Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes

Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes

Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel <i>SYT1</i> and <i>DDR2</i> Rearrangements

<div><p>Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including <i>MDM2</i>, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (<i>NAV3</i>, <i>SYT1</i>, <i>PAWR</i>) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified <i>DDR2</i>, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as <i>MDM2</i> and <i>DDR2</i>.</p></div

GeneGo pathway analysis of genes containing SNV, gene fusions or amplifications.

<p>GeneGo pathway analysis of genes containing SNV, gene fusions or amplifications.</p

Circos plot of validated genetic variation in a well-differentiated liposarcoma.

<p>Inner-most circle contains validated structural rearrangements of fusion genes with translocations indicated in purple, and intra-chromosomal rearrangements indicated in orange. The middle ring contains the aCGH plot with copy number loss indicated in green and copy number gain in red; each orange ring corresponds to a log2 value of 1. The outer-most ring indicates validated, damaging single nucleotide variants.</p

Validated functionally damaging SNV.

<p>Validated functionally damaging SNV.</p

Depiction of genomic rearrangement hotspot on chromosome 12.

<p>We identified and further characterized a putative transposable element (LOC100507498) located on the (-) strand, within the PAWR-SYT1-NAV3 gene cluster (<b>3A</b>). The LOC100507498 and closely related sequences were characterized by comparing both nucleotide (<b>3B</b>,top) and translated (<b>3B</b>,bottom) sequences to known families of repetitive elements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#s2" target="_blank">Methods</a>). Highly conserved sequence domains/motifs are color coded by known families of repetitive elements (Legend). Overall, these sequences exhibited the highest similarity to the L1 retrotransposon and Alu repeat elements (domain hit counts and similarity score). Sequence alignments of LOC100507498 (*) with known L1 elements <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Pickeral1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Goodier1" target="_blank">[33]</a> exhibited the highest overall homology to Class 3 L1 elements as described by Pickeral et al. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone-0087113-t001" target="_blank">Table 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Pickeral1" target="_blank">[32]</a>) and in addition to the 5′-GGAG and 3′-AATA signature motifs, LOC100507498 carries several ‘AATGTTTA’ motifs that suggest multiple rounds of L1-mediated transduction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Goodier1" target="_blank">[33]</a>. The LOC100507498 locus resides within a genomic region that is deleted in the Tumor (T) sample, but present in the Normal (N) genome (<b>3C</b>).</p

DNA validated fusion genes.

<p>DNA validated fusion genes.</p