Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Subclonal Cancer Driver Mutations Are Prevalent in the Unresected Peritumoral Edema of Adult Diffuse Gliomas.

Adult diffuse gliomas commonly recur regardless of therapy. As recurrence typically arises from the peritumoral edema adjacent to the resected bulk tumor, the profiling of somatic mutations from infiltrative malignant cells within this critical, unresected region could provide important insights into residual disease. A key obstacle has been the inability to distinguish between next-generation sequencing (NGS) noise and the true but weak signal from tumor cells hidden among the noncancerous brain tissue of the peritumoral edema. Here, we developed and validated True2 sequencing to reduce NGS-associated errors to <1 false positive/100 kb panel positions while detecting 97.6% of somatic mutations with an allele frequency ≥0.1%. True2 was then used to study the tumor and peritumoral edema of 22 adult diffuse gliomas including glioblastoma, astrocytoma, oligodendroglioma, and NF1-related low-grade neuroglioma. The tumor and peritumoral edema displayed a similar mutation burden, indicating that surgery debulks these cancers physically but not molecularly. Moreover, variants in the peritumoral edema included unique cancer driver mutations absent in the bulk tumor. Finally, analysis of multiple samples from each patient revealed multiple subclones with unique mutations in the same gene in 17 of 22 patients, supporting the occurrence of convergent evolution in response to patient-specific selective pressures in the tumor microenvironment that may form the molecular foundation of recurrent disease. Collectively, True2 enables the detection of ultralow frequency mutations during molecular analyses of adult diffuse gliomas, which is necessary to understand cancer evolution, recurrence, and individual response to therapy. SIGNIFICANCE: True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies.

Detection of circulating tumor DNA without a tumor-informed search using next-generation sequencing is a prognostic biomarker in pancreatic ductal adenocarcinoma.

The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ2=5.4, P = 0.021). Guiding ctDNA detection in pre-operative ccfDNA based on mutations present in tumor DNA yielded a similar survival analysis. Detection of ctDNA in the post-operative ccfDNA with or without tumor-informed guidance was not associated with outcomes. Therefore, the detection of PDAC-derived ctDNA during a broad and untargeted survey of ccfDNA with NGS may be a valuable, non-invasive, prognostic biomarker to integrate into the clinical assessment and management of patients prior to surgery.

Leveraging the Fragment Length of Circulating Tumour DNA to Improve Molecular Profiling of Solid Tumour Malignancies with Next-Generation Sequencing: A Pathway to Advanced Non-invasive Diagnostics in Precision Oncology?

Circulating cell-free DNA (ccfDNA) has emerged as a promising diagnostic tool in oncology. Identification of tumour-derived ccfDNA (i.e. circulating tumour DNA [ctDNA]) provides non-invasive access to a malignancy's molecular landscape to diagnose, inform therapeutic strategies, and monitor treatment efficacy. Current applications of ccfDNA to detect somatic mutations, however, have been largely constrained to tumour-informed searches and identification of common mutations because of the interaction between ctDNA signal and next-generation sequencing (NGS) noise. Specifically, the low allele frequency of ctDNA associated with non-metastatic and early-stage lesions may be indistinguishable from artifacts that accrue during sample preparation and NGS. Thus, using ccfDNA to achieve non-invasive and personalized molecular profiling to optimize individual patient care is a highly sought goal that remains limited in clinical practice. There is growing evidence, however, that further advances in the field of ccfDNA diagnostics may be achieved by improving detection of somatic mutations through leveraging the inherently shorter fragment lengths of ctDNA compared to non-neoplastic ccfDNA. Here, the origins and rationale for seeking to improve the mutation-based detection of ctDNA by using ccfDNA size profiling are reviewed. Subsequently, in vitro and in silico methods to enrich for a target ccfDNA fragment length are detailed to identify current practices and provide perspective into the potential of using ccfDNA size profiling to impact clinical applications in oncology.

Heterozygous variants that disturb the transcriptional repressor activity of FOXP4 cause a developmental disorder with speech/language delays and multiple congenital abnormalities.

