by normal antigen and amidolytic activity levels but low anticoagulant activity. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining amplification by the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis of the amplified fragments, and direct sequencing of fragments displaying altered melting behavior. We detected five novel mutations. Three were located in the C-terminal part of the propeptide encoded by exon III: Arginine (Arg)-5 to tryptophan (Trp), Arg-1 to histidine (His), and Arg-1 to cysteine (Cys) mutations. The two others, located in exon IX, affected Arg 229 and serine (Ser) 252, which were respectively replaced by glutamine (Gln) and asparagine (Asn). DNA studies of the other exons from affected individuals showed no other abnormalities. These novel mutations provide further insight into the importance of the affected amino acids located close to the active site, near Asp 257, one of the three amino acids of the catalytic triad. The low anticoagulant activity of the abnormal protein C indicated that Arg 229 and Ser 252 play a key role during the interaction between protein C and its cofactor protein S, phospholipids, or factors Va and VIIIa. The Arg-1 to Cys mutation led to the dimerization of protein C with another plasmatic component, as evidenced by the presence in the plasma of a high molecular weight form of protein C that disappeared after reduction. No molecular mass abnormalities were observed in heavy and light chains of all other protein C mutants. In the five families explored, 9 (64%) of the 14 subjects bearing the mutations reported thrombotic events. This suggests that the protein C amino acids affected by the mutations are very important for the in vivo expression of the antithrombotic properties of protein C.

Noval Mutation in Four Saudi Families with Glanzmann Thrombasthenia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1136-1136
Author(s):  
Tarek Owaidah ◽  
Hala Abalkhail ◽  
Abdulrahman Al Musa ◽  
Hasan Mosmali ◽  
Albanyan Abdulmajeed ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Stop Codon ◽  
Direct Sequencing ◽  
Family Members ◽  
Premature Stop Codon ◽  
Type I ◽  
Bleeding Tendency ◽  
Coding Region ◽  
Glanzmann Thrombasthenia ◽  
Exon 1

Abstract Abstract 1136 Introduction: Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation and variable bleeding tendency. Inherited genetic mutations in integrin alpha IIb and beta3 (ITGA2B, ITGB3) result in a heterogeneity of the thrombasthenia phenotypes. It is phenotypically expressed in homozygotes or compound heterozygotes, given that 50% of normal aIIbb3 is sufficient to guarantee unimpaired platelet function that result in asymptomatic carriers. Defects in ITGB3 result in failure of binding of B3 and alpha IIb. These defects had been reported in Arabs (Iraqi Jews). We are reporting some results of Saudi GT genotype project. Materials & Methods: In this study, we analyzed the entire coding region ITGB3 gene using polymerase chain reaction (PCR) and direct sequencing with primers specifically designed to amplify the coding region of exon 1–15 and exon /Intron boundaries in a cohort of 51 GT patients diagnosed and treated in our institute. Results: Out of 51 cases from 20 families had mutational screening of the ITGB3 gene with the aim to detect the causative pathogenic mutations to enable the pre-symptomatic diagnosis in at risk family members. In this study we detect 1 novel germline mutation c.2190delC (p.Ser703fs) in exon 13. The mutation is predicted to result in premature stop codon and protein truncation. The mutation was detected in 6 patients in homozygous stat (3 males and 3 females). Three tested samples from the patients family members detected the mutation in heterozygous state and all of them were asymptomatic with normal PFA and Intact expression of Platelet Glycoprotiens CD41(Gpllb), CD42a(GPIX), CD42b(GPlb), and CD61(Gpllla). All the GT patients with this mutation were type I GT with Prolonged PFA and complete absence of CD41(Gpllb) and CD61(Gpllla) glycoprotein. Conclusion: The result of this study represents the first Molecular analysis of ITGB3 gene in Saudi Arabia and displays the existence of novel pathogenic and possibly a founder effect in Saudi families. Disclosures: No relevant conflicts of interest to declare.

Characterization of the mouse thyrotrophin-releasing hormone receptor gene: an exon corresponds to a deletion in the rat cDNA

1993 ◽  
Vol 11 (2) ◽  
pp. 141-149 ◽  
Author(s):  
S M Duthie ◽  
P L Taylor ◽  
K A Eidne
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Stop Codon ◽  
Amino Acid Sequences ◽  
R Gene ◽  
Coding Region ◽  
Thyrotrophin Releasing Hormone ◽  
Exon 2 ◽  
Exon 4

ABSTRACT The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5′ untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3′ UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3′ UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3′ UTR, allowing transcription to continue into the 3′ UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA. This would then result in an alternative 412 amino acid version of the mouse TRH-R protein, with 95% homology to the rat TRH-R. This study focused on the structural differences in the intracellular COOH-terminal tail of the receptor, which is known to be a functionally important domain in other members of the G protein-coupled receptor family. We have also recently characterized the human TRH-R cDNA, which revealed a third variant at the COOH terminus. Comparisons between mouse, rat and human TRH-Rs show that the amino acid sequences are virtually identical. However, significant differences between these species exist at the COOH terminus, with each TRH-R having a unique form of the COOH-terminal tail, beginning at exactly the same site and encoding 1, 20 and 6 amino acids in the mouse, rat and human respectively.

