Rare novel variants in the ZIC3 gene cause X-linked heterotaxy

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Rare novel variants in the ZIC3 gene cause X-linked heterotaxy

Variants in the ZIC3 gene are rare, but have demonstrated their profound clinical significance in X-linked heterotaxy, affecting in particular male patients with abnormal arrangement of thoracic and visceral organs. Several reports have shown relevance of ZIC3 gene variants in both familial and sporadic cases and with a predominance of mutations detected in zinc-finger domains. No studies so far have assessed the functional consequences of ZIC3 variants in an in vivo model organism. A study population of 348 patients collected over more than 10 years with a large variety of congenital heart disease including heterotaxy was screened for variants in the ZIC3 gene. Functional effects of three variants were assessed both in vitro and in vivo in the zebrafish. We identified six novel pathogenic variants (1,7%), all in either male patients with heterotaxy (n=5) or a female patient with multiple male deaths due to heterotaxy in the family (n=1). All variants were located within the zinc-finger domains or leading to a truncation before these domains. Truncating variants showed abnormal trafficking of mutated ZIC3 proteins, whereas the missense variant showed normal trafficking. Overexpression of wild-type and mutated ZIC protein in zebrafish showed full non-functionality of the two frame-shift variants and partial activity of the missense variant compared with wild-type, further underscoring the pathogenic character of these variants. Concluding, we greatly expanded the number of causative variants in ZIC3 and delineated the functional effects of three variants using in vitro and in vivo model systems

Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants

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