PURPOSE: Heterozygous pathogenic variants in various FOXP genes cause specific developmental disorders. The phenotype associated with heterozygous variants in FOXP4 has not been previously described. METHODS: We assembled a cohort of eight individuals with heterozygous and mostly de novo variants in FOXP4: seven individuals with six different missense variants and one individual with a frameshift variant. We collected clinical data to delineate the phenotypic spectrum, and used in silico analyses and functional cell-based assays to assess pathogenicity of the variants. RESULTS: We collected clinical data for six individuals: five individuals with a missense variant in the forkhead box DNA-binding domain of FOXP4, and one individual with a truncating variant. Overlapping features included speech and language delays, growth abnormalities, congenital diaphragmatic hernia, cervical spine abnormalities, and ptosis. Luciferase assays showed loss-of-function effects for all these variants, and aberrant subcellular localization patterns were seen in a subset. The remaining two missense variants were located outside the functional domains of FOXP4, and showed transcriptional repressor capacities and localization patterns similar to the wild-type protein. CONCLUSION: Collectively, our findings show that heterozygous loss-of-function variants in FOXP4 are associated with an autosomal dominant neurodevelopmental disorder with speech/language delays, growth defects, and variable congenital abnormalities.

VPS4A Mutations in Humans Cause Syndromic Congenital Dyserythropoietic Anemia due to Cytokinesis and Trafficking Defects.

The Congenital Dyserythropoietic Anemia (CDA) Registry was established with the goal to facilitate investigations of natural history, biology, and molecular pathogenetic mechanisms of CDA. Three unrelated individuals enrolled in the registry had a syndrome characterized by CDA and severe neurodevelopmental delay. They were found to have missense mutations in VPS4A, a gene coding for an ATPase that regulates the ESCRT-III machinery in a variety of cellular processes including cell division, endosomal vesicle trafficking, and viral budding. Bone marrow studies showed binucleated erythroblasts and erythroblasts with cytoplasmic bridges indicating abnormal cytokinesis and abscission. Circulating red blood cells were found to retain transferrin receptor (CD71) in their membrane, demonstrating that VPS4A is critical for normal reticulocyte maturation. Using proband-derived induced pluripotent stem cells (iPSCs), we have successfully modeled the hematologic aspects of this syndrome in vitro, recapitulating their dyserythropoietic phenotype. Our findings demonstrate that VPS4A mutations cause cytokinesis and trafficking defects leading to a human disease with detrimental effects to erythropoiesis and neurodevelopment.

Somalier: rapid relatedness estimation for cancer and germline studies using efficient genome sketches.

BACKGROUND: When interpreting sequencing data from multiple spatial or longitudinal biopsies, detecting sample mix-ups is essential, yet more difficult than in studies of germline variation. In most genomic studies of tumors, genetic variation is detected through pairwise comparisons of the tumor and a matched normal tissue from the sample donor. In many cases, only somatic variants are reported, which hinders the use of existing tools that detect sample swaps solely based on genotypes of inherited variants. To address this problem, we have developed Somalier, a tool that operates directly on alignments and does not require jointly called germline variants. Instead, Somalier extracts a small sketch of informative genetic variation for each sample. Sketches from hundreds of germline or somatic samples can then be compared in under a second, making Somalier a useful tool for measuring relatedness in large cohorts. Somalier produces both text output and an interactive visual report that facilitates the detection and correction of sample swaps using multiple relatedness metrics. RESULTS: We introduce the tool and demonstrate its utility on a cohort of five glioma samples each with a normal, tumor, and cell-free DNA sample. Applying Somalier to high-coverage sequence data from the 1000 Genomes Project also identifies several related samples. We also demonstrate that it can distinguish pairs of whole-genome and RNA-seq samples from the same individuals in the Genotype-Tissue Expression (GTEx) project. CONCLUSIONS: Somalier is a tool that can rapidly evaluate relatedness from sequencing data. It can be applied to diverse sequencing data types and genome builds and is available under an MIT license at github.com/brentp/somalier .

The stochastic nature of errors in next-generation sequencing of circulating cell-free DNA.

Challenges with distinguishing circulating tumor DNA (ctDNA) from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Here we show early and random PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding, removal of artifacts due to clonal hematopoiesis of indeterminate potential, and suppression of patterned errors. We also demonstrate sample duplicates are necessary to eliminate the stochastic noise associated with NGS. Integration of sample duplicates into NGS analytics may broaden ctDNA applications by removing NGS-related errors that confound identification of true very low frequency variants during searches for ctDNA without a priori knowledge of specific mutations to target.

