Abstract TP269: Distinct Molecular Mechanisms of Htra1 Mutants in Manifesting Heterozygotes With Carasil

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Hiroaki Nozaki ◽  
Taisuke Kato ◽  
Megumi Nihonmatsu ◽  
Yohei Saito ◽  
Ikuko Mizuta ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Molecular Mechanisms ◽  
Small Vessel Disease ◽  
Gel Filtration ◽  
Gene Mutations ◽  
Missense Mutations ◽  
Inherited Disease ◽  
Wild Type ◽  
Vessel Disease ◽  
Activation Cascade

Introduction: Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an autosomal recessive inherited cerebral small vessel disease (CSVD), involves severe leukoaraiosis, multiple lacunar infarcts, early-onset alopecia, and spondylosis deformans. High-temperature requirement serine peptidase A1 (HTRA1) gene mutations cause CARASIL by decreasing HTRA1 protease activity. Although CARASIL is a recessive inherited disease, heterozygous mutations in the HTRA1 gene were recently identified in 11 families with CSVD. Because CSVD is frequently observed in elderly individuals, it is unclear which mutants truly contribute to CSVD pathogenesis. Here, we found heterozygous mutations in the HTRA1 gene in individuals with CSVD and investigated the differences in biochemical characteristics between these mutant HTRA1s and mutant HTRA1s observed in homozygotes. Methods: We recruited 113 unrelated index patients with clinically diagnosed CSVD. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. Results: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in CARASIL (A252T and V297M) did not inhibit wild- type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer- associated HTRA1 activation. Conclusions: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.

COL4A1 is a Novel Causative Gene Responsible for Congenital Hemolytic Anemia, Representing Characteristic Clinical Course in Infants

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 934-934
Author(s):  
Hiromi Ogura ◽  
Shouichi Ohga ◽  
Takako Aoki ◽  
Taiju Utsugisawa ◽  
Hidehiro Takahashi ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Small Vessel Disease ◽  
Gene Mutations ◽  
Basement Membranes ◽  
Red Cell ◽  
Missense Mutations ◽  
Causative Gene ◽  
Congenital Hemolytic Anemia

Abstract We have been working on the differential diagnosis of congenital hemolytic anemia, but even with extensive analysis of hemoglobin, red cell membrane and enzymes, approximately 40% of patients remained to be diagnosed. In this study, we analyzed 17 undiagnosed hemolytic anemia subjects under the age of 1 by whole-exome sequencing, and identified COL4A1 gene mutations in 5 cases (29.4%). All patients were de novo cases without family histories and exhibited moderate to severe neonatal hemolytic anemia: Hgb, 5.2-9.3 g/dl; MCV, 90.0-126.9; MCHC, 29.9-32.7; and reticulocyte count, 9.2-33.0%. Either schizocytes or poikilocytes were observed in peripheral blood smears of 3 cases, suggesting that the microangiopathy was attributable to hemolysis. Previous reports showed that mutation of COL4A1 accounts for brain small-vessel disease characterized by stroke and eye abnormalities and the most characteristic complications of the present cases were congenital anomaly in the central nervous system, such as porencephaly, schizencephaly, congenital hydrocephalus, cataracts or paraventricular calcification, as reported previously. Hemolytic anemia became less severe within 2 months after birth, and all cases no longer required red cell transfusion after Day 50. COL4A1 encodes subtype 1 of type IV collagen, which is most abundantly expressed in basement membranes, including the vasculature. The COL4A1 gene mutations identified in the cases were all novel missense mutations except one, located in exons 26, 27, 37, 38 and 51. Although the pathophysiological significance of the mutations remains unclear, COL4A1 is the first identified causative gene responsible for congenital hemolytic anemia without intrinsic defects of red blood cells, and mutation of COL4A1 is the most prevalent cause of neonatal hemolytic anemia. Disclosures No relevant conflicts of interest to declare.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL)

2021 ◽  
pp. practneurol-2021-003058
Author(s):  
Rhea YY Tan ◽  
Anna M Drazyk ◽  
Kathryn Urankar ◽  
Clare Bailey ◽  
Stefan Gräf ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Small Vessel Disease ◽  
Postmortem Brain ◽  
Small Vessel ◽  
Retrospective Diagnosis ◽  
Vessel Disease ◽  
Brain Scan ◽  
Mr Brain ◽  
Retinal Drusen ◽  
Brain Examination

