Genotypic analysis of the female BPH/5 mouse, a model of superimposed preeclampsia

Animal models that recapitulate human diseases and disorders are widely used to investigate etiology, diagnosis, and treatment of those conditions in people. Disorders during pregnancy are particularly difficult to explore as interventions in pregnant women are not easily performed. Therefore, models that allow for pre-conception investigations are advantageous for elucidating the mechanisms involved in adverse pregnancy outcomes that are responsible for both maternal and fetal morbidity, such as preeclampsia. The Blood Pressure High (BPH)/5 mouse model has been used extensively to study the pathogenesis of preeclampsia. The female BPH/5 mouse is obese with increased adiposity and borderline hypertension, both of which are exacerbated with pregnancy making it a model of superimposed preeclampsia. Thus, the BPH/5 model shares traits with a large majority of women with pre-existing conditions that predisposes them to preeclampsia. We sought to explore the genome of the BPH/5 female mouse and determine the genetic underpinnings that may contribute to preeclampsia-associated phenotypes in this model. Using a whole genome sequencing approach, we are the first to characterize the genetic mutations in BPH/5 female mice that make it unique from the closely related BPH/2 model and the normotensive background strain, C57Bl/6. We found the BPH/5 female mouse to be uniquely different from BPH/2 and C57Bl/6 mice with a genetically complex landscape. The majority of non-synonymous consequences within the coding region of BPH/5 females were missense mutations found most abundant on chromosome X when comparing BPH/5 and BPH/2, and on chromosome 8 when comparing BPH/5 to C57Bl/6. Genetic mutations in BPH/5 females largely belong to immune system-related processes, with overlap between BPH/5 and BPH/2 models. Further studies examining each gene mutation during pregnancy are warranted to determine key contributors to the BPH/5 preeclamptic-like phenotype and to identify genetic similarities to women that develop preeclampsia.

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Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome

Blood ◽

10.1182/blood.v81.9.2339.2339 ◽

1993 ◽

Vol 81 (9) ◽

pp. 2339-2347 ◽

Cited By ~ 70

Author(s):

SD Wright ◽

K Michaelides ◽

DJ Johnson ◽

NC West ◽

EG Tuddenham

Keyword(s):

Normal Subjects ◽

Immunoblot Analysis ◽

Platelet Glycoprotein ◽

Bleeding Tendency ◽

Missense Mutations ◽

Coding Region ◽

Giant Platelets ◽

Double Heterozygosity ◽

Induced Aggregation ◽

Bernard Soulier Syndrome

Abstract Bernard-Soulier syndrome (BSS) giant platelets have defective and/or deficient glycoprotein (GP) Ib/IX complexes, causing absent ristocetin- induced aggregation, defective interaction with von Willebrand factor, morphologic abnormality, and a clinical bleeding tendency. Recently several mutations have been described in the platelet GPIb alpha gene in individuals exhibiting the BSS phenotype. We have studied a family with classical BSS, and have excluded lesions at the GPIb alpha locus by restriction fragment length polymorphism linkage analysis. Analysis of the genes for two other components of the platelet GPIb:IX complex, namely GPIb beta and GPIX, showed two different missense mutations in the coding region of the GPIX gene: an A-->G transition in codon 21 results in conversion of an aspartic acid to glycine and an A-->G change in codon 45 converts an asparagine residue to serine. Three affected individuals are doubly heterozygous for these mutations, which alter conserved residues in or flanking the GPIX leucine-rich glycoprotein motif. Both mutations create new recognition sites for the enzyme Fnu 4H1; therefore, this enzyme was used to screen 60 normal subjects (120 alleles). Neither mutation was detected in any subject other than direct relatives of the affected individuals. Although low levels of GPIb were demonstrable by both flow cytometry and immunoblot analysis in an affected individual's platelets, there was no evidence of GPIX immunoreactivity. We propose that expression of abnormal GPIX prevents stable assembly of the GPIb/IX complex, causing BSS in the doubly heterozygous individuals in this family.

