ABSTRACT
At least 14 causative genes have been identified for both syndromic and nonsyndromic forms of thoracic aortic aneurysm/dissection (TAA), an important cause of death in the industrialized ...world. Molecular confirmation of the diagnosis is increasingly important for gene‐tailored patient management but consecutive, conventional molecular TAA gene screening is expensive and labor‐intensive. To circumvent these problems, we developed a TAA gene panel for next‐generation sequencing of 14 TAA genes. After validation, we applied the assay to 100 Marfan patients. We identified 90 FBN1 mutations, 44 of which were novel. In addition, Multiplex ligation‐dependent probe amplification identified large deletions in six of the remaining samples, whereas false‐negative results were excluded by Sanger sequencing of FBN1, TGFBR1, and TGFBR2 in the last four samples. Subsequently, we screened 55 syndromic and nonsyndromic TAA patients. We identified causal mutations in 15 patients (27%), one in each of the six following genes: ACTA2, COL3A1, TGFBR1, MYLK, SMAD3, SLC2A10 (homozygous), two in NOTCH1, and seven in FBN1. We conclude that our approach for TAA genetic testing overcomes the intrinsic hurdles of consecutive Sanger sequencing of all candidate genes and provides a powerful tool for the elaboration of clinical phenotypes assigned to different genes.
Thoracic aortic aneurysm (TAA) is an important cause of death in the industrialized world. Both syndromic and non‐syndromic forms of TAA exists and to date 14 causative genes have been identified. To shorten the turnaround time, increase mutation‐uptake and reduce the overall cost of molecular testing, we developed a next generation sequencing panel of 14 TAA genes (ACTA2, COL3A1, EFEMP2, FBN1, FLNA, MYH11, MYLK, NOTCH1, SKI, SLC2A10, SMAD3, TGFB2, TGFBR1 and TGFBR2) that can be tested simultaneously.
A large family is described with gray platelet syndrome due to an autosomal dominant inheritance pattern related to a dominant-negative mutation in
GFI1B
. The mutation leads to a loss in gene ...repression during megakaryocyte development.
Platelets are formed through fragmentation of megakaryocytes that reside in the bone marrow.
1
,
2
Platelet alpha granules, which are by far the most abundant platelet organelles, store proteins that stimulate platelet adhesiveness, hemostasis, and wound healing.
3
,
4
The gray platelet syndrome is an inherited bleeding disorder characterized by defective production of alpha granules.
5
,
6
Patients with this syndrome have reduced numbers of larger-than-normal platelets, and on light microscopy these platelets have a typical gray appearance caused by the lack of alpha granules. For a final diagnosis, the lack of alpha granules must be confirmed by means of electron microscopy.
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Clinically, . . .
Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related ...TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as
, and
. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g.,
) or in syndromic (e.g.,
) or non-syndromic (e.g.,
) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (
= 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (
= 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was
(
= 0.002), with 2.5% (
= 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of
mutations to the etiology of the BAV/TAA phenotype.
Bicuspid aortic valve (BAV) is the most common congenital heart defect (CHD), affecting 1-2% of the population. BAV is associated with thoracic aortic aneurysms (TAAs). Deleterious copy number ...variations (CNVs) were found previously in up to 10% of CHD cases. This study aimed at unravelling the contribution of deleterious deletions or duplications in 95 unrelated BAV/TAA patients. Seven unique or rare CNVs were validated, harbouring protein-coding genes with a role in the cardiovascular system. Based on the presence of overlapping CNVs in patients with cardiovascular phenotypes in the DECIPHER database, the identification of similar CNVs in whole-exome sequencing data of 67 BAV/TAA patients and suggested topological domain involvement from Hi-C data, supportive evidence was obtained for two genes (DGCR6 and TBX20) of the seven initially validated CNVs. A rare variant burden analysis using next-generation sequencing data from 637 BAV/TAA patients was performed for these two candidate genes. This revealed a suggestive genetic role for TBX20 in BAV/TAA aetiology, further reinforced by segregation of a rare TBX20 variant with the phenotype within a BAV/TAA family. To conclude, our results do not confirm a significant contribution for deleterious CNVs in BAV/TAA as only one potentially pathogenic CNV (1.05%) was identified. We cannot exclude the possibility that BAV/TAA is occasionally attributed to causal CNVs though, or that certain CNVs act as genetic risk factors by creating a sensitised background for BAV/TAA. Finally, accumulative evidence for TBX20 involvement in BAV/TAA aetiology underlines the importance of this transcription factor in cardiovascular disease.
