Fryns type mesomelic dysplasia of the upper limbs caused by inverted duplications of the HOXD gene cluster

Abstract

The HoxD cluster is critical for vertebrate limb development. Enhancers located in both the telomeric and centromeric gene deserts flanking the cluster regulate the transcription of HoxD genes. In rare patients, duplications, balanced translocations or inversions misregulating HOXD genes are responsible for mesomelic dysplasia of the upper and lower limbs. By aCGH, whole-genome mate-pair sequencing, long-range PCR and fiber fluorescent in situ hybridization, we studied patients from two families displaying mesomelic dysplasia limited to the upper limbs. We identified microduplications including the HOXD cluster and showed that microduplications were in an inverted orientation and inserted between the HOXD cluster and the telomeric enhancers. Our results highlight the existence of an autosomal dominant condition consisting of isolated ulnar dysplasia caused by microduplications inserted between the HOXD cluster and the telomeric enhancers. The duplications likely disconnect the HOXD9 to HOXD11 genes from their regulatory sequences. This presumptive loss-offunction may have contributed to the phenotype. In both cases, however, these rearrangements brought HOXD13 closer to telomeric enhancers, suggesting that the alterations derive from the dominant-negative effect of this digit-specific protein when ectopically expressed during the early development of forearms, through the disruption of topologically associating domain structure at the HOXD locus.

MESOMELIC DYSPLASIAS ASSOCIATED WITH THEHOXDLOCUS ARE CAUSED BY REGULATORY REALLOCATIONS

“…In the case where the duplicated segment would also be inverted, HOXD13 would even be in closer contact with multiple proximal limb enhancers (Supplementary Figure 5b, top). A similar reasoning applies for the two families described in Le Caignec et al (2019). In family 1, the duplicated and inverted copy of HOXD13 is now in close contact with proximal enhancers (Supplementary Figure 5c, top).…”

“…Human mesomelic dysplasias have been associated with the HOXD locus (Fujimoto et al, 1998; Le Caignec et al, 2019; Peron et al, 2018; Spitz et al, 2002; Ventruto et al, 1983)(Figure 1a), but the gene bodies were not affected. Instead, the physical relationship with the flanking regulatory regions was modified, suggesting a potential impact of chromosomal rearrangements upon the long-range regulation of these genes during early limb development (Andrey et al, 2013; Kragesteen et al, 2018; Le Caignec et al, 2019). While the murine Ulnaless X-ray-induced inversion is an excellent proxy for these conditions, the severity of its effects and the early homozygous lethality, perhaps due to a breakpoint in the Lnpk gene (Figure 1a, Ulnaless ), made the use of these mice difficult for further analyses and genome editing.…”

“…In both cases, the duplicated copy of HOXD13 (purple pin) is moved away of the distal enhancers, which continue to activate the native HOXD13 copy, and positioned at the vicinity of the proximal enhancers, certainly leading to its ectopic expression in proximal limbs and concurrent mesomelic dysplasia. (c) In the two families reported by Le Caignec et al (2019), the same explanatory framework can be used. In the first family (top), a double duplication with one inversion brings HOXD13 right next to the proximal limb enhancers, while distal enhancers are still involved in the regulation of the native HOXD13 in distal cells.…”

“…Several human families displaying shortened and bent forearm bones have been reported with large chromosomal rearrangements in the q31 band of chromosome 2, a region containing the HOXD gene cluster (Cho et al, 2010; Kantaputra et al, 2010; Le Caignec et al, 2019; Peron et al, 2018). Although they are correlated, the potential involvement of HOX genes in causing these limb dysmorphias (mesomelic dysplasias) has never been confirmed, despite various studies in mice indicating that some Hox mutations can reproduce this condition.…”

“…(a) The top panel shows a map of the human HOXD locus with the positions of mapped chromosomal rearrangements (black bars) leading to mesomelic dysplasia. The numbers inside the black bars refer to the original references: 1 (Kantaputra et al, 2010), 2 (Peron et al, 2018), 3 (Cho et al, 2010), 4 (Le Caignec et al, 2019). The panels below (grey box) depict the wild type mouse locus (top) and the structure of the Hoxd inν2 inverted allele.…”