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Identification of Bruton's tyrosine kinase (Btk) gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: a nationwide study of Btk deficiency in Japan

Blood ◽

10.1182/blood.v88.2.561.bloodjournal882561 ◽

1996 ◽

Vol 88 (2) ◽

pp. 561-573 ◽

Cited By ~ 80

Author(s):

S Hashimoto ◽

S Tsukada ◽

M Matsushita ◽

T Miyawaki ◽

Y Niida ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Kinase Activity ◽

Protein Analysis ◽

Kinase Assay ◽

Bruton’S Tyrosine Kinase ◽

Missense Mutations ◽

Coding Region ◽

Bruton's Tyrosine Kinase ◽

Nationwide Study

Deficiencies of Bruton's tyrosine kinase (Btk) have been implicated in the pathogenesis of human X-linked agammaglobulinemia (XLA). The distinctive phenotype observed in B-cell deficiency indicates the crucial role of Btk in B-cell development. This report describes a nationwide study of Btk deficiency in Japan, covering 51 XLA patients (35 independent families). Along with the identification of mutations, the resulting protein products were characterized by an in vitro kinase assay and a Western blot analysis. Thirty-one of the families were found to have mutations in the coding region of Btk. Although mutations were not found in the cDNA of 4 families, the Btk transcripts of these patients were greatly reduced. The identification of several novel missense mutations, in combination with the result of other studies, clarified the presence of two (missense) mutation hot spots, one in the SH1 and the other in the PH domain. The absence of kinase activity seen in 32 of the families underscored the importance of Btk protein analysis as a diagnostic indicator of XLA. The protein analysis also clarified the different effects of missense mutations on kinase activity and protein stability.

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Analysis of the gene coding for steroidogenic factor 1 (SF1, NR5A1) in a cohort of 50 Egyptian patients with 46,XY disorders of sex development

Acta Endocrinologica ◽

10.1530/eje-13-0965 ◽

2014 ◽

Vol 170 (5) ◽

pp. 759-767 ◽

Cited By ~ 13

Author(s):

Sally Tantawy ◽

Inas Mazen ◽

Hala Soliman ◽

Ghada Anwar ◽

Abeer Atef ◽

...

Keyword(s):

Adrenal Insufficiency ◽

Direct Sequencing ◽

Disorders Of Sex Development ◽

Missense Mutations ◽

Coding Region ◽

Steroidogenic Factor 1 ◽

Sex Development ◽

Gene Coding ◽

Future Fertility

ObjectiveSteroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamic–pituitary–gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD.DesignClinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum.MethodsMolecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected.ResultsThree novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Müllerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism.ConclusionWe identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5–15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

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MUNC13–4 Mutations in Patients with Hemophagocytic Lymphohistiocytosis Are Scattered over the Functional Domains of the Protein.

Blood ◽

10.1182/blood.v108.11.1249.1249 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1249-1249

Author(s):

Alessandra Santoro ◽

Sonia Cannella ◽

Giovanna Bossi ◽

Federico Gallo ◽

Antonino Trizzino ◽

...

Keyword(s):

