Molecular phenotyping and functional assessment of smooth muscle-like cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.

Circulating biomarkers of cardiovascular disease are related to aneurysm volume in abdominal aortic aneurysm.

BACKGROUND: Surveillance programs in abdominal aortic aneurysms (AAA) are mainly based on imaging and leave room for improvement to timely identify patients at risk for AAA growth. Many biomarkers are dysregulated in patients with AAA, which fuels interest in biomarkers as indicators of disease progression. We examined associations of 92 cardiovascular disease (CVD)-related circulating biomarkers with AAA and sac volume. METHODS: In a cross-sectional analysis, we separately investigated (1) 110 watchful waiting (WW) patients (undergoing periodic surveillance imaging without planned intervention) and (2) 203 patients after endovascular aneurysm repair (EVAR). The Cardiovascular Panel III (Olink Proteomics AB, Sweden) was used to measure 92 CVD-related circulating biomarkers. We used cluster analyses to investigate protein-based subphenotypes, and linear regression to examine associations of biomarkers with AAA and sac volume on CT scans. RESULTS: Cluster analyses revealed two biomarker-based subgroups in both WW and EVAR patients, with higher levels of 76 and 74 proteins, respectively, in one subgroup versus the other. In WW patients, uPA showed a borderline significant association with AAA volume. Adjusting for clinical characteristics, there was a difference of -0.092 (-0.148, -0.036) loge mL in AAA volume per SD uPA. In EVAR patients, after multivariable adjustment, four biomarkers remained significantly associated with sac volume. The mean effects on sac volume per SD difference were: LDLR: -0.128 (-0.212, -0.044), TFPI: 0.139 (0.049, 0.229), TIMP4: 0.110 (0.023, 0.197), IGFBP-2: 0.103 (0.012, 0.194). CONCLUSION: LDLR, TFPI, TIMP4, and IGFBP-2 were independently associated with sac volume after EVAR. Subgroups of patients with high levels of the majority of CVD-related biomarkers emphasize the intertwined relationship between AAA and CVD.ClinicalTrials.gov Identifier: NCT03703947.

State-of-the art review: Noncompaction cardiomyopathy in pediatric patients.

Noncompaction cardiomyopathy (NCCM) is a disease characterized by hypertrabeculation, commonly hypothesized due to an arrest in compaction during fetal development. In 2006, NCCM was classified as a distinct form of cardiomyopathy (CMP) by the American Heart Association. NCCM in childhood is more frequently familial than when diagnosed in adulthood and is associated with other congenital heart diseases (CHDs), other genetic CMPs, and neuromuscular diseases (NMDs). It is yet a rare cardiac diseased with an estimated incidence of 0.12 per 100.000 in children up to 10 years of age. Diagnosing NCCM can be challenging due to non-uniform diagnostic criteria, unawareness, presumed other CMPs, and presence of CHD. Therefore, the incidence of NCCM in children might be an underestimation. Nonetheless, NCCM is the third most common cardiomyopathy in childhood and is associated with heart failure, arrhythmias, and/or thromboembolic events. This state-of-the-art review provides an overview on pediatric NCCM. In addition, we discuss the natural history, epidemiology, genetics, clinical presentation, outcome, and therapeutic options of NCCM in pediatric patients, including fetuses, neonates, infants, and children. Furthermore, we provide a simple classification of different forms of the disease. Finally, the differences between the pediatric population and the adult population are described.

A Large-Scale Full GBA1 Gene Screening in Parkinson's Disease in the Netherlands.

