Dataset Information

Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants.

ABSTRACT: The phenotypic heterogeneity of Lamin A/C (LMNA) variants renders it difficult to classify them. As a consequence, many LMNA variants are classified as variant of unknown significance (VUS). A number of studies reported different types of visible nuclear abnormalities in LMNA-variant carriers, such as herniations, honeycomb-like structures and irregular Lamin staining. In this study, we used lamin A/C immunostaining and nuclear DAPI staining to assess the number and type of nuclear abnormalities in primary dermal fibroblast cultures of laminopathy patients and healthy controls. The total number of abnormal nuclei, which includes herniations, honeycomb-structures, and donut-like nuclei, was found to be the most discriminating parameter between laminopathy and control cell cultures. The percentage abnormal nuclei was subsequently scored in fibroblasts of 28 LMNA variant carriers, ranging from (likely) benign to (likely) pathogenic variant. Using this method, 27 out of 28 fibroblast cell cultures could be classified as either normal (n?=?14) or laminopathy (n?=?13) and no false positive results were obtained. The obtained specificity was 100% (CI 40-100%) and sensitivity 77% (46-95%). We conclude that assessing the percentage of abnormal nuclei is a quick and reliable method, which aids classification or confirms pathogenicity of identified LMNA variants causing formation of aberrant lamin A/C protein.

SUBMITTER: van Tienen FHJ

PROVIDER: S-EPMC6460565 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants.

van Tienen Florence H J FHJ Lindsey Patrick J PJ Kamps Miriam A F MAF Krapels Ingrid P IP Ramaekers Frans C S FCS Brunner Han G HG van den Wijngaard Arthur A Broers Jos L V JLV

European journal of human genetics : EJHG 20181112 3

The phenotypic heterogeneity of Lamin A/C (LMNA) variants renders it difficult to classify them. As a consequence, many LMNA variants are classified as variant of unknown significance (VUS). A number of studies reported different types of visible nuclear abnormalities in LMNA-variant carriers, such as herniations, honeycomb-like structures and irregular Lamin staining. In this study, we used lamin A/C immunostaining and nuclear DAPI staining to assess the number and type of nuclear abnormalities ...[more]

PMID: 30420677

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Project description:Genetic variants in low-density lipoprotein receptor (LDLR) are known to cause familial hypercholesterolemia (FH), occurring in up to 1 in 200 people (Youngblom E. et al. 1993 and Nordestgaard BG et al. 34:3478-3490a, 2013) and leading to significant risk for heart disease. Clinical genomics testing using high-throughput sequencing is identifying novel genomic variants of uncertain significance (VUS) in individuals suspected of having FH, but for whom the causal link to the disease remains to be established (Nordestgaard BG et al. 34:3478-3490a, 2013). Unfortunately, experimental data about the atomic structure of the LDL binding domains of LDLR at extracellular pH does not exist. This leads to an inability to apply protein structure-based methods for assessing novel variants identified through genetic testing. Thus, the ambiguities in interpretation of LDLR variants are a barrier to achieving the expected clinical value for personalized genomics assays for management of FH. In this study, we integrated data from the literature and related cellular receptors to develop high-resolution models of full-length LDLR at extracellular conditions and use them to predict which VUS alter LDL binding. We believe that the functional effects of LDLR variants can be resolved using a combination of structural bioinformatics and functional assays, leading to a better correlation with clinical presentation. We have completed modeling of LDLR in two major physiologic conditions, generating detailed hypotheses for how each of the 1007 reported protein variants may affect function. KEY MESSAGES: • Hundreds of variants are observed in the LDLR, but most lack interpretation. • Molecular modeling is aided by biochemical knowledge. • We generated context-specific 3D protein models of LDLR. • Our models allowed mechanistic interpretation of many variants. • We interpreted both rare and common genomic variants in their physiologic context. • Effects of genomic variants are often context-specific.

Project description:The LMNA gene encodes lamin A and lamin C, which play important roles in nuclear organization. Pathogenic variants in LMNA cause laminopathies, a group of disorders with diverse phenotypes. There are two main groups of disease-causing variants: missense variants affecting dimerization and intermolecular interactions, and heterozygous substitutions activating cryptic splice sites. These variants lead to different disorders, such as dilated cardiomyopathy and Hutchinson-Gilford progeria (HGP). Among these, the phenotypic terms for LMNA-associated cardiocutaneous progeria syndrome (LCPS), which does not alter lamin A processing and has an older age of onset, have been described. Here, we present the workup of an LMNA variant of uncertain significance, NM_170707.2 c. 4G>A, p.(Glu2Lys), in a 36-year-old female with severe calcific aortic stenosis, a calcified mitral valve, premature aging, and a family history of similar symptoms. Due to the uncertainty of in silico predictions for this variant, an assessment of nuclear morphology was performed using the immunocytochemistry of stable cell lines to indicate whether the p.(Glu2Lys) had a similar pathogenic mechanism as a previously described pathogenic variant associated with LCPS, p.Asp300Gly. Indirect immunofluorescence analysis of nuclei from stable cell lines showed abnormal morphology, including lobulation and occasional ringed nuclei. Relative to the controls, p.Glu2Lys and p.Asp300Gly nuclei had significantly (p < 0.001) smaller average nuclear areas than controls (mean = 0.10 units, SD = 0.06 for p.Glu2Lys; and mean = 0.09 units, SD = 0.05 for p.Asp300Gly versus mean = 0.12, SD = 0.05 for WT). After functional studies and segregation studies, this variant was upgraded to likely pathogenic. In summary, our findings suggest that p.Glu2Lys impacts nuclear morphology in a manner comparable to what was observed in p.Asp300Gly cells, indicating that the variant is the likely cause of the LCPS segregating within this family.

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Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants.

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Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants.

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