Hemophagocytic Lymphohistiocytosis ◽

Stop Codon ◽

Functional Domains ◽

Functional Domain ◽

Missense Mutations ◽

Coding Region ◽

Cycle Sequencing ◽

Familial Form ◽

Protein Functional Domains

Abstract Mutations of PRF1 and MUNC13–4 genes, involved with cellular cytotoxicity, are associated with the familial form of Hemophagocytic Lymphohistiocytosis (HLH) type 2 (FHL2) and 3 (FHL3). In all patients with HLH, in which PRF1 mutations had been excluded, we screened MUNC13–4 mutations. The 32 exons and their proximal flanking regions of MUNC13–4 gene were sequenced by “cycle sequencing” approach (BigDye Terminator, Applied Biosystems). Of 60 patients, 21 had MUNC13–4 mutations. 5 were reported mutations: 753+1G>T, 817C>T (R273X); 1389+1G>A; 1822del 12 bp (del V608-A611); 2346–9delGGAG (R782FsX12). Of 16 novel mutations 2 (2212C>T and 2650C>T) introduce a stop codon (at Q738 and Q884); 4 cause frameshift: 441delA (P147fsX14), 532delC (Q178fsX70), 3082delC (L1028fsX), and 3226insG (H1076fsX51). Of 8 missense mutations [175G>A (A59T), 419T>C (I140T), 610A>G (M204B), 1241G>T (R414L), 1847A>G (E616G), 2039G>C (R680P), 2570T>G (F857C), 2782C>T (R928C)], 6 fall within the protein functional domains; each of the 2 falling outside was associated with 2 additional pathogenic mutations. The remaining novel 1992+5 G>A and 2448insC −12 fall outside the coding region but are close enough to induce alternative splicing. We identified polymorphisms 279C>T and 3198A>G. The 2599A>G (K867E) transition was found in several unrelated families, including one homozygous parent. To assess its frequency we studied 50 consecutive newborns, of which 64% were heterozygous. To understand the effects of the mutations we analysed Munc13–4 protein expression from CTL and/or NK cells by western blot using an antibody raised against amino acids 1–262. Trace amounts of protein could be detected only in 5 patients. Analysis of granule polarisation in 2 patients with trace amounts of Munc13–4 protein showed many more docked granules visible than in controls, consistent with a block in granule secretion in these patients. Median age at diagnosis of FHL3 was 6.5 months, but 8/21 (38%) patients were diagnosed when older than 5 years, with one young adult of 18 years. CNS disease was present in 10 patients; NK was markedly reduced or absent in all patients tested. MUNC13–4 mutations were found in 35% of our HLH patients non type-2. Mutations were almost entirely different from those reported so far and scattered over different exons, but in far most cases they fall within the protein functional domain. Since these patients may develop the disease during adolescence or even later on, not only pediatric but also as adults hematologists should include FHL-2 and 3 in the differential diagnosis of children and young adults with fever, cytopenia, splenomegaly and hypercytokinemia.

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Double Genetic Mutations in PML-Rara Fusion Gene Confirmed in a Patient Showing Resistance to All-Trans Retinoic Acid and Arsenic-Trioxide Therapy.

Blood ◽

10.1182/blood.v114.22.1743.1743 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1743-1743 ◽

Cited By ~ 1

Author(s):

Emi Goto ◽

Akihiro Tomita ◽

Akihide Atsumi ◽

Hitoshi Kiyoi ◽

Tomoki Naoe

Keyword(s):

Drug Resistance ◽

Retinoic Acid ◽

Disease Progression ◽

Research Funding ◽

Fusion Gene ◽

Leukemia Cells ◽

Genetic Mutations ◽

Missense Mutations ◽

All Trans Retinoic Acid ◽

The Relationship

Abstract Abstract 1743 Poster Board I-769 Background Molecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO. Aims Analyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy. Results Eight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression. Conclusions Double genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy. Disclosures Kiyoi: Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

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New mutations in SERPING1 gene of Brazilian patients with hereditary angioedema

Biological Chemistry ◽

10.1515/hsz-2015-0222 ◽

2016 ◽

Vol 397 (4) ◽

pp. 337-344 ◽

Cited By ~ 9

Author(s):

Nathália Cagini ◽

C.L. Veronez ◽

R.N. Constantino-Silva ◽

Márcia Buzolin ◽

Renan Paulo Martin ◽

...