BACKGROUND: The most common genetic risk factor for Parkinson's disease known is a damaging variant in the GBA1 gene. The entire GBA1 gene has rarely been studied in a large cohort from a single population. The objective of this study was to assess the entire GBA1 gene in Parkinson's disease from a single large population. METHODS: The GBA1 gene was assessed in 3402 Dutch Parkinson's disease patients using next-generation sequencing. Frequencies were compared with Dutch controls (n = 655). Family history of Parkinson's disease was compared in carriers and noncarriers. RESULTS: Fifteen percent of patients had a GBA1 nonsynonymous variant (including missense, frameshift, and recombinant alleles), compared with 6.4% of controls (OR, 2.6; P < 0.001). Eighteen novel variants were detected. Variants previously associated with Gaucher's disease were identified in 5.0% of patients compared with 1.5% of controls (OR, 3.4; P < 0.001). The rarely reported complex allele p.D140H + p.E326K appears to likely be a Dutch founder variant, found in 2.4% of patients and 0.9% of controls (OR, 2.7; P = 0.012). The number of first-degree relatives (excluding children) with Parkinson's disease was higher in p.D140H + p.E326K carriers (5.6%, 21 of 376) compared with p.E326K carriers (2.9%, 29 of 1014); OR, 2.0; P = 0.022, suggestive of a dose effect for different GBA1 variants. CONCLUSIONS: Dutch Parkinson's disease patients display one of the largest frequencies of GBA1 variants reported so far, consisting in large part of the mild p.E326K variant and the more severe Dutch p.D140H + p.E326K founder allele. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC. on behalf of International Parkinson and Movement Disorder Society.

Risk of abdominal aortic aneurysm (AAA) among male and female relatives of AAA patients.

Sex affects the presentation, treatment, and outcomes of abdominal aortic aneurysm (AAA). Although AAAs are less prevalent in women, at least in the general population, women with an AAA have a poorer prognosis in comparison to men. Sex differences in the genetic predisposition for aneurysm disease remain to be established. In this study we investigated the familial risk of AAA for women compared to men. All living AAA patients included in a 2004-2012 prospective database were invited to the multidisciplinary vascular/genetics outpatient clinic between 2009 and 2012 for assessment of family history using detailed questionnaires. AAA risk for male and female relatives was calculated separately and stratified by sex of the AAA patients. Families of 568 AAA patients were investigated and 22.5% of the patients had at least one affected relative. Female relatives had a 2.8-fold and male relatives had a 1.7-fold higher risk than the estimated sex-specific population risk. Relatives of female AAA patients had a higher aneurysm risk than relatives of male patients (9.0 vs 5.9%, p = 0.022), corresponding to 5.5- and 2.0-fold increases in aneurysm risk in the female and male relatives, respectively. The risk for aortic aneurysm in relatives of AAA patients is higher than expected from population risk. The excess risk is highest for the female relatives of AAA patients and for the relatives of female AAA patients. These findings endorse targeted AAA family screening for female and male relatives of all AAA patients.

Postnatal Outcome of Fetal Left Ventricular Hypertrabeculation/Noncompaction.

Left ventricular hypertrabeculation/noncompaction (LVHT) is a cardiac abnormality of unknown etiology. Prenatal diagnosis of LVHT can be established by fetal echocardiography. A review of 106 published cases showed that 46 cases with prenatally diagnosed LVHT were alive 0.5-120 months after birth. Since the course of cases with prenatally LVHT after publication is unknown, we aimed to collect follow-up-information. Information regarding vital status, cardiac and extracardiac morbidity was gathered by contacting the authors of the 46 cases. Fourteen of the 28 authors answered and gave information about 18 cases (six females, seven males, five gender-unknown, age 18 months to 10 years, mean follow-up 60 months). No differences were found between the 18 cases with follow-up and the 28 cases without follow-up regarding age, gender, cardiac or extracardiac comorbidities, and interventions. Three of the 18 cases had died subsequently from heart failure, osteosarcoma, and enterocolitis, respectively. Mutations or chromosomal abnormalities were found in six of the seven examined patients, extracardiac abnormalities in nine patients. Three patients received a pacemaker because of complete AV block, and two patients underwent heart transplantation. Cardiac surgical or interventional procedures were carried out in four patients. None suffered from malignant arrhythmias or had a cardioverter-defibrillator implanted. Based on the limited information, there are indications that cases with fetal diagnosis of LVHT have a continuing morbidity and mortality, even if they receive appropriate care. Since fetal LVHT is frequently associated with genetic abnormalities, further research about survival and underlying genetic causes is needed.

First genetic analysis of aneurysm genes in familial and sporadic abdominal aortic aneurysm.

