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Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.

The craniofacial skeleton is made up of the neurocranium, which encases the brain, and the viscerocranium, which includes the bones of the face and cranial base. At birth, the human craniofacial skeleton is comprised of 44 bones that develop via both endochondral and intramembranous ossification, depending on the bone. Undifferentiated mesenchyme separates the bones for varying periods of postnatal life, playing a key role in regulating cranioskeletal growth. Appositional bone growth at the suture margins is regulated by a complex crosstalk between growth factor signaling pathways, some of which function more prominently in specific sutures. In this chapter, we review the embryology and current understanding of the molecular genetic basis of premature fusions (synostosis) of both the calvarial and facial sutures gleaned from genetic studies in humans and model organisms such as the mouse. In addition, we reassess the spectrum of mutations found in FGFR2, the most prominent factor implicated in craniofacial synostosis, and propose a new model for explaining the impact of many of the common amino acid substitutions that result in pathology. Finally, advancements that promise to enable use of mesenchymal stem cells in tissue engineering-based approaches are discussed with respect to prospects for more effective treatment of these craniofacial conditions.

Auriculocondylar syndrome (ACS) is a rare, autosomal-dominant craniofacial malformation syndrome characterized by variable micrognathia, temporomandibular joint ankylosis, cleft palate, and a characteristic “question-mark” ear malformation. Careful phenotypic characterization of severely affected probands in our cohort suggested the presence of a mandibular patterning defect resulting in a maxillary phenotype (i.e., homeotic transformation). We used exome sequencing of five probands and identified two novel (exclusive to the patient and/or family studied) missense mutations in PLCB4 and a shared mutation in GNAI3 in two unrelated probands. In confirmatory studies, three additional novel PLCB4 mutations were found in multigenerational ACS pedigrees. All mutations were confirmed by Sanger sequencing, were not present in more than 10,000 control chromosomes, and resulted in amino-acid substitutions located in highly conserved protein domains. Additionally, protein-structure modeling demonstrated that all ACS substitutions disrupt the catalytic sites of PLCB4 and GNAI3. We suggest that PLCB4 and GNAI3 are core signaling molecules of the endothelin-1-distal-less homeobox 5 and 6 (EDN1-DLX5/DLX6) pathway. Functional studies demonstrated a significant reduction in downstream DLX5 and DLX6 expression in ACS cases in assays using cultured osteoblasts from probands and controls. These results support the role of the previously implicated EDN1-DLX5/6 pathway in regulating mandibular specification in other species, which, when disrupted, results in a maxillary phenotype. This work defines the molecular basis of ACS as a homeotic transformation (mandible to maxilla) in humans.

Publisher Summary The regulation of heme biosynthesis in animals has been a topic of interest for many years. It is generally agreed that the first enzyme of the heme pathway, 5-aminolevulinate synthase, determines the rate of heme biosynthesis and the regulation of this enzyme is, therefore, a major focus of this chapter. The heme biosynthetic pathway is present in all cell types except mature erythrocytes. The final step of heme biosynthesis occurs in mitochondria and the heme is then utilized for the formation of different hemoproteins located in mitochondria, microsomes, peroxisomes, the cytosol, and probably the nucleus. Heme can control the biosynthesis of some proteins. In erythroid cells, heme controls the translation of proteins, notably α- and β-globin chains, by modulating the activity of a specific kinase. All nucleated animal cells must synthesize heme for incorporation into respiratory cytochromes, but erythroid and liver cells have the highest rates of heme synthesis. Erythroid cells synthesize about 90% of the total heme in the body for assembly into hemoglobin. Although the bulk of heme in the liver is made in situ , the liver may also obtain some heme from serum haptoglobin–hemoglobin and heme–hemopexin complexes, following intravascular hemolysis. All enzymes of the heme biosynthetic pathway, except for protoporphyrinogen oxidase, have been cloned from higher vertebrates. The genes encoding these enzymes are located on different chromosomes.