Molecular mechanisms of antithrombin deficiency in two Chinese families

2005 ◽  
Vol 94 (12) ◽  
pp. 1172-1176 ◽  
Author(s):  
Rong-Fu Zhou ◽  
Qi-Hua Fu ◽  
Wen-Bin Wang ◽  
Shuang Xie ◽  
Jin Dai ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Direct Sequencing ◽  
Type I ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Chinese Families ◽  
Real Time Quantitative Pcr ◽  
Intracellular Degradation ◽  
Phenotype Analysis ◽  
At Deficiency

SummaryWe investigated the molecular mechanisms responsible for type I congenital antithrombin (AT) deficiency in two unrelated Chinese pedigrees manifesting multiple site venous thrombosis. Phenotype analysis showed both probands had almost 50% of normal AT levels. Direct sequencing of amplified DNA revealed 2757C>T in proband 1 and 13328G>A in proband 2, predicting a heterozygous Thr98Ile (T98I) and Ala404Thr (A404T), respectively. No proband had 20210A allele or factorV Leiden mutation. Transient expression of complementary DNA coding for the mutations in COS-7 cells showed impaired secretion of the mutant molecules. Real-time quantitative PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT-I98 mutant were stained diffusely without perinuclear enhancement and cells expressing AT-T404 mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. We conclude that the impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular pathogenesis of AT deficiency caused by T98I and A404T mutation for the two families, respectively.

scholarly journals Protein S deficiency type I: identification of point mutations in 9 of 10 families

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3444-3451 ◽  
Author(s):  
S Mustafa ◽  
I Pabinger ◽  
C Mannhalter
Keyword(s):  
Stop Codon ◽  
Direct Sequencing ◽  
Protein S ◽  
Type I ◽  
Missense Mutations ◽  
Protein S Deficiency ◽  
Site Mutation ◽  
Frame Shift ◽  
S Deficiency ◽  
Deficiency Type

We identified potentially causative mutations in the active protein S gene (PROS 1) by direct sequencing of PROS 1-specific polymerase chain reaction (PRC) products of all 15 exons, including exon-intron boundaries in 10 families with hereditary protein S deficiency type I. Seven different mutations were found in 9 of 10 families, including one frame shift mutation, a previously published splice site mutation (both occurring in two unrelated families), four missense mutations, and a stop codon at the beginning of exon 12. In family studies, cosegregation of the mutation with the disease could be demonstrated for five mutations; for two missense mutations, this was not possible due to limited family data. All seven mutations were the only abnormalities identified in the respective index patients and were absent in 44 to 62 normal individuals. Therefore, they most likely represent the causal gene defects. For five mutations, analysis of ectopic RNA could be performed. Mutant transcripts were present in the case of the frame shift and three of the missense mutations, while no mutant RNA could be detected in the case of the stop codon.

scholarly journals Analysis of genetic and clinical characteristics of a Chinese Kallmann syndrome cohort with ANOS1 mutations

2017 ◽  
Vol 177 (4) ◽  
pp. 389-398 ◽  
Author(s):  
Min Nie ◽  
Hongli Xu ◽  
Rongrong Chen ◽  
Jiangfeng Mao ◽  
Xi Wang ◽  
...  
Keyword(s):  
Clinical Characteristics ◽  
Clinical Presentation ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Kallmann Syndrome ◽  
Chinese Patients ◽  
Missense Mutations ◽  
The Novel ◽  
Novel Mutations ◽  
Functional Relevance

Objective To analyze ANOS1 gene mutations in a large Chinese Kallmann syndrome (KS) cohort and to characterize the clinical presentation of the disease in patients with ANOS1 mutations. Patients and methods Chinese patients with KS, including 187 sporadic and 23 pedigree cases were recruited. Patients’ ANOS1 gene sequences were analyzed by direct sequencing of PCR-amplified products. In silico analysis was used to assess functional relevance of newly identified missense mutations. Patients’ clinical characteristics were analyzed retrospectively. Result(s) Fifteen nonsynonymous rare ANOS1 variants were found in 13 out of 187 sporadic and 8 out of 23 familial IHH probands. Seven novel (C86F, C90Y, C151W, Y379X, c.1062 + 1G > A, Y579L fs 591X, R597X) and eight recurrent ANOS1 mutations (S38X, R257X, R262X, R423X, R424X, V560I, c.1843-1G > A, p.R631X) were identified. All the novel mutations were predicted to be pathogenic. The prevalence of cryptorchidism was high (38.1%) and occurred in patients with different kind of ANOS1 mutations, while the patients with the same mutation did not present with cryptorchidism uniformly. Conclusion(s) The prevalence of ANOS1 gene mutations is low in sporadic KS patients, but is much higher in familial KS patients. In the present study, we identify seven novel ANOS1 mutations, including two mutations in the CR domain, which are probably pathogenic. These mutations expand the ANOS1 mutation spectrum and provide a foundation for prenatal diagnosis and genetic counseling.

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