Keyword(s):

Functional Analysis ◽

Genetic Analysis ◽

Hereditary Angioedema ◽

Autosomal Dominant ◽

Missense Mutations ◽

Inherited Disease ◽

Coding Region ◽

Serping1 Gene ◽

Brazilian Cohort ◽

New Mutations

Abstract Hereditary Angioedema is an autosomal dominant inherited disease leading to oedema attacks with variable severity and localization predominantly caused by C1-INH deficit. More than 400 mutations have been already identified, however no genetic analysis of a Brazilian cohort of HAE patients with C1-INH deficiency has been published. Our aim was to perform genetic analysis of C1-INH gene (SERPING1) in Brazilian HAE patients. We screened the whole SERPING1 coding region from 30 subjects out of 16 unrelated families with confirmed diagnosis of HAE due to C1-INH deficiency. Clinical diagnosis was based on symptoms and quantitative and/or functional analysis of C1-INH. We identified fifteen different mutations among which eight were not previously described according to databases. We found five small deletions (c.97_115del19; c.553delG; c.776_782del7; c.1075_1089del15 and c.1353_1354delGA), producing frameshifts leading to premature stop codons; seven missense mutations (c.498C>A; c.550G>C; c.752T>C; c.889G>A; c.1376C>A; c.1396C>T; c.1431C>A); one nonsense mutation (c.1480C>T), and two intronic alterations (c.51+1G>T; c.51+2T>C). Despite the small number of participants in this study, our results show mutations not previously identified in SERPING1 gene. This study represents the first Brazilian HAE cohort evaluated for mutations and it introduces the possibility to perform genetic analysis in case of need for differential diagnosis.

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An unusual adenine phosphoribosyltransferase pseudogene is syntenic with its functional gene and is flanked by highly polymorphic DNAs.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4161 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4161-4167 ◽

Cited By ~ 11

Author(s):

M K Dush ◽

J A Tischfield ◽

S A Khan ◽

E Feliciano ◽

J M Sikela ◽

...

Keyword(s):

Dna Sequences ◽

Ecori Fragment ◽

Chromosome 8 ◽

Functional Gene ◽

Direct Repeats ◽

Coding Region ◽

Adenine Phosphoribosyltransferase ◽

Protein Coding ◽

Dna Library ◽

Processed Pseudogenes

A mouse adenine phosphoribosyltransferase (aprt) pseudogene that had previously been recovered from a BALB/c sperm DNA library possessed several unusual features. Its nucleotide sequence, like that of other processed pseudogenes, was colinear with its corresponding mRNA, but it was truncated at its 3' end and lacked a poly(A) tail. The pseudogene was 82% homologous with corresponding regions of the functional gene and had incurred mutations that included transitions, transversions, deletions, and a point insertion. Even though the pseudogene was truncated within the protein-coding region of the corresponding functional gene, it was flanked at both ends by 13-base-pair direct repeats. Curiously, the direct repeats exhibited homology to APRT mRNA at the site of pseudogene divergence. The pseudogene appeared to be common to BALB/c and A/J mice, but it was contained on a 3-kilobase EcoRI fragment in the former strain and a 4.5-kilobase EcoRI fragment in the latter. The BALB/c and apparently the A/J pseudogene both mapped to chromosome 8, which also contains the functional aprt gene. The DNA sequences immediately surrounding the pseudogene in the two strains appeared to be similar, suggesting that the BALB/c and A/J pseudogenes are allelic. However, DNA sequences more distal to the pseudogene in the two strains appeared to vary. Thus, the EcoRI polymorphism was not due to simple loss of an EcoRI site, but was more complex. The pattern of flanking restriction sites was different for each of several enzymes, consistent with extensive DNA rearrangement. Double digests of BALB/c and A/J genomic DNAs revealed complex polymorphisms on both sides of the pseudogene. The results were consistent with insertion, deletion, or other rearrangement of DNA sequences that flank the pseudogene and suggest that this region of mouse chromosome 8 may be a region active for mutation or recombination.