Genetic causes for abdominal aortic aneurysm (AAA) have not been identified and the role of genes associated with familial thoracic aneurysms in AAA has not been explored. We analyzed nine genes associated with familial thoracic aortic aneurysms, the vascular Ehlers-Danlos gene COL3A1 and the MTHFR p.Ala222Val variant in 155 AAA patients. The thoracic aneurysm genes selected for this study were the transforming growth factor-beta pathway genes EFEMP2, FBN1, SMAD3, TGBF2, TGFBR1, TGFBR2, and the smooth muscle cells genes ACTA2, MYH11 and MYLK. Sanger sequencing of all coding exons and exon-intron boundaries of these genes was performed. Patients with at least one first-degree relative with an aortic aneurysm were classified as familial AAA (n = 99), the others as sporadic AAA. We found 47 different rare heterozygous variants in eight genes: two pathogenic, one likely pathogenic, twenty-one variants of unknown significance (VUS) and twenty-three unlikely pathogenic variants. In familial AAA we found one pathogenic and segregating variant (COL3A1 p.Arg491X), one likely pathogenic and segregating (MYH11 p.Arg254Cys), and fifteen VUS. In sporadic patients we found one pathogenic (TGFBR2 p.Ile525Phefs*18) and seven VUS. Thirteen patients had two or more variants. These results show a previously unknown association and overlapping genetic defects between AAA and familial thoracic aneurysms, indicating that genetic testing may help to identify the cause of familial and sporadic AAA. In this view, genetic testing of these genes specifically or in a genome-wide approach may help to identify the cause of familial and sporadic AAA.

Familial Ehlers-Danlos syndrome with lethal arterial events caused by a mutation in COL5A1.

Different forms of Ehlers-Danlos syndrome (EDS) exist, with specific phenotypes and associated genes. Vascular EDS, caused by heterozygous mutations in the COL3A1 gene, is characterized by fragile vasculature with a high risk of catastrophic vascular events at a young age. Classic EDS, caused by heterozygous mutations in the COL5A1 or COL5A2 genes, is characterized by fragile, hyperextensible skin and joint laxity. To date, vessel rupture in four unrelated classic EDS patients with a confirmed COL5A1 mutation has been reported. We describe familial occurrence of a phenotype resembling vascular EDS in a mother and her two sons, who all died at an early age from arterial ruptures. Diagnostic Sanger sequencing in the proband failed to detect aberrations in COL3A1, COL1A1, COL1A2, TGFBR1, TGFBR2, SMAD3, and ACTA2. Next, the proband's DNA was analyzed using a next-generation sequencing approach targeting 554 genes linked to vascular disease (VASCULOME project). A novel heterozygous mutation in COL5A1 was detected, resulting in an essential glycine substitution at the C-terminal end of the triple helix domain (NM_000093.4:c.4610G>T; p.Gly1537Val). This mutation was also present in DNA isolated from autopsy material of the index's brother. No material was available from the mother, but the mutation was excluded in her parents, siblings and in the father of her sons, suggesting that the COL5A1 mutation occurred in the mother's genome de novo. In conclusion, we report familial occurrence of lethal arterial events caused by a COL5A1 mutation.

Familial abdominal aortic aneurysm is associated with more complications after endovascular aneurysm repair.

OBJECTIVE: A familial predisposition to abdominal aortic aneurysms (AAAs) is present in approximately one-fifth of patients. Nevertheless, the clinical implications of a positive family history are not known. We investigated the risk of aneurysm-related complications after endovascular aneurysm repair (EVAR) for patients with and without a positive family history of AAA. METHODS: Patients treated with EVAR for intact AAAs in the Erasmus University Medical Center between 2000 and 2012 were included in the study. Family history was obtained by written questionnaire. Familial AAA (fAAA) was defined as patients having at least one first-degree relative affected with aortic aneurysm. The remaining patients were considered sporadic AAA. Cardiovascular risk factors, aneurysm morphology (aneurysm neck, aneurysm sac, and iliac measurements), and follow-up were obtained prospectively. The primary end point was complications after EVAR, a composite of endoleaks, need for secondary interventions, aneurysm sac growth, acute limb ischemia, and postimplantation rupture. Secondary end points were specific components of the primary end point (presence of endoleak, need for secondary intervention, and aneurysm sac growth), aneurysm neck growth, and overall survival. Kaplan-Meier estimates for the primary end point were calculated and compared using log-rank (Mantel-Cox) test of equality. A Cox-regression model was used to calculate the independent risk of complications associated with fAAA. RESULTS: A total of 255 patients were included in the study (88.6% men; age 72 ± 7 years, median follow-up 3.3 years; interquartile range, 2.2-6.1). A total of 51 patients (20.0%) were classified as fAAA. Patients with fAAA were younger (69 vs 72 years; P = .015) and were less likely to have ever smoked (58.8% vs 73.5%; P = .039). Preoperative aneurysm morphology was similar in both groups. Patients with fAAA had significantly more complications after EVAR (35.3% vs 19.1%; P = .013), with a twofold increased risk (adjusted hazard ratio, 2.1; 95% confidence interval, 1.2-3.7). Secondary interventions (39.2% vs 20.1%; P = .004) and aneurysm sac growth (20.8% vs 9.5%; P = .030) were the most important elements accounting for the difference. Furthermore, a trend toward more type I endoleaks during follow-up was observed (15.6% vs 7.4%; P = .063) and no difference in overall survival. CONCLUSIONS: The current study shows that patients with a familial form of AAA develop more aneurysm-related complications after EVAR, despite similar AAA morphology at baseline. These findings suggest that patients with fAAA form a specific subpopulation and create awareness for a possible increase in the risk of complications after EVAR.