Targeted gene panel sequencing for the rapid diagnosis of acutely ill infants.

BACKGROUND: Exome/genome sequencing (ES/GS) have been recently used in neonatal and pediatric/cardiac intensive care units (NICU and PICU/CICU) to diagnose and care for acutely ill infants, but the effectiveness of targeted gene panels for these purposes remains unknown. METHODS: RapSeq, a newly developed panel targeting 4,503 disease-causing genes, was employed on selected patients in our NICU/PICU/CICU. Twenty trios were sequenced from October 2015 to March 2017. We assessed diagnostic yield, turnaround times, and clinical consequences. RESULTS: A diagnosis was made in 10/20 neonates (50%); eight had de novo variants (ASXL1, CHD, FBN1, KMT2D, FANCB, FLNA, PAX3), one was a compound heterozygote for CHAT, and one had a maternally inherited GNAS variant. Preliminary reports were generated by 9.6 days (mean); final reports after Sanger sequencing at 16.3 days (mean). In all positive infants, the diagnosis changed management. In a case with congenital myasthenia, diagnosis and treatment occurred at 17 days versus 7 months in a historical control. CONCLUSIONS: This study shows that a gene panel that includes the majority of known disease-causing genes can rapidly identify a diagnosis in a large number of tested infants. Due to simpler deployment and interpretation and lower costs, this approach might represent an alternative to ES/GS in the NICU/PICU/CICU.

DPAGT1 Deficiency with Encephalopathy (DPAGT1-CDG): Clinical and Genetic Description of 11 New Patients.

Pathogenic mutations in DPAGT1 cause a rare type of a congenital disorder of glycosylation termed DPAGT1-CDG or, alternatively, a milder version with only myasthenia known as DPAGT1-CMS. Fourteen disease-causing mutations in 28 patients from 10 families have previously been reported to cause the systemic form, DPAGT1-CDG. We here report on another 11 patients from 8 families and add 10 new mutations. Most patients have a very severe disease course, where common findings are pronounced muscular hypotonia, intractable epilepsy, global developmental delay/intellectual disability, and early death. We also present data on three affected females that are young adults and have a somewhat milder, stable disease. Our findings expand both the molecular and clinical knowledge of previously published data but also widen the phenotypic spectrum of DPAGT1-CDG.

Automated size selection for short cell-free DNA fragments enriches for circulating tumor DNA and improves error correction during next generation sequencing.

Circulating tumor-derived cell-free DNA (ctDNA) enables non-invasive diagnosis, monitoring, and treatment susceptibility testing in human cancers. However, accurate detection of variant alleles, particularly during untargeted searches, remains a principal obstacle to widespread application of cell-free DNA in clinical oncology. In this study, isolation of short cell-free DNA fragments is shown to enrich for tumor variants and improve correction of PCR- and sequencing-associated errors. Subfractions of the mononucleosome of circulating cell-free DNA (ccfDNA) were isolated from patients with melanoma, pancreatic ductal adenocarcinoma, and colorectal adenocarcinoma using a high-throughput-capable automated gel-extraction platform. Using a 128-gene (128 kb) custom next-generation sequencing panel, variant alleles were on average 2-fold enriched in the short fraction (median insert size: ~142 bp) compared to the original ccfDNA sample, while 0.7-fold reduced in the fraction corresponding to the principal peak of the mononucleosome (median insert size: ~167 bp). Size-selected short fractions compared to the original ccfDNA yielded significantly larger family sizes (i.e., PCR duplicates) during in silico consensus sequence interpretation via unique molecular identifiers. Increments in family size were associated with a progressive reduction of PCR and sequencing errors. Although consensus read depth also decreased at larger family sizes, the variant allele frequency in the short ccfDNA fraction remained consistent, while variant detection in the original ccfDNA was commonly lost at family sizes necessary to minimize errors. These collective findings support the automated extraction of short ccfDNA fragments to enrich for ctDNA while concomitantly reducing false positives through in silico error correction.

Pathogenic ASXL1 somatic variants in reference databases complicate germline variant interpretation for Bohring-Opitz Syndrome.