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Utility of Next Generation Sequencing in Prognostication and Therapeutic Decision Making in Cytogenetically Normal AML with DNMT3A Mutations

Blood ◽

10.1182/blood.v128.22.2886.2886 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2886-2886

Author(s):

Arjun Datt Law ◽

Mahadeo A. Sukhai ◽

Mariam Thomas ◽

Andrea Arruda ◽

Narmin Ibrahimova ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Clinical Characteristics ◽

Normal Karyotype ◽

Gene Interactions ◽

Missense Mutations ◽

Newly Diagnosed ◽

Coding Region ◽

Advisory Committees ◽

Cytogenetically Normal Aml

Abstract Background: The prognostication of cytogenetically normal AML (CN-AML) continues to evolve with the use of NGS-based risk stratification. Emerging data indicate that the presence of additional mutations in good- and intermediate-risk patients (as defined by conventional cytogenetic and molecular analyses) changes the behavior of their disease, suggesting that more personalized treatment approaches are needed. Methods: We analyzed the mutational profile of newly diagnosed AML patients with DNMT3A mutations (N = 48) seen at our center and described their clinical characteristics and associated mutations. These patients were identified as part of the Advanced Genomics in Leukemia (AGILE) clinical project currently underway at the Princess Margaret Cancer Centre, Toronto. NGS molecular profiling was performed using the TruSight Myeloid Sequencing Panel (TMSP; Illumina) on the MiSeq benchtop genome sequencer (Illumina). This process permitted profiling of 54 genes (39 in the hotspot region; 15 complete coding region coverage) using amplicon-based library preparation and sequencing by synthesis. 160 patients with newly diagnosed AML were evaluated for this project from February 2015 to February 2016. All were analyzed in parallel by the standard cytogenetic and molecular (NPM1, FLT3-ITD/TKD) diagnostic algorithm. Results: 48 unique patients were identified bearing mutated DNMT3A. Of these, 31 patients had a normal karyotype. Their clinical characteristics are depicted in Table 1. The R882X DNMT3A variant was detected in 12 patients. A total of 100 additional mutations were identified on sequencing (Figure1). The most common associated mutations were in NPM1 (38.7%) followed by IDH1 (29%), RUNX1 (25.8%) and TET2 (22.6%). PTPN11 mutations were identified in 6 patients, 75% of which also had mutated NPM1. Two patients were found to have biallelic CEBPA mutations. Potential gene-gene interactions were also examined and led to the identification of subgroups such as NPM1-FLT3-DNMT3A mutated (n=4), DNMT3A-IDH2R140 (n=3) and DNMT3A-IDH2R172 (n=3) based on recent data by Papaemmanuil, et al (New England Journal of Medicine, 2016) defining these subgroups as being prognostically relevant.( Patients >60 years of age had more frequent mutations in NRAS, BCOR, BCORL1 and TET2. NRAS and BCOR mutations were mutually exclusive with NPM1. RUNX1, IDH2, SRSF2 and U2AF1 mutations were also seen exclusively in the NPM1 negative group. Eligible patients (n=25) received induction therapy with daunorubicin and cytarabine leading to CR1 in 18 patients (72%). Primary induction failure occurred in 6 cases (24%). All 6 cases were NPM1 negative and had missense mutations in DNMT3A including 3 R882X variants. (Figure 2) Additional mutations were identified in IDH1, RUNX1 and/or TET2 in all 6 patients. During the median follow up of 8 months (range 1 - 15 months), 4 patients relapsed, 2 of which had mutated NPM1 with wild type FLT3-ITD. Sixteen patients are alive at this point and 12 are in CR, six having received allogeneic stem cell transplants. One patient with relapsed disease entered a clinical trial of an IDH1 inhibitor Conclusion: Genomic analysis is increasingly recognized as a vital adjunct to conventional diagnostic and prognostic approaches. With ongoing advancements in technology leading to increasing cost effectiveness and decreased turnaround times, the use of NGS is likely to become an up-front investigation resulting in a more personalized approach to therapy. Also, unique patient subgroups defined by gene-gene interactions can be identified to further predict clinical behavior and potentially identify druggable targets for therapy. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding. Schimmer:Novartis: Honoraria. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kamel-Reid:BMS: Research Funding. Schuh:Amgen: Membership on an entity's Board of Directors or advisory committees.