Lower atherosclerotic burden in familial abdominal aortic aneurysm.

OBJECTIVE: Despite the apparent familial tendency toward abdominal aortic aneurysm (AAA) formation, the genetic causes and underlying molecular mechanisms are still undefined. In this study, we investigated the association between familial AAA (fAAA) and atherosclerosis. METHODS: Data were collected from a prospective database including AAA patients between 2004 and 2012 in the Erasmus University Medical Center, Rotterdam, The Netherlands. Family history was obtained by written questionnaire (93.1% response rate). Patients were classified as fAAA when at least one affected first-degree relative with an aortic aneurysm was reported. Patients without an affected first-degree relative were classified as sporadic AAA (spAAA). A standardized ultrasound measurement of the common carotid intima-media thickness (CIMT), a marker for generalized atherosclerosis, was routinely performed and patients' clinical characteristics (demographics, aneurysm characteristics, cardiovascular comorbidities and risk factors, and medication use) were recorded. Multivariable linear regression analyses were used to assess the mean adjusted difference in CIMT and multivariable logistic regression analysis was used to calculate associations of increased CIMT and clinical characteristics between fAAA and spAAA. RESULTS: A total of 461 AAA patients (85% men, mean age, 70 years) were included in the study; 103 patients (22.3%) were classified as fAAA and 358 patients (77.7%) as spAAA. The mean (standard deviation) CIMT in patients with fAAA was 0.89 (0.24) mm and 1.00 (0.29) mm in patients with spAAA (P = .001). Adjustment for clinical characteristics showed a mean difference in CIMT of 0.09 mm (95% confidence interval, 0.02-0.15; P = .011) between both groups. Increased CIMT, smoking, hypertension, and diabetes mellitus were all less associated with fAAA compared with spAAA. CONCLUSIONS: The current study shows a lower atherosclerotic burden, as reflected by a lower CIMT, in patients with fAAA compared with patients with spAAA, independent of common atherosclerotic risk factors. These results support the hypothesis that although atherosclerosis is a common underlying feature in patients with aneurysms, atherosclerosis is not the primary driving factor in the development of fAAA.

Increased vascular smooth muscle cell senescence in aneurysmal Fibulin-4 mutant mice.

Aortic aneurysms are dilatations of the aorta that can rupture when left untreated. We used the aneurysmal Fibulin-4R/R mouse model to further unravel the underlying mechanisms of aneurysm formation. RNA sequencing of 3-month-old Fibulin-4R/R aortas revealed significant upregulation of senescence-associated secretory phenotype (SASP) factors and key senescence factors, indicating the involvement of senescence. Analysis of aorta histology and of vascular smooth muscle cells (VSMCs) in vitro confirmed the senescent phenotype of Fibulin-4R/R VSMCs by revealing increased SA-ß-gal, p21, and p16 staining, increased IL-6 secretion, increased presence of DNA damage foci and increased nuclei size. Additionally, we found that p21 luminescence was increased in the dilated aorta of Fibulin-4R/R|p21-luciferase mice. Our studies identify a cellular aging cascade in Fibulin-4 aneurysmal disease, by revealing that Fibulin-4R/R aortic VSMCs have a pronounced SASP and a senescent phenotype that may underlie aortic wall degeneration. Additionally, we demonstrated the therapeutic effect of JAK/STAT and TGF-ß pathway inhibition, as well as senolytic treatment on Fibulin-4R/R VSMCs in vitro. These findings can contribute to improved therapeutic options for aneurysmal disease aimed at reducing senescent cells.