Individuals with multiple relatives with colorectal cancer (CRC) and/or a relative with early-onset CRC have an increased risk of developing CRC. They are eligible for preventive measures, such as ...surveillance by regular colonoscopy and/or genetic counselling. Currently, most at-risk individuals do not follow the indicated follow-up policy. In a new guideline on familial and hereditary CRC, clinicians have new tasks in calculating, interpreting, and communicating familial CRC risk. This will lead to better recognition of individuals at an increased familial CRC risk, enabling them to take effective preventive measures. This trial compares two implementation strategies (a common versus an intensive implementation strategy), focussing on clinicians' risk calculation, interpretation, and communication, as well as patients' uptake of the indicated follow-up policy.
A clustered randomized controlled trial including an effect, process, and cost evaluation will be conducted in eighteen hospitals. Nine hospitals in the control group will receive the common implementation strategy (i.e., dissemination of the guideline). In the intervention group, an intensive implementation strategy will be introduced. Clinicians will receive education and tools for risk calculation, interpretation, and communication. Patients will also receive these tools, in addition to patient decision aids. The effect evaluation includes assessment of the number of patients for whom risk calculation, interpretation, and communication is performed correctly, and the number of patients following the indicated follow-up policy. The actual exposure to the implementation strategies and users' experiences will be assessed in the process evaluation. In a cost evaluation, the costs of the implementation strategies will be determined.
The results of this study will help determine the most effective method as well as the costs of improving the recognition of individuals at an increased familial CRC risk. It will provide insight into the experiences of both patients and clinicians with these strategies.The knowledge gathered in this study can be used to improve the recognition of familial and hereditary CRC at both the national and international level, and will serve as an example to improve care for patients and their relatives worldwide. Our results may also be useful in improving healthcare in other diseases.
ClinicalTrials.gov NCT00929097.
ABSTRACT
Marfan syndrome (MFS) is caused by mutations in the FBN1 (fibrillin‐1) gene, but approximately 10% of MFS cases remain genetically unsolved. Here, we report a new FBN1 mutation in an MFS ...family that had remained negative after extensive molecular genomic DNA FBN1 testing, including denaturing high‐performance liquid chromatography, Sanger sequencing, and multiplex ligation‐dependent probe amplification. Linkage analysis in the family and cDNA sequencing of the proband revealed a deep intronic point mutation in intron 56 generating a new splice donor site. This mutation results in the integration of a 90‐bp pseudo‐exon between exons 56 and 57 containing a stop codon, causing nonsense‐mediated mRNA decay. Although more than 90% of FBN1 mutations can be identified with regular molecular testing at the genomic level, deep intronic mutations will be missed and require cDNA sequencing or whole‐genome sequencing.
Marfan syndrome is caused by mutations in the fibrillin‐1 (FBN1) gene. A large family presenting with MFS features could not be explained after repetitive clinical diagnostic testing. After FBN1 cDNA sequencing, the aberrant inclusion of a pseudo‐exon between exon 56 and 57 was revealed. Deep intronic mutations are missed using current diagnostic tools. Their detection requires cDNA sequencing or whole genome sequencing.
A reduced heparan sulphate (HS) expression in the glomerular basement membrane of patients with overt diabetic nephropathy is associated with an increased glomerular heparanase expression. We ...investigated the possible association of urinary heparanase activity with the development of proteinuria in patients with Type 1 diabetes (T1D), Type 2 diabetes (T2D), or membranous glomerulopathy (MGP) as non-diabetic disease controls.
Heparanase activity, albumin, HS and creatinine were measured in the urine of patients with T1D (n=58) or T2D (n=31), in patients with MGP (n=52) and in healthy controls (n=10). Heparanase messenger RNA (mRNA) expression in leukocytes was determined in a subgroup of patients with T1D (n=19).
Urinary heparanase activity was increased in patients with T1D and T2D, which was more prominent in patients with macroalbuminuria, whereas no activity could be detected in healthy controls. Albuminuria levels were associated with increased urinary heparanase activity in diabetic patients (r=0.20; P<0.05) but not in patients with MGP (r=0.11; P=0.43). A lower urinary heparanase activity was observed in diabetic patients treated with inhibitors of the renin-angiotensin-aldosterone system (RAAS), when compared to diabetic patients treated with other anti-hypertensives. Additionally, urinary heparanase activity was associated with age in T1D and MGP. In MGP, heparanase activity and β2-microglobulin excretion correlated. In patients with T1D, no differences in heparanase mRNA expression in leukocytes could be observed.
Urinary heparanase activity is increased in diabetic patients with proteinuria. However, whether increased heparanase activity is a cause or consequence of proteinuria requires additional research.