The clinical interpretation of genetic variants has come to rely heavily on reference population databases such as the Exome Aggregation Consortium (ExAC) database. Pathogenic variants in genes associated with severe, pediatric-onset, highly penetrant, autosomal dominant conditions are assumed to be absent or rare in these databases. Exome sequencing of a 6-year-old female patient with seizures, developmental delay, dysmorphic features, and failure to thrive identified an ASXL1 variant previously reported as causative of Bohring-Opitz syndrome (BOS). Surprisingly, the variant was observed seven times in the ExAC database, presumably in individuals without BOS. Although the BOS phenotype fit, the presence of the variant in reference population databases introduced ambiguity in result interpretation. Review of the literature revealed that acquired somatic mosaicism of ASXL1 variants (including pathogenic variants) during hematopoietic clonal expansion can occur with aging in healthy individuals. We examined all ASXL1 truncating variants in the ExAC database and determined most are likely somatic. Failure to consider somatic mosaicism may lead to the inaccurate assumption that conditions like BOS have reduced penetrance, or the misclassification of potentially pathogenic variants.

A continuous-infusion dynamic MRI model at 3.0 Tesla for the serial quantitative evaluation of microvascular proliferation in an animal model of glioblastoma multiforme.

PURPOSE: To develop a continuous-infusion dynamic MRI technique to characterize tumor-associated microvascular proliferation (MVP) in a rat brain model of glioblastoma multiforme. METHODS: The proposed model assumes effects due to tumor-associated MVP (eg, vascular permeability, Ktrans ; intravascular plasma fraction, vp ) cannot be individually separated and solves for a single parameter (kvasc ) that quantifies the T1 -weighted contrast enhancement from dynamic images acquired during continuous contrast agent (CA) infusion. Untreated C6 tumor-bearing animals (N = 6) were serially imaged on postoperative days (PODs) 14 and 18 with a 3 Tesla clinical scanner utilizing a dynamic spatial and temporal resolution of 0.38 × 0.38 × 1.5 mm3 and 3.47 s, respectively. RESULTS: An association was present between PODs 14 and 18 for median tumor kvasc (Pearson's r = 0.94, P = 0.0052) and CA concentration ([CA], derived from pre- and postcontrast R1 maps; r = 0.94, P = 0.0054). On POD 18, there was a voxel-based association between kvasc and [CA] within each tumor (0.45 < r < 0.82, P < 0.001). However, voxel-based subregions demonstrated a reduced association between kvasc and [CA] (N = 5; -0.08 < r < 0.22, P > 0.05) or an inverse association (N = 1; r = -0.28, P = 0.001), indicating differences between locations of vascular permeability and subsequent CA pooling in tumors. CONCLUSION: The continuous-infusion method may provide a quantitative measure for characterizing and monitoring tumor-associated MVP. Magn Reson Med 78:1824-1838, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Fragment Length of Circulating Tumor DNA.

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

Detecting the effects of Fabry disease in the adult human brain with diffusion tensor imaging and fast bound-pool fraction imaging.

BACKGROUND: To identify quantitative MRI parameters associated with diffusion tensor imaging (DTI) and fast bound-pool fraction imaging (FBFI) that may detect alterations in gray matter and/or white matter in adults with Fabry disease, a lysosomal storage disorder. MATERIALS AND METHODS: Twelve healthy controls (mean age ± standard deviation: 48.0 ± 12.4 years) and 10 participants with Fabry disease (46.7 ± 12.9 years) were imaged at 3.0 Tesla. Whole-brain parametric maps of diffusion tensor metrics (apparent diffusion coefficient [ADC] and fractional anisotropy [FA]) and the bound-pool fraction (f) were acquired. Mean voxel values of parametric maps from regions-of-interest within gray and white matter structures were compared between cases and controls using the independent t-test. Spearman's rho was used to identify associations between parametric maps and age. RESULTS: Compared with controls, the left thalamus of Fabry participants had an increase in FA (0.29 ± 0.02 versus 0.33 ± 0.05, respectively; P = 0.030) and a trend toward an increase in ADC (0.73 ± 00.02 versus 0.76 ± 0.03 µm(2) /s, respectively; P = 0.082). The left posterior white matter demonstrated a reduction in f (10.45 ± 0.37 versus 9.00 ± 1.84%, respectively; P = 0.035), an increase in ADC (0.78 ± 0.04 versus 0.94 ± 0.19 µm(2) /s, respectively; P = 0.024), and a trend toward a reduction in FA (0.42 ± 0.07 versus 0.36 ± 0.08, respectively; P = 0.052). Among all parameters, only f measured in the left posterior white matter was significantly associated with age in Fabry participants (rho = -0.71; P = 0.022). CONCLUSION: Parameters derived from DTI and FBFI detect Fabry-related changes in the adult human brain, particularly in the posterior white matter where reductions in myelin density as measured by FBFI appear age related.