Targeted proteomics and metabolomics for biomarker discovery in abdominal aortic aneurysm and post-EVAR sac volume.

BACKGROUND AND AIMS: Abdominal aortic aneurysm (AAA) patients undergo uniform surveillance programs both leading up to, and following surgery. Circulating biomarkers could play a pivotal role in individualizing surveillance. We applied a multi-omics approach to identify relevant biomarkers and gain pathophysiological insights. MATERIALS AND METHODS: In this cross-sectional study, 108 AAA patients and 200 post-endovascular aneurysm repair (post-EVAR) patients were separately investigated. We performed partial least squares regression and ingenuity pathway analysis on circulating concentrations of 96 proteins (92 Olink Cardiovascular-III panel, 4 ELISA-assays) and 199 metabolites (measured by LC-TQMS), and their associations with CT-based AAA/sac volume. RESULTS: The median (25th-75th percentile) maximal diameter was 50.0 mm (46.0, 53.0) in the AAA group, and 55.4 mm (45.0, 64.2) in the post-EVAR group. Correcting for clinical characteristics in AAA patients, the aneurysm volume Z-score differed 0.068 (95 %CI: (0.042, 0.093)), 0.066 (0.047, 0.085) and -0.051 (-0.064, -0.038) per Z-score valine, leucine and uPA, respectively. After correcting for clinical characteristics and orthogonalization in the post-EVAR group, the sac volume Z-score differed 0.049 (0.034, 0.063) per Z-score TIMP-4, -0.050 (-0.064, -0.037) per Z-score LDL-receptor, -0.051 (-0.062, -0.040) per Z-score 1-OG/2-OG and -0.056 (-0.066, -0.045) per Z-score 1-LG/2-LG. CONCLUSIONS: The branched-chain amino acids and uPA were related to AAA volume. For post-EVAR patients, LDL-receptor, monoacylglycerols and TIMP-4 are potential biomarkers for sac volume. Additionally, distinct markers for sac change were identified.

The proprotein convertase FURIN is a novel aneurysm predisposition gene impairing TGF-ß signaling.

AIM: Aortic aneurysms (AA) frequently involve dysregulation of transforming growth factor ß (TGF-ß)-signaling in the aorta. Here, FURIN was tested as aneurysm predisposition gene given its role as proprotein convertase in pro-TGF-ß maturation. METHODS AND RESULTS: Rare FURIN variants were detected by whole-exome sequencing of 781 unrelated aortic aneurysm patients and affected relatives. Thirteen rare heterozygous FURIN variants occurred in 3.7% (29) unrelated index AA patients, of which 72% had multiple aneurysms or a dissection.FURIN maturation and activity of these variants were decreased in vitro. Patient-derived fibroblasts showed decreased pro-TGF-ß processing, phosphorylation of downstream effector SMAD2 and kinases ERK1/2, and steady-state mRNA levels of the TGF-ß-responsive ACTA2 gene. In aortic tissue, collagen and fibrillin fibers were affected. One variant (R745Q), observed in 10 unrelated cases, affected TGF-ß signaling variably, indicating effect modification by individual genetic backgrounds. CONCLUSION: FURIN is a novel, frequent genetic predisposition for abdominal-, thoracic-, and multiple aortic or middle sized artery aneurysms in older patients, by affecting intracellular TGF-ß signaling, depending on individual genetic backgrounds.

Vascular Ehlers-Danlos Syndrome: A Comprehensive Natural History Study in a Dutch National Cohort of 142 Patients.