A 44-year-old Caucasian man presented with seizures and cognitive impairment. He had marked retinal drusen, and MR brain scan showed features of cerebral small vessel disease; he was diagnosed with a leukoencephalopathy of uncertain cause. He died at the age of 46 years and postmortem brain examination showed widespread small vessel changes described as a vasculopathy of unknown cause. Seven years postmortem, whole-genome sequencing identified a homozygous nonsense HTRA1 mutation (p.Arg302Ter), giving a retrospective diagnosis of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy.

Abstract 38: New Mutations Linked to Cerebral Autosomal Recessive Arteriopathy With Subcortical Infarcts and Leukoencephalopathy in Africa and North America

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Adeola Olowu ◽  
Spence Septien ◽  
Alka Khera ◽  
Worthy Warnack ◽  
Ty Shang
Keyword(s):  
Autosomal Recessive ◽  
Clinical Presentation ◽  
Small Vessel Disease ◽  
Hereditary Disease ◽  
Small Vessel ◽  
Heterozygous Mutation ◽  
Homozygous Mutation ◽  
Stroke Work ◽  
Vessel Disease ◽  
Asian Populations

Introduction: Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare hereditary disease. It is linked to mutations in the high-temperature requirement A serine peptidase 1 gene ( HTRA1 ). The clinical presentation is characterized by cerebral small vessel disease, alopecia, and spondylosis. CARASIL was initially thought to be a recessive disorder and exclusively exist in Asian populations. The paradigm of CARASIL has recently expanded. Genetically confirmed heterozygous mutations and manifestation in other ethnicities were reported. A few cases were reported in Hispanic and Caucasian populations but none in the African population. Here we report a new homozygous mutation in a Hispanic male and a known heterozygous mutation in an African female. Methods: Patients underwent routine ischemic stroke work up and risk factor management. MRI brain results were consistent with severe small vessel disease out of proportion to age. Genetic testing for vascular dementia for NOTCH-3 and HTRA1 in Patient 1 and only HTRA1 in Patient 2 were performed through Mayo Clinic. Results: Table 1. Patient Characteristics and HTRA1 Mutations Discussion: The prevalence of CARASIL is unknown but probably underestimated. Since the recognition of heterozygous HTRA1 mutation in CARASIL, more cases of heterozygous HTRA1 mutations have been reported. One of the clinical CARASIL triad -alopecia was not reported or observed in our cases. Cases of CARASIL without alopecia have been reported. Thus, CARASIL should still be suspected in the appropriate clinical presentation even if there is no classic triad or in non-Asian populations. Our cases expanded CARASIL mutations and affected populations. Being more aware of the broad clinical presentation of CARASIL can lead to early diagnosis.

Missense mutations in the human β fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1336-1341 ◽  
Author(s):  
Stefano Duga ◽  
Rosanna Asselta ◽  
Elena Santagostino ◽  
Sirous Zeinali ◽  
Tatjana Simonic ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Missense Mutations ◽  
Wild Type ◽  
Large Deletions ◽  
Congenital Afibrinogenemia ◽  
Low Levels ◽  
A Chain ◽  
Mutation Mechanisms

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen A-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bβ-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bβ fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion.

A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4700-4706 ◽  
Author(s):  
Ilia Voskoboinik ◽  
Marie-Claude Thia ◽  
Joseph A. Trapani
Keyword(s):  
Functional Analysis ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Autosomal Recessive ◽  
Missense Mutations ◽  
Wild Type ◽  
Lytic Function ◽  
Functional Defect ◽  
Target Cell Membrane ◽  
And Function ◽  
Prf1 Gene

Abstract Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms. (Blood. 2005;105:4700-4706)

scholarly journals SAMHD1Gene Mutations Are Associated with Cerebral Large-Artery Atherosclerosis