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Two Novel Disease-Causing Mutations in the LDLR of Familial Hypercholesterolemia

Frontiers in Genetics ◽

10.3389/fgene.2021.762587 ◽

2021 ◽

Vol 12 ◽

Author(s):

Haochang Hu ◽

Tian Shu ◽

Jun Ma ◽

Ruoyu Chen ◽

Jian Wang ◽

...

Keyword(s):

Familial Hypercholesterolemia ◽

High Throughput Sequencing ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Bioinformatic Analysis ◽

Genetic Mutations ◽

Missense Mutations ◽

Autosomal Dominant Disorder ◽

Lipid Clinic

As an autosomal dominant disorder, familial hypercholesterolemia (FH) is mainly caused by pathogenic mutations in lipid metabolism-related genes. The aim of this study is to investigate the genetic mutations in FH patients and verify their pathogenicity. First of all, a pedigree investigation was conducted in one family diagnosed with FH using the Dutch Lipid Clinic Network criteria. The high-throughput sequencing was performed on three family members to explore genetic mutations. The effects of low-density lipoprotein receptor (LDLR) variants on their expression levels and activity were further validated by silico analysis and functional studies. The results revealed that LDLC levels of the proband and his daughter were abnormally elevated. The whole-exome sequencing and Sanger sequencing were used to confirm that there were two LDLR missense mutations (LDLR c.226 G > C, c.1003 G > T) in this family. Bioinformatic analysis (Mutationtaster) indicated that these two mutations might be disease-causing variants. In vitro experiments suggested that LDLR c.226 G > C and c.1003 G > T could attenuate the uptake of Dil-LDL by LDLR. In conclusion, the LDLR c.226 G > C and c.1003 G > T variants might be pathogenic for FH by causing uptake dysfunction of the LDLR.

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Six Different Point Mutations in Seven Danish Families with Symptomatic Protein C Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653749 ◽

1995 ◽

Vol 73 (02) ◽

pp. 186-193 ◽

Cited By ~ 5

Author(s):

Bent Lind ◽

Marianne Schwartz ◽

Sixtus Thorsen

Keyword(s):

Splice Site ◽

Protein C ◽

Family Members ◽

Point Mutations ◽

Patient Specific ◽

Missense Mutations ◽

Coding Region ◽

Protein C Deficiency ◽

Epidermal Growth Factor Domain ◽

Increased Risk

SummarySix different point mutations of the protein C gene are described in seven Danish families with protein C deficiency associated with an increased risk of venous thromboembolism. All affected family members are heterozygotes for the mutated protein C genotype. One mutation is a G2992→A transition at position +5 in the 5’ splice site of intron D. The other five mutations affect the protein coding region. One is a Cl432→T transition in exon III converting the highly conserved Arg15 to Trp in the Gla-domain. Another mutation is a G3157→C transversion in exon V converting the non-conserved Gly72 to Arg in the epidermal growth factor domain. The remaining three mutations are located in non-conserved amino acid positions in exon IX and affect the serine proteinase domain. The first is a G8559→C transversion converting Gly282 to Arg. The second is a C8571→T transition (present in two families) converting Arg286 to Cys. The third is a C8695→T transition converting Pro327 to Leu. In each family the protein C deficiency cosegregates or probably cosegregates (one family, G8559→C) with the mutation. All affected family members exhibit a reduction of both the antigen and the functional plasma concentration of protein C to approximately 50% of normal indicating that the mutated protein C is not present (type 1 deficiency) or only present in low amounts in plasma. Agarose gel electrophoresis followed by Western blotting shows that the Arg15→Trp substitution is associated with a normal as well as an abnormal migrating plasma protein C band. This provides positive evidence for that both the normal and mutated alleles are expressed (type 2 deficiency). The five other mutations are associated with only one band with the mobility of normal protein C. In one family a novel G1390→A transition converting the normal Ala1 to Thr was demonstrated. This mutation is not linked to the patient specific G8559→C transversion. In conclusion one splice site mutation and five different missense mutations of the protein C gene are described.

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