BACKGROUND: Vascular Ehlers-Danlos syndrome (vEDS) is a rare connective tissue disorder with a high risk for arterial, bowel, and uterine rupture, caused by heterozygous pathogenic variants in COL3A1. The aim of this cohort study is to provide further insights into the natural history of vEDS and describe genotype-phenotype correlations in a Dutch multicenter cohort to optimize patient care and increase awareness of the disease. METHODS: Individuals with vEDS throughout the Netherlands were included. The phenotype was charted by retrospective analysis of molecular and clinical data, combined with a one-time physical examination. RESULTS: A total of 142 individuals (50% female) participated the study, including 46 index patients (32%). The overall median age at genetic diagnosis was 41.0 years. More than half of the index patients (54.3%) and relatives (53.1%) had a physical appearance highly suggestive of vEDS. In these individuals, major events were not more frequent (P=0.90), but occurred at a younger age (P=0.01). A major event occurred more often and at a younger age in men compared with women (P<0.001 and P=0.004, respectively). Aortic aneurysms (P=0.003) and pneumothoraces (P=0.029) were more frequent in men. Aortic dissection was more frequent in individuals with a COL3A1 variant in the first quarter of the collagen helical domain (P=0.03). CONCLUSIONS: Male sex, type and location of the COL3A1 variant, and physical appearance highly suggestive of vEDS are risk factors for the occurrence and early age of onset of major events. This national multicenter cohort study of Dutch individuals with vEDS provides a valuable basis for improving guidelines for the diagnosing, follow-up, and treatment of individuals with vEDS.

Aneurysmal disease is associated with lower carotid intima-media thickness than occlusive arterial disease.

OBJECTIVE: Patients with aneurysmal and occlusive arterial disease have overlapping cardiovascular risk profiles. The question remains how atherosclerosis is related to the formation of aortic aneurysms. Common carotid artery intima-media thickness (CIMT) is an easily accessible and objective marker of early atherosclerosis. The aim of the current study was to investigate whether there is a difference in atherosclerotic burden as measured by CIMT between patients with aneurysmal and those with occlusive arterial disease. METHODS: From 2004 to 2011, the CIMT was measured using B-mode ultrasound scanning in patients undergoing vascular surgery for aortic aneurysmal or occlusive arterial disease at the Erasmus University Medical Center. Cardiovascular risk factors, comorbidities, and medication were recorded. Patients treated for combined aneurysmal and occlusive arterial disease and patients diagnosed with a genetic aneurysm syndrome were excluded. Univariable and multivariable analyses were used to calculate differences in CIMT between aneurysmal and occlusive arterial disease. RESULTS: In total, 904 patients were included in the study: 502 patients with aneurysmal disease (85% male; mean age, 72 years) and 402 patients with occlusive arterial disease (65% male; mean age, 64 years). The mean (standard deviation) CIMT in patients with aneurysmal disease was 0.97 (0.29) mm and was 1.07 (0.38) mm in patients with occlusive arterial disease (P < .001). Adjustment for cardiovascular risk factors, comorbidities, and medication showed a mean difference in CIMT of 0.15 mm (95% confidence interval, 0.10-0.20; P < .001). CONCLUSIONS: The current study shows a lower CIMT in patients with aneurysmal disease than in those with occlusive arterial disease, indicating a lower atherosclerotic burden in patients with aneurysmal disease. These findings endorse the idea that additional pathogenic mechanisms are involved in aortic aneurysm formation. Further studies are needed to clarify the role of atherosclerosis in aortic aneurysm formation.

The clinical and pathological phenotype of C9ORF72 hexanucleotide repeat expansions.

There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 ± 8.3 years (range 39-76), and disease duration 7.6 ± 4.6 years (range 1-22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation.

Presence of ATM protein and residual kinase activity correlates with the phenotype in ataxia-telangiectasia: a genotype-phenotype study.

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with multisystem involvement and cancer predisposition, caused by mutations in the A-T mutated (ATM) gene. To study genotype-phenotype correlations, we evaluated the clinical and laboratory data of 51 genetically proven A-T patients, and additionally measured ATM protein expression and kinase activity. Patients without ATM kinase activity showed the classical phenotype. The presence of ATM protein, correlated with slightly better immunological function. Residual kinase activity correlated with a milder and essentially different neurological phenotype, absence of telangiectasia, normal endocrine and pulmonary function, normal immunoglobulins, significantly lower X-ray hypersensitivity in lymphocytes, and extended lifespan. In these patients, cancer occurred later in life and generally consisted of solid instead of lymphoid malignancies. The genotypes of severely affected patients generally included truncating mutations resulting in total absence of ATM kinase activity, while patients with milder phenotypes harbored at least one missense or splice site mutation resulting in expression of ATM with some kinase activity. Overall, the phenotypic manifestations in A-T show a continuous spectrum from severe classical childhood-onset A-T to a relatively mild adult-onset disorder, depending on the presence of ATM protein and kinase activity. Each patient is left with a tremendously increased cancer risk.