2015 ◽  
Vol 2015 ◽  
pp. 1-8
Author(s):  
Wei Li ◽  
Baozhong Xin ◽  
Junpeng Yan ◽  
Ying Wu ◽  
Bo Hu ◽  
...  
Keyword(s):  
Small Vessel Disease ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Splice Site Mutation ◽  
Large Artery ◽  
Missense Mutations ◽  
Site Mutation ◽  
Chinese Han ◽  
Functionally Impaired

Background. To investigate whether one or moreSAMHD1gene mutations are associated with cerebrovascular disease in the general population using a Chinese stroke cohort.Methods. Patients with a Chinese Han background (N=300) diagnosed with either cerebral large-artery atherosclerosis (LAA,n=100), cerebral small vessel disease (SVD,n=100), or other stroke-free neurological disorders (control,n=100) were recruited. Genomic DNA from the whole blood of each patient was isolated, and direct sequencing of theSAMHD1gene was performed. Both wild type and mutant SAMHD1 proteins identified from the patients were expressed inE. coliand purified; then their dNTPase activities and ability to form stable tetramers were analysedin vitro.Results. Three heterozygous mutations, including two missense mutations c.64C>T (P22S) and c.841G>A (p.E281K) and one splice site mutation c.696+2T>A, were identified in the LAA group with a prevalence of 3%. No mutations were found in the patients with SVD or the controls (p=0.05). The mutant SAMHD1 proteins were functionally impaired in terms of their catalytic activity as a dNTPase and ability to assemble stable tetramers.Conclusions. HeterozygousSAMHD1gene mutations might cause genetic predispositions that interact with other risk factors, resulting in increased vulnerability to stroke.

scholarly journals The first Greek case of heterozygous cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy: An atypical clinico-radiological presentation

2017 ◽  
Vol 30 (6) ◽  
pp. 583-585 ◽  
Author(s):  
Anastasia Bougea ◽  
George Velonakis ◽  
Nikolaos Spantideas ◽  
Evangelos Anagnostou ◽  
George Paraskevas ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Small Vessel Disease ◽  
Protease Gene ◽  
Unknown Aetiology ◽  
Dominant Inheritance ◽  
Vessel Disease ◽  
Recessive Form ◽  
Subsequent Loss ◽  
Biallelic Mutations ◽  
Serine Protease Gene

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) was previously considered a rare, early-onset recessive form of small-vessel disease (SVD) caused by biallelic mutations in the serine protease gene HTRA1 with subsequent loss of its activity. However, very recently, there is growing interest of research showing heterozygous HTRA1 mutations as causes of SVD with a dominant inheritance pattern. This first Greek heterozygous CARASIL case with unusual clinico-radiological presentation extends our very recent knowledge on how heterozygous CARASIL mutations may be associated with cerebral SVD. Our findings highlight heterozygous HTRA1 mutations as an important cause of familial SVD, and that screening of HTRA1 should be considered in all patients with a hereditary SVD of unknown aetiology.

ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria

Blood ◽  
2011 ◽  
Vol 118 (6) ◽  
pp. 1443-1451 ◽  
Author(s):  
Jordi To-Figueras ◽  
Sarah Ducamp ◽  
Jerome Clayton ◽  
Celia Badenas ◽  
Constance Delaby ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Gene Mutations ◽  
Wild Type ◽  
Aminolevulinate Synthase ◽  
Sequence Variations ◽  
Congenital Erythropoietic Porphyria ◽  
New Type ◽  
Terminal Amino ◽  
Exon 11 ◽  
Increased Activity

AbstractMutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.

scholarly journals International Evaluation of MIC Distributions and Epidemiological Cutoff Value (ECV) Definitions for Fusarium Species Identified by Molecular Methods for the CLSI Broth Microdilution Method

2015 ◽  
Vol 60 (2) ◽  
pp. 1079-1084 ◽  
Author(s):  
A. Espinel-Ingroff ◽  
A. L. Colombo ◽  
S. Cordoba ◽  
P. J. Dufresne ◽  
J. Fuller ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fusarium Species ◽  
Gene Mutations ◽  
The United States ◽  
Wild Type ◽  
Content Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Relationship ◽  
Dna Pcr

