Genomic and dental investigations of individuals and families with non-syndromic cleft lip and/ or cleft palate, Van der Woude and popliteal pterygium syndromes

Van der Woude syndrome (VWS) is the most common oral cleft syndrome, accounting for two percent of all cleft lip and/or cleft palate (CL/P) cases. The main characteristics of VWS are lip pits (LP), cleft lip (CL), cleft lip and palate (CLP), cleft palate (CP) and/or hypodontia (H). Popliteal pterygium syndrome (PPS) has the same orofacial characteristics as VWS, combined with systemic anomalies. In 2002, the gene responsible for VWS/ PPS was found to be the interferon regulatory factor 6 gene (IRF6), located on chromosome 1, regio q32.2 The gene encodes for a transcription factor containing both a DNA binding domain and a protein binding domain. Non-syndromic (NS) cleft lip with or without cleft palate (CL±P) occurs without associated malformations in any other organs in approximately 70% of the affected subjects. The inheritance pattern for NSCL/P is complex, with a probable co-segregation of several genes and environmental factors. The IRF6 gene has been proposed to be part of the cause of NSCL/P. The aim of this thesis was to investigate individuals and families, mostly of Swedish and Finnish origin, with NSCL/P, VWS and PPS, with regard to their phenotypes, including dental anomalies, to detect mutations of the IRF6 gene in the syndromic cases and to investigate whether the IRF6 gene is responsible for the cleft phenotype in the nonsyndromic individuals. In Study I, 129 individuals affected with NS unilateral (U) CL±P were analysed for dental characteristics. Malformed lateral incisors were common in NSUCL, while hypodontia was more common in the NSUCLP phenotype. In the total material, hypodontia was found in 29.5% inside and in 15.5% outside the region of the cleft. Most of the existing lateral incisors were positioned distal to the cleft in both the primary and the permanent dentition. Study II revealed IRF6 gene mutation in 59% of the 17 VWS/ PPS families studied using direct sequencing of all exons of the gene. In Study III, the IRF6 gene was investigated in 17 Swedish NSCL/P families, using direct sequencing of the gene, in one affected and one healthy individual of each family. We could not detect any mutation in the protein-coding region of the gene. However, two noncoding SNPs – rs861019, a non-coding polymorphism in exon 2, and rs7552506, located in intron 3 – showed an association with the NSCLP phenotype. In Study IV, we tested two SNPs of IRF6, rs642961 in the promoter and rs2235371 in exon 7 (Val274Ile), for association with our entire sample set of NSCL/P, VWS and PPS families (119 families). In all but the Finnish VWS/ PPS families, the “A” allele of rs642961 was identified as a risk allele; transmission to an affected child occurred in a large majority on the same chromosome as the detected IRF6 mutation. The SNP rs642961, located in the AP-2a binding site in the promoter of the IRF6 gene, has previously been shown to be associated with NSCL±P but our results do not support this. However, we did find a significant risk (p=0.013) for transmission of the G-C haplotype (rs642961-rs2235371) to affected individuals in the NSCP subgroup of Swedish families. Of the 16 VWS/ PPS families found to have a mutation in IRF6 (Studies II and IV), 31% had a de novo mutation, that is, a mutation occurring in the proband only and not in the healthy parents. To conclude, NSCL/P is a complex anomaly, where disturbed dental development is a frequent finding. NSCL/P is not dependent on a single gene, as in Mendelian inherited VWS. Dividing our material into sub-phenotypes resulted in rather small groups, but we did find a significant risk with a haplotype of IRF6 in the NSCP group, and also an association for two SNPs of the IRF6 gene with NSCLP. Our results on NSCL/P emphasize the need for additional evaluation of the IRF6 gene and other genes/modifiers, to further clarify their roles in the development of the NSCL/P phenotype

Taurodontism in the first permanent molars in Van der Woude syndrome compared to isolated cleft palate

Objectives: To analyse prevalence, pattern, and severity of taurodontism in individuals with Van der Woude syndrome (VWS) exhibiting cleft palate and compare with aged-matched non-syndromic cleft palate (NSCP) and non-cleft controls. Materials and methods: One hundred and seventy-eight dental panoramic tomographs (DPTs) (105 girls and 73 boys) consisting of 42 VWS patients (x = 8.55 +/- 1.02 years), 42 NSCP patients (x = 8.59 +/- 1.02 years), and 94 normative non-cleft children (x = 8.79 +/- 1.16 years) were assessed and their first permanent molars evaluated. Measurement 3 of the taurodontism index developed by Shifman and Chanannel with the Tulensalo modification was used. Prevalence, pattern, and severity were compared between groups. Statistical differences were determined by one-way analysis of variance and Fisher test. Repeatability was calculated by Cohens Kappa test. Results: The prevalence of taurodontic molars was 59.5% in VWS, 45.2% in NSCP, and 26.6% in non-cleft controls.The prevalence and severity of taurodontism in VWS and NSCP were significantly higher than in non-cleft children in all first permanent molars. There was no significant difference in prevalence and severity between VWS and NSCP. The odds for having taurodontism in the VWS group was approximately double compared to the NSCP group. Most of the taurodontic molars showed hypotaurodontism and taurodontism occurred bilaterally more frequently than unilaterally. Conclusion: This study shows a higher prevalence of taurodontism in VWS and NSCP. Most taurodontic molars are hypotaurodontic and most occur bilaterally.Peer reviewe