A human MYBPC3 mutation appearing about 10 centuries ago results in a hypertrophic cardiomyopathy with delayed onset, moderate evolution but with a risk of sudden death.

BACKGROUND: Hypertrophic Cardiomyopathy (HCM) is a genetically heterogeneous disease. One specific mutation in the MYBPC3 gene is highly prevalent in center east of France giving an opportunity to define the clinical profile of this specific mutation. METHODS: HCM probands were screened for mutation in the MYH7, MYBPC3, TNNT2 and TNNI3 genes. Carriers of the MYBPC3 IVS20-2A>G mutation were genotyped with 8 microsatellites flanking this gene. The age of this MYBPC3 mutation was inferred with the software ESTIAGE. The age at first symptom, diagnosis, first complication, first severe complication and the rate of sudden death were compared between carriers of the IVS20-2 mutation (group A) and carriers of all other mutations (group B) using time to event curves and log rank test. RESULTS: Out of 107 HCM probands, 45 had a single heterozygous mutation in one of the 4 tested sarcomeric genes including 9 patients with the MYBPC3 IVS20-2A>G mutation. The IVS20-2 mutation in these 9 patients and their 25 mutation carrier relatives was embedded in a common haplotype defined after genotyping 4 polymorphic markers on each side of the MYBPC3 gene. This result supports the hypothesis of a common ancestor. Furthermore, we evaluated that the mutation occurred about 47 generations ago, approximately at the 10th century.We then compared the clinical profile of the IVS20-2 mutation carriers (group A) and the carriers of all other mutations (group B). Age at onset of symptoms was similar in the 34 group A cases and the 73 group B cases but group A cases were diagnosed on average 15 years later (log rank test p = 0.022). Age of first complication and first severe complication was delayed in group A vs group B cases but the prevalence of sudden death and age at death was similar in both groups. CONCLUSION: A founder mutation arising at about the 10th century in the MYBPC3 gene accounts for 8.4% of all HCM in center east France and results in a cardiomyopathy starting late and evolving slowly but with an apparent risk of sudden death similar to other sarcomeric mutations.

Manifest disease, risk factors for sudden cardiac death, and cardiac events in a large nationwide cohort of predictively tested hypertrophic cardiomyopathy mutation carriers: determining the best cardiological screening strategy.

AIMS: We investigated the presence of a clinical diagnosis of hypertrophic cardiomyopathy (HCM), risk factors for sudden cardiac death (SCD), and cardiac events during follow-up in predictively tested-not known to have a clinical diagnosis of HCM before the DNA test-carriers of a sarcomeric gene mutation and associations with age and gender to determine the best cardiological screening strategy. METHODS AND RESULTS: One hundred and thirty-six (30%) of 446 mutation carriers were diagnosed with HCM at one or more cardiological evaluation(s). Male gender and higher age were associated with manifest disease. Incidence of newly diagnosed manifest HCM was <10% per person-year under the age of 40 years and >10% in older carriers, although numbers were small in carriers <15 years. Twenty-three percent of carriers, with and without manifest disease, had established risk factor(s) for SCD (no significant difference). During an average follow-up of 3.5 ± 1.7 years two carriers, both with manifest disease, died suddenly (0.13% per person-year). A high-risk status for SCD (≥2 risk factors and manifest HCM) was present in 17 carriers during follow-up (2.4% per person-year). Age but not gender was associated with a high-risk status for SCD. CONCLUSION: Thirty percent of carriers had or developed manifest HCM after predictive DNA testing and risk factors for SCD were frequently present. Our data suggest that the SCD risk is low and risk stratification for SCD can be omitted in carriers without manifest disease and that frequency of cardiological evaluations can possibly be decreased in carriers between 15 and 40 years as long as hypertrophy is absent.

Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness.

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with ß-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. ß-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.