Prediction of high-risk plaque development and plaque progression with the carotid atherosclerosis score.

OBJECTIVES: The goal of this prospective study was to evaluate the carotid atherosclerosis score (CAS) for predicting the development of high-risk plaque features and plaque burden progression. BACKGROUND: Previous studies have shown that carotid intraplaque hemorrhage (IPH) and a disrupted luminal surface (DLS), as identified by using magnetic resonance imaging, are associated with greater risk for cerebrovascular events. On the basis of data from a large cross-sectional study, a scoring system was developed to determine which plaque features are associated with the presence of IPH and DLS. However, the predictive value of CAS has not been previously tested in a prospective, longitudinal study. METHODS: A total of 120 asymptomatic subjects with 50% to 79% carotid stenosis underwent carotid magnetic resonance imaging scans at baseline and 3 years thereafter. Presence of IPH and DLS, wall volume, maximum wall thickness, and maximum percent lipid-rich necrotic core area were measured at both time-points. Baseline CAS values were calculated on the basis of previously published criteria. RESULTS: Of the 73 subjects without IPH or DLS at baseline, 9 (12%) developed 1 or both of these features during follow-up. There was a significant increasing trend between CAS and the development of new DLS (p < 0.001) and with plaque burden progression (p = 0.03) but not with the development of new IPH (p = 0.3). Percent carotid stenosis was not significantly associated with new DLS (p = 0.2), new IPH (p = 0.1), or plaque progression (p = 0.6). CONCLUSIONS: CAS was found to have a significant increasing relationship with incident DLS and plaque progression in this prospective study. CAS can potentially provide improved risk stratification beyond luminal stenosis.

Asymptomatic methylmalonic acidemia in a homozygous MUT mutation (p.P86L).

Deficiency in methylmalonyl-coenzyme A mutase (MCM) is associated with accumulation of methylmalonic acid (MMA) and clinical outcomes that include early death and neurological impairment. Reported here are two unrelated patients with a homozygous p.P86L mutation in the MUT gene, which encodes MCM, diagnosed following newborn screening. This is the first description of a homozygous mutation in the N-terminal extended segment of the MCM apoenzyme. Both in vitro and in vivo testing did not find a response to supplemental hydroxocobalamin. After discontinuation of hydroxocobalamin in one patient, serum MMA level remained elevated but stable, while urine MMA increased. Both patients have remained asymptomatic with normal development. The observed homozygous p.P86L mutation in the N-terminal extended segment may yield reduced MCM activity and is refractory to hydroxocobalamin supplementation, while not inducing a metabolically unstable phenotype. These genotype-phenotype associations further enhance the understanding of methylmalonic acidemia, which will continue to improve patient care.

Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.

The ß-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and ß-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of ß-tropomyosin-related myopathies through the identification of a novel ß-tropomyosin mutation in two clinical contexts not previously associated with ß-tropomyosin. The first clinical phenotype is core-rod myopathy, with a ß-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of ß-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric ß-tropomyosin, a change predicted to alter protein-protein binding between ß-tropomyosin and other molecules and to disturb head-to-tail polymerization of ß-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del ß-tropomyosin in the developing zebrafish. p.K7del ß-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail ß-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel ß-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with ß-tropomyosin and pathogenic data from the first animal model of ß-tropomyosin-related myopathies.

Characterization of distensibility, plaque burden, and composition of the atherosclerotic carotid artery using magnetic resonance imaging.

