Differential Diagnosis between Marfan Syndrome and Loeys–Dietz Syndrome Type 4: A Novel Chromosomal Deletion Covering TGFB2

Marfan syndrome (MFS) and Loeys–Dietz syndrome type 4 (LDS4) are two hereditary connective tissue disorders. MFS displays ectopia lentis as a distinguishing, characterising feature, and thoracic aortic ectasia, aneurysm, dissection, and systemic features as manifestations overlapping with LDS4. LDS4 is characterised by the presence of hypertelorism, cleft palate and/or bifid uvula, with possible ectasia or aneurysms in other arteries. The variable age of onset of clinical manifestations makes clinical diagnosis more difficult. In this study, we report the case of a patient with Marfan syndrome diagnosed at our centre at the age of 33 on the basis of typical clinical manifestations of this syndrome. At the age of 38, the appearance of ectasia of the left common iliac artery and tortuosity of the iliac arteries suggested the presence of LDS4. Next Generation Sequencing (NGS) analysis, followed by Array-CGH, allowed the detection of a novel chromosomal deletion including the entire TGFB2 gene, confirming not only the clinical suspicion of LDS4, but also the clinical phenotype associated with the haploinsufficiency mechanism, which is, in turn, associated with the deletion of the entire gene. The same mutation was detected in the two young sons. This emblematic case confirms that we must be very careful in the differential diagnosis of these two pathologies, especially before the age of 40, and that, in young subjects suspected to be affected by MFS in particular, we must verify the diagnosis, extending genetic analysis, when necessary, to the search for chromosomal alterations. Recently, ectopia lentis has been reported in a patient with LDS4, confirming the tight overlap between the two syndromes. An accurate revision of the clinical parameters both characterising and overlapping the two pathologies is highly desirable.

Quantitative analysis of CRISPR-del for complete gene knockout in human diploid cells

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel- based gene disruption. In this study, we optimized CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform complete gene knockout in non-transformed human diploid RPE1 cells. By improving several procedures, the optimized CRISPR-del allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the ability of this method to introduce a large chromosomal deletion enables the generation of a model cell line having a bi- allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the optimized CRISPR-del is a high-throughput method for performing complete gene knockout in RPE1 cells.

FIP1L1-PDGFRA fusion gene in a patient with asthma and rhinitis

Eosinophilia in asthma or rhinitis is usually considered to be reactive to the allergic diseases. We report a 33-year-old man with asthma and rhinitis, and progressive hypereosinophilia. Fluorescence in situ hybridization analysis detected interstitial chromosomal deletion at 4q12 in cells of the bone marrow. The patient was diagnosed as myeloproliferative neoplasm with a FIP1L1-PDGFRA fusion gene, and successfully treated with the tyrosine kinase inhibitor, imatinib. Clonal expansion of eosinophils due to the FIP1L1-PDGFRA fusion gene could underlie refractory mechanisms in patients with bronchial asthma or allergic rhinitis.