Dominant Mutations in GRHL3 Cause Van der Woude Syndrome and Disrupt Oral Periderm Development

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6+/−;Grhl3+/−) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS

Dominant Mutations in GRHL3 Cause Van der Woude Syndrome and Disrupt Oral Periderm Development

Peer reviewe

Key outcomes in childbirth : Development of a perinatal core outcome set for management of labor and delivery at or near term

Introduction: Consistency and relevance of perinatal outcome measures are necessary basics for obstetric research, audit, and clinical counseling. Still, there is an unwarranted variation in reported perinatal outcomes, which impairs research synthesis, validity, and implementation, as well as clinical benchmarking and longitudinal comparisons. The aim of this study was to develop a short-term perinatal (fetal and neonatal) Core Outcome Set to be used in research and quality assurance of management of labor and delivery at or near term. Material and methods: The methods were guided by the Core Outcome Measures in Effectiveness Trials Initiative Handbook. The project was prospectively registered on July 2, 2020 in the Core Outcome Measures in Effectiveness Trials (COMET) data base (reference number 1593). A list of potential outcomes was created based on a systematic review of studies evaluating interventions for peripartum management at or near term (>= 34 weeks of gestation), including decisions regarding timing and type of onset of labor, intrapartum care, and mode of delivery. The list was entered into a two-round Delphi survey with predefined consensus criteria. Participants (n = 67) included clinicians, researchers, lay persons with experience of childbirth (patient representatives), and other stakeholders. A consensus meeting was held to reach a final agreement. Results: Response rates were 82.1% (55/67) and 92.7% (51/55) for the first and second Delphi rounds, respectively. In total, 17 outcomes were included in the final core outcome set, reflecting mortality, health or morbidity, including asphyxia, central nervous system status, infection, neonatal resuscitation and admission, breastfeeding and mother-infant interaction, operative delivery due to fetal distress, as well as birthweight and gestational age. Two of these outcomes were suggested by patient representatives. Conclusions: The Swedish Perinatal Core Outcome Set (SPeCOS) study involved a broad circle of relevant stakeholders and reached consensus on a minimal set of perinatal outcomes that should be collected and reported in a standardized way in all future studies on management of labor and delivery at or near term, regardless of the specific population or condition studied. This could improve obstetric research, evidence synthesis, uptake, implementation, and adherence, as well as clinical practice, audit, and comparisons in childbirth care

Validation of reported dentoalveolar relationships in the Swedish Quality Registry for Cleft Lip and Palate

Objectives: The present study validated data that had been reported to the Swedish Quality Registry for Cleft Lip and Palate (CLP) under new requirements from 2016, when use of the 5-year-old (5YO) and the Modified Huddart and Bodenham (MHB) indices for rating occlusion in children born with unilateral CLP (UCLP) was introduced. Materials and methods: The sample included blinded study casts (n = 97) and photos (n = 4) of 5-year-old children who had been born with UCLP in 2009−2011 and were enrolled at one of six cleft centres in Sweden. Fourteen orthodontists from the centres assessed the patients (n = 101) using the 5YO and the MHB indices. Median 5YO and MHB scores of the 14 assessments were compared with original registry data (n = 61). Each centre devised code keys to protect the identities of their patients in the registry. Results: Interrater agreement among the 14 orthodontists was good for the 5YO index (quadratic-weighted kappa: 0.72−0.92) and the MHB index (intraclass correlation coefficient: 0.991−0.994). Comparisons of median 5YOs for each identifiable child with their registry data (n = 61) found total agreement for 70.5 per cent. Comparisons between median MHBs and registry data showed very good or good agreement in 93.4 per cent of the cases. Limitations: Two teams lost their code keys, which reduced the sample to 61 patients. Conclusions: The dentoalveolar outcome data in the CLP registry was trustworthy. There was good agreement among the Swedish cleft teams assessing the 5YO and MHB indices in children born with UCLP at age 5 years