PURPOSE: Arterial distensibility is a marker that can measure vessel wall functional and structural changes resulting from atherosclerosis with applications including estimation of mechanical properties of the wall. We sought to assess the feasibility of using magnetic resonance imaging (MRI) to include wall distensibility in the characterization of atherosclerotic carotid arteries and to analyze the relationship between distensibility and morphological and compositional plaque features. METHODS: Five healthy volunteers were imaged with a multiple-slice CINE MR sequence twice, within 24 h, to determine the interscan reproducibility of distensibility measurements. Twenty-one subjects with >15% carotid stenosis and the five healthy volunteers were imaged using a multicontrast carotid MRI protocol to characterize arterial wall morphology and composition. Normalized wall index (wall area∕total vessel area), maximum wall thickness and, if present, percentages of wall area occupied by calcification and lipid-rich necrotic core were determined. A multiple-slice CINE MR sequence was added to the multicontrast protocol to measure the distensibility coefficient (DC) at several locations spanning the bifurcation. The intraclass correlation coefficient (ICC) and the coefficient of variation were used to assess the reproducibility of DC measurements made on the healthy subjects. The DC was compared between arterial segments and between the healthy and diseased groups. Furthermore, within the diseased group, DC was correlated to plaque morphology and composition at each location as well as that averaged over the plaque. RESULTS: Distensibility measurements were highly reproducible: ICC (95% confidence interval) was 0.998 (0.96-1.0) for the common carotid segment and 0.990 (0.92-1.0) for the internal carotid segment. In healthy volunteers, we found significantly higher distensibility in the common segment of the carotid artery compared to the internal carotid segment (mean ± SD = 4.56 ± 1.02 versus 3.56 ± 1.32 × 10(-5)∕Pa; p < 0.05). However, no segmental differences were seen in the diseased group (3.25 ± 1.84 versus 3.26 ± 1.60 × 10(-5)∕Pa; p = 0.607). Location-to-location changes in DC were not found to correlate to changes in the local plaque morphology or composition nor were average DC found to be associated with aggregate plaque features. CONCLUSIONS: These results demonstrate the feasibility of MRI to measure distensibility in the carotid artery and to presumably detect changes in distensibility due to age and∕or disease. The results suggest that the effect of atherosclerosis on local distensibility may not strongly depend upon the specific underlying plaque features in mild to moderate stenotic carotid lesions though more diffuse or nonlocal changes in arterial distensibility could not be ruled out.

Sustained acceleration in carotid atherosclerotic plaque progression with intraplaque hemorrhage: a long-term time course study.

OBJECTIVES: This study sought to determine the immediate and long-term effects of intraplaque hemorrhage (IPH) on plaque progression in the carotid artery. BACKGROUND: Previous studies have associated IPH in the carotid artery with more rapid plaque progression. However, the time course and long-term effect remain unknown. Carotid magnetic resonance imaging is a noninvasive imaging technique that has been validated with histology for the accurate in vivo detection of IPH and measurement of plaque burden. METHODS: Asymptomatic subjects with 50% to 79% carotid stenosis underwent carotid magnetic resonance imaging at baseline and then serially every 18 months for a total of 54 months. Subjects with IPH present in at least 1 carotid artery at 54 months were selected. Subsequently, presence/absence of IPH and wall volume were determined independently in all time points for both sides. A piece-wise progression curve was fit by using a linear mixed model to compare progression rates described as annualized changes in wall volume between periods defined by their relationship to IPH development. RESULTS: From 14 subjects who exhibited IPH at 54 months, 12 arteries were found to have developed IPH during the study period. The progression rates were -20.5 ± 13.1, 20.5 ± 13.6, and 16.5 ± 10.8 mm(3)/year before, during, and after IPH development, respectively. The progression rate during IPH development tended to be higher than the period before (p = 0.080) but comparable to the period after (p = 0.845). The progression rate in the combined period during/after IPH development was 18.3 ± 6.5 mm(3)/year, which indicated significant progression (p = 0.008 compared with a slope of 0) and was higher than the period before IPH development (p = 0.018). No coincident ischemic events were noted for new IPH. CONCLUSIONS: The development of IPH posed an immediate and long-term promoting effect on plaque progression. IPH seems to alter the biology and natural history of carotid atherosclerosis. Early identification of patients with IPH may prove invaluable in optimizing management to minimize future sequelae.