ABSTRACTThe CLSI epidemiological cutoff values (ECVs) of antifungal agents are available for variousCandidaspp.,Aspergillusspp., and the Mucorales. However, those categorical endpoints have not been established forFusariumspp., mostly due to the difficulties associated with collecting sufficient CLSI MICs for clinical isolates identified according to the currently recommended molecular DNA-PCR-based identification methodologies. CLSI MIC distributions were established for 53Fusarium dimerumspecies complex (SC), 10F. fujikuroi, 82F. proliferatum, 20F. incarnatum-F. equisetiSC, 226F. oxysporumSC, 608F. solaniSC, and 151F. verticillioidesisolates originating in 17 laboratories (in Argentina, Australia, Brazil, Canada, Europe, Mexico, and the United States). According to the CLSI guidelines for ECV setting, ECVs encompassing ≥97.5% of pooled statistically modeled MIC distributions were as follows: for amphotericin B, 4 μg/ml (F. verticillioides) and 8 μg/ml (F. oxysporumSC andF. solaniSC); for posaconazole, 2 μg/ml (F. verticillioides), 8 μg/ml (F. oxysporumSC), and 32 μg/ml (F. solaniSC); for voriconazole, 4 μg/ml (F. verticillioides), 16 μg/ml (F. oxysporumSC), and 32 μg/ml (F. solaniSC); and for itraconazole, 32 μg/ml (F. oxysporumSC andF. solaniSC). Insufficient data precluded ECV definition for the other species. Although these ECVs could aid in detecting non-wild-type isolates with reduced susceptibility to the agents evaluated, the relationship between molecular mechanisms of resistance (gene mutations) and MICs still needs to be investigated forFusariumspp.

Diverse Functional Implications of ADAMTS13 Gene Mutations in Patients with TTP and Congenital Deficiency.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 513-513 ◽  
Author(s):  
Roberta Donadelli ◽  
Federica Banterla ◽  
Cristina Capoferri ◽  
Miriam Galbusera ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Proteolytic Activity ◽  
Signal Sequence ◽  
Gene Mutations ◽  
Kinetic Studies ◽  
Missense Mutations ◽  
Wild Type ◽  
Small Vessels ◽  
Congenital Deficiency ◽  
Reported Data

Abstract Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels that is associated with deficiency of the VWF cleaving protease ADAMTS13, causing life-threatening disseminated microvascular thrombosis. Here we report four missense mutations (V88M, G1239V, R1219W, R1123C) in three patients with congenital ADAMTS13 deficiency. The three subjects carrying the mutations had less than 10% normal ADAMTS13 plasma antigen levels (measured by ELISA), which is consistent with previously reported data showing that most ADAMTS13 mutations in TTP patients result in impaired secretion of the protein. To evaluate whether the small amount of the mutant protease present in patients’ plasma had proteolytic activity, we cloned the ADAMTS13 cDNA in the pMT/Bip/His Drosophila expression vector and introduced the respective mutations by directed mutagenesis technique using wild-type cDNA as a template. These wild-type and mutant constructs were stably transfected into S2 Drosophila cells under the influence of the Drosophila BiP protein signal sequence, which allows the protein to be secreted into the medium and overcomes impaired secretion caused by the mutations. The induction of the histidine-tagged ADAMTS13 recombinants following the addition of copper was analyzed by Western blotting using an anti-hexahistidine monoclonal antibody. Equal amounts of recombinant ADAMTS13 proteins were used to evaluate proteolytic activity of recombinant proteins by cleavage of the rVWF A1-A2-A3 substrate. The proteolytic carboxyl terminal product of about 30 kDa was visualized by Western blot with a mouse monoclonal antibody directed against an epitope contained within the A3 domain of VWF. All the four missense mutations exhibited reduced activity: V88M:40%, G1239V: 66%, R1219W: 62% and R1123C: 64% of wild type activity . The results were also confirmed by collagen binding assay. For the the V88M mutation (located in the metalloprotease domain), decreased activity was confirmed by kinetic studies. In conclusion, mutant ADAMTS13 proteins found in patients with TTP, besides defective secretion, also have reduced protease activity. The mechanisms of the deficiency will be the matter of further studies.

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