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Genetics
2017 Jan 21;2081:283-296. doi: 10.1534/genetics.117.300535.
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Identification of Isthmin 1 as a Novel Clefting and Craniofacial Patterning Gene in Humans.
Lansdon LA, Darbro BW, Petrin AL, Hulstrand AM, Standley JM, Brouillette RB, Long A, Mansilla MA, Cornell RA, Murray JC, Houston DW, Manak JR.
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Orofacial clefts are one of the most common birth defects, affecting 1-2 per 1000 births, and have a complex etiology. High-resolution array-based comparative genomic hybridization has increased the ability to detect copy number variants (CNVs) that can be causative for complex diseases such as cleft lip and/or palate. Utilizing this technique on 97 nonsyndromic cleft lip and palate cases and 43 cases with cleft palate only, we identified a heterozygous deletion of Isthmin 1 in one affected case, as well as a deletion in a second case that removes putative 3' regulatory information. Isthmin 1 is a strong candidate for clefting, as it is expressed in orofacial structures derived from the first branchial arch and is also in the same "synexpression group" as fibroblast growth factor 8 and sprouty RTK signaling antagonist 1a and 2, all of which have been associated with clefting. CNVs affecting Isthmin 1 are exceedingly rare in control populations, and Isthmin 1 scores as a likely haploinsufficiency locus. Confirming its role in craniofacial development, knockdown or clustered randomly interspaced short palindromic repeats/Cas9-generated mutation of isthmin 1 in Xenopus laevis resulted in mild to severe craniofacial dysmorphologies, with several individuals presenting with median clefts. Moreover, knockdown of isthmin 1 produced decreased expression of LIM homeobox 8, itself a gene associated with clefting, in regions of the face that pattern the maxilla. Our study demonstrates a successful pipeline from CNV identification of a candidate gene to functional validation in a vertebrate model system, and reveals Isthmin 1 as both a new human clefting locus as well as a key craniofacial patterning gene.
Figure 4: In situ hybridization of ism1 expression in late stage Xenopus laevis embryos.
Embryos are shown in a lateral (A-D) or anterior (A’-D’) view with the cement gland (cg) labeled for ventral orientation. (A) Stage 28 embryo showing strong expression in the branchial arches (ba), midbrain-hindbrain boundary (mhb), ear placode (e), and tailbud (tb) with the yellow arrowheads marking the primitive mouth. (B) Stage 33 embryos showing decreased expression in the branchial arches (ba), midbrain-hindbrain boundary (mhb) and tailbud (tb), with concentrated expression surrounding the primitive mouth (arrowhead) and expression in the somites (s). (C-D) Stage 37 and 39 embryos showing continued concentrated ism1 expression surrounding the primitive mouth (arrowhead).
Figure 6: Morpholino knockdown of ism1 in Xenopus laevis embryos results in clefting phenotypes. (A) Quantification of embryos injected with 24ng control morpholino (Ctrl MO) versus 24ng ism1 translation-blocking (ATG) MO shows craniofacial anomalies and clefting phenotypes in the ism1-altered group. (B) Faces of stage 43 embryos which are uninjected (top) or injected with 24ng ism1 ATG MO and exhibiting a cleft (bottom). Mouths have been outlined in red. (C) Phalloidin (red) and E-cadherin (green) staining to detect cell boundaries and epithelial cells, respectively, of the mouth of an uninjected (top) or 24ng ism1 ATG MO injected
(bottom) embryos.
Figure 7: Expression of lhx8 decreases with knockdown of ism1. Embryos are shown in an anterior view with the cement gland (cg) labeled for ventral orientation. Control uninjected (Un) or embryos pricked with the injection needle on half (HP) show strong lhx8 expression in the first branchial arch at stage 28 (A-D) surrounding the primitive mouth (yellow arrowhead) as well as in the maxillary prominences (green arrowhead). Knockdown of ism1 with 12ng ATG MO in half of the animal results in undetectable (E) or decreased (F) branchial arch expression of lhx8, especially in the maxillary prominence.
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