Gray platelet syndrome (GPS) is a hereditary, usually autosomal recessive bleeding disorder caused by defective production of α-granules in platelets. GPS patients show reduced numbers of platelets ...that are larger and have a typical gray appearance under light microscopy, caused by the lack of α-granules. We describe a large family with an autosomal dominant type of GPS characterized by mild to severe bleeding complications. In addition to large gray platelets, other GPS-associated phenomena like myelofibrosis, thrombocytopenia, and low platelet factor 4 expression were observed in affected individuals. Histopathological examination of a BM biopsy from a patient showed a cellular marrow with increased numbers of megakaryocytes that were pleomorphic in size and shape. Megakaryocytes clustered along BM sinuses and showed dysmorphic stretched features.
To determine the disease causing mutation we performed linkage analysis and identified a candidate locus on chromosome 9q34 with a LOD score of 3.9. We considered GFI1B (Growth Factor Independence 1B), located within this region, an excellent candidate gene because of its function as a transcriptional repressor in megakaryocyte development. Sequence analysis identified a nonsense mutation in GFI1B exon 6 (c.859C>T, p.Gln287*) that completely co-segregated with the GPS disease in this family. The mutated transcript predicts a 44 amino acid C-terminally truncated protein, GFI1BTr. The truncation is located within zinc finger 5 of GFI1B, deleting all of its four amino acids that directly interact with DNA. Luciferase gene reporter assays showed that GFI1BTr was unable to repress gene expression. Importantly, GFI1BTr inhibited gene repression mediated by wild type GFI1B, indicating that the mutant interferes with wild type GFI1B in a dominant-negative manner. To validate that GFI1BTr adversely affects normal GFI1B, we expressed the mutant in mouse bone marrow cells followed by induction of megakaryocytic differentiation. Compared to control cells, GFI1BTr-positive megakaryocytes showed dysplastic features including hypolobulation of the nuclei, irregular contours and multiple separate nuclei, that were very similar to those observed in patient cells. This indicates that GFI1BTr causes megakaryocytic abnormalities and that it functions in a dominant-negative manner.
GFI1B silencing inhibits the development of human megakaryocyte colonies in vitro. We observed that megakaryocyte colony forming cells were significantly more frequent in patient bone marrow compared to controls. In addition, patient-derived megakaryocyte colonies were significantly larger compared to controls. Immunophenotypic analyses of peripheral blood showed no differences in myeloid and erythroid lineages and the platelet markers GP3B, ITGA2B and ITGB3 among affected an non-affected individuals. However, within the ITGA2B/CD41-positive platelet population, 5 of 6 affected members showed a marked decrease in the platelet surface membrane glycoprotein 1b-α (GP1BA/CD42b), compared to unaffected members. In addition, a strong expression of CD34, which is usually confined to immature hematopoietic progenitors, was detected on platelets from all studied affected individuals. Immunostaining of a BM biopsy from a patient showed the presence of ITGB3/CD61 positive megakaryocytes that intensely expressed CD34. Electron microscopy analysis showed megakaryocytes with few, small, irregularly shaped and centrally located α-granules characterized by an extensive peripheral cytoplasm with irregular proplatelets, largely devoid of cell organelles. To test whether these abnormalities were cell intrinsic, we stimulated CD34+ cells from two patients to differentiate along the megakaryocytic lineage in vitro. Megakaryocytic cells showed dysplastic features reminiscent of those observed in the bone marrow aspirates. In addition, increased CD34 and decreased GP1BA/CD42b expression were observed on megakaryocytes, indicating that GFI1BTr-induced abnormalities are intrinsic to the cell. In summary, we have identified GFI1B as a causative gene in autosomal dominant GPS. GFI1BTr acts in a dominant-negative manner over wild type GFI1B and affects the development of megakaryocytes and platelets, demonstrating a pivotal role of GFI1B in governing normal megakaryopoiesis and platelet production.
No relevant conflicts of interest to declare.
During cancer genetic counseling, different items which counselors consider important are discussed. However, relatively little empirical evidence exists regarding the needs and preferences of ...counselees. In this study needs and preferences were assessed from counselees with a personal and/or family history of colorectal cancer (CRC), who were referred for genetic counseling regarding CRC. They received a slightly modified version of the QUOTE-GENE
ca
questionnaire prior to their first visit to the Hereditary Cancer Clinic. Response rate was 60 % (48/80 participants). Counselees rated the importance of 45 items assessing their needs and preferences regarding the content and process of genetic counseling. Participants rated the items regarding discussion of information about their familial CRC risk (100 %) and preventive options (98 %) as important or very important. Fewer participants rated items concerning general information on genetics as important. Sensitive communication during counseling was considered very important by a large percentage of counselees. Generally, no major differences were seen between participants in relation to individual characteristics. Our data suggest that focusing on familial CRC risk and surveillance options, in combination with sensitive communication may lead to better satisfaction with genetic counseling.
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