All PhD Theses

K.D. Khandelwal

Genetic mechanisms of orofacial clefting


A scientific essay in Medical Sciences

DOCTORAL THESIS defended in public on 26th of June 2018


Orofacial clefts (OFCs) are congenital structural anomalies of the lip, the alveolus and/or the palate, which affect around 1 in 700 live births. They are characterized by incomplete formation and/or fusion of structures separating the oral and the nasal cavities. OFCs have a complex etiology with multiple genetic and environmental factors that can lead to the disruption of normal embryonic development of the lip and palate. This thesis aimed to use novel genomic approaches to decipher the genetics of OFC.

Chapter 1 is a brief introduction about the normal development of the lip and palate structures in humans, underlying cellular processes and molecular mechanisms, and how perturbations in these mechanisms result in orofacial abnormalities. The two forms of OFCs, syndromic and nonsyndromic and their genetic models are then discussed. Finally, the chapter gives the outline of the thesis, highlighting the various approaches used to decipher the genetics of OFC.

Chapter 2 reviews the various genomic approaches used for studying OFC. It discusses the genomewide approaches used in genetic studies of OFC including copy number variations (CNVs), genomewide linkage studies, genome-wide association studies (GWAS), exome sequencing (ES), and the identification of genetic variants in non-coding regulatory elements. The applicability of these approaches and future perspectives of genomic studies in OFCs are also discussed. 

Chapter 3 describes a study to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy-number variants were assessed in three OFC families using microarray analysis. A cohort of 1072 individuals affected with OFC and 706 population-based controls were analysed using Molecular Inversion Probes (MIPs). Partial deletions of TP63 were identified in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant (p.Ser189Serfs*6) and a nonsense variant (p.Gln333*) that introduces a premature stop codon in the DNA-binding domain. In addition, the first missense variants were identified in the oligomerization domain (p.Val405Met), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant negative. All these variants and deletions uncover a dosage dependent function of p63 and a loss-offunction mechanism behind nonsyndromic OFC. These findings suggest modifications to the previous model that loss-of-function alleles in TP63 do not give rise to any clinical phenotype. 

Chapter 4 describes a study that aims at identifying novel IRF6 mutations in orofacial cleft patients. Targeted multiplex sequencing using MIPs was performed in 1072 OFC patients, 67 patients with tooth agenesis (TA), and 706 controls, which included IRF6 and other genes as well. Three potentially pathogenic de novo mutations of IRF6 (Pro380Gln, Asp225His and Arg9Trp) were identified in OFC patients. In addition, three rare missense variants were identified (Phe375Ser, Thr129Leufs*3 and Arg35Cys), for which pathogenicity could not unequivocally be shown. Retrospective investigation of the patients with these variants revealed the presence of lip pits in one of the patients with a de novo mutation (Asp225His) suggesting a Van der Woude syndrome phenotype, while in other patients lip pits were identified. These findings add to the increasing evidence that rare IRF6 variants play a role in the etiology of nonsyndromic OFCs. Moreover, this study highlights the need for deep clinical phenotyping and medical history of families that present at genetic counselling and the advantage of a “genotype first” approach when studying large patient cohorts.

Chapter 5 describes a study that aims to identify a novel genetic cause of TA and/or OFC by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients. WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. A frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), respectively, in the patient with TA and OFC and in the patient with severe TA only. After deleterious mutations were identified in LRP6, all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo. This study showed that LRP6 mutations cause severe TA with or without other dental anomalies such as ankylosis, enamel defects, and tooth-shape anomalies. However, the genetic data do not provide evidence that rare monogenic LRP6 mutations underlie nonsyndromic orofacial clefts. 

Chapter 6 reports the identification of a de novo variant in CHUK in a patient with an EEC/AEC syndromelike phenotype and additional hypogammaglobulinemia. Neither pathogenic mutations in TP63 nor CNVs at the TP63 locus were identified. Exome sequencing revealed de novo heterozygous variants in CHUK, PTGER4, and IFIT2. The variant in CHUK appeared to be most relevant for the EEC/AEC-like phenotype. CHUK is a direct target gene of p63 and encodes a component of the IKK complex that plays a key role in NF-κB pathway activation. The identified CHUK variant (g.101980394T>C; c.425A>G; p.His142Arg) was located in the kinase domain which is responsible for the phosphorylation activity of the protein. The variant may affect CHUK function and thus contribute to the disease phenotype in three ways: 1) the variant exhibits a dominant negative effect and results in an inactive IKK complex that affects the canonical NF-κB pathway; 2) it affects the feedback loop of the canonical and noncanonical NF-κB pathways that are CHUK kinase activity-dependent; 3) it disrupts NF-κB independent epidermal development that is often p63-dependent. The difference in zygosity of the mutations and potentially affected pathways could explain the less severe ectodermal dysplasia phenotype and the additional immune-related phenotype in the present patient, as compared to Cocoon syndrome and Bartsocas-Papas syndrome, which are caused by recessively manifesting CHUK mutations. The PTGER4 variant could have a role, alone or in combination with the CHUK variant, in the immune and growth-related phenotypes present in the patient. Further functional analysis of this variant would be necessary to confirm its contribution to the observed phenotypes in the patient. These findings support the testing of CHUK for mutations in patients with the combination of EEC/AEC syndrome-like and immune-related phenotypes, such as one presented here to affirm the role of CHUK further. 

Chapter 7 presents a systematic genome-wide analysis to identify functional variants in p63-bound non-coding regulatory elements involved in orofacial clefting. p63 binding sites containing a common polymorphism in the core p63 recognition motif were selected based on four criteria: (I) Motifs that are in proximity to the OFC-related genes, (II) Motifs that overlap with the IRF6 binding sites, (III) Motifs that lie in the OFC-associated GWAS loci, and (IV) Motifs that lie upstream to ARHGAP29/ in ABCA4 gene. Transactivation assays revealed 14 binding sites responsive to p63. Four of these p63 binding sites, which were either co-bound by IRF6 or present in an OFC-associated locus, were also tested in a transgenic zebrafish reporter assay to assess how they drive developmental gene expression. One binding site in ABCA4, BS abca4.5, was found to be active in neural tissues and in ventral cells that will form facial structures in zebrafish. The data presented in this report provides a list of p63 binding sites which could act at non-coding regulatory elements controlling the expression of novel or known OFC genes. The variants present in such binding sites could disrupt the ability of p63 to recognize the DNA and its binding and hence, the regulation of distantly or nearby located OFC-related genes.

Chapter 8 is a general discussion of the major contributions of the work, methodological problems and the limitations, and future perspectives.