Thymopoietin or TMPO (indicated by its alternative gene symbol, LAP2, in this work) has been proposed as a candidate disease gene for dilated cardiomyopathy (DCM), since a LAP2 product associates with nucleoplasmic lamins A/C, which are encoded by the DCM gene LMNA. We developed a study to screen for genetic mutations in LAP2 in a large collection of DCM patients and families. A total of 113 subjects from 88 families (56 with familial DCM (FDC) and 32 with sporadic DCM) were screened for LAP2 mutations using denaturing high-performance liquid chromatography and sequence analysis. We found a single putative mutation affecting the LAP2alpha isoform in one FDC pedigree. The mutation predicts an Arg690Cys substitution (c.2068C>T; p.R690C) located in the C-terminal domain of the LAP2alpha protein, a region that is known to interact with lamin A/C. RT-PCR, Western blot analyses, and immunolocalization revealed low-level LAP2alpha expression in adult cardiac muscle, and localization to a subset of nuclei. Mutated Arg690Cys LAP2alpha expressed in HeLa cells localized to the nucleoplasm like wild-type LAP2alpha, with no effect on peripheral and nucleoplasmic lamin A distribution. However, the in vitro interaction of mutated LAP2alpha with the pre-lamin A C-terminus was significantly compromised compared to the wild-type protein. LAP2 mutations may represent a rare cause of DCM. The Arg690Cys mutation altered the observed LAP2alpha interaction with A-type lamins. Our finding implicates a novel nuclear lamina-associated protein in the pathogenesis of genetic forms of dilated cardiomyopathy.

X-linked dilated cardiomyopathy (XLCM) was first described in 1987 and associated with dystrophin gene (DMD) mutations a decade later in one of the original two families. Here we report long-term follow-up of the second family (XLCM-2), for which a DMD mutation was never found. Analysis of the lysosome-associated membrane protein-2 (LAMP-2) gene detected a novel mutation, confirming a diagnosis of Danon disease. The broad phenotype in this family included dilated and hypertrophic cardiomyopathy, cardiac pre-excitation, skeletal myopathy with high serum creatine kinase, cognitive impairment (in males), and a pigmentary retinopathy in affected females. Cardiac biopsy specimens showed extensive vacuolar changes in an affected adult male. Remarkably, the skeletal muscle biopsy in a 13-month-old mutation-carrying male showed no vacuolization by standard histology. We conclude that XLCM may be the presenting sign of Danon disease and, in the presence of a familial history of HCM, pre-excitation, skeletal muscle involvement and retinal pigmentary dystrophy should prompt LAMP-2 clinical testing. Furthermore, the absence of vacuolar myopathy in biopsies from young patients may not exclude Danon disease.

SUMMARY Semiautomated methods are used to measure elongated, curved and complex branching profiles and isolated perimeter segments in monochrome video images with a general‐purpose analysis system. These methods are used to make the major primary measurements of bone histomorphometry. Accuracy and reproducibility of the image acquisition, processing and measurement system is documented by measuring a semicircular standard of known dimensions. Semiautomated applications of the Ar/Le method for measuring areas and perimeters, and calculating lengths and widths of osteoid seams, lengths of mineralization labels and mineral apposition rate, wall width, indirect measurements of eroded, osteoclastic and osteoblastic perimeters without tracing, and measurement of mineralized or total cancellous bone area and perimeter gave values comparable to measurements of the same parameters by tracing or grid counting techniques with equal or better reproducibility and much greater efficiency. Intraindividual variation in measuring multiple bone biopsies was comparable to that reported with current standard methods. Major sources of variability for semiautomated methods were image magnification and selection of profile edges by thresholding, and sources of variability for manual methods are image magnification, numbers of orthogonal intercepts, tracing speed and accuracy of the algorithm used to measure traced pixels. Semiautomated methods are accurate, reproducible and rapid methods suitable for bone histomorphometry.

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