‘Location, Location, Location’: a spatial approach for rare variant analysis and an application to a study on non-syndromic cleft lip with or without cleft palate

Motivation: For the analysis of rare variants in sequence data, numerous approaches have been suggested. Fixed and flexible threshold approaches collapse the rare variant information of a genomic region into a test statistic with reduced dimensionality. Alternatively, the rare variant information can be combined in statistical frameworks that are based on suitable regression models, machine learning, etc. Although the existing approaches provide powerful tests that can incorporate information on allele frequencies and prior biological knowledge, differences in the spatial clustering of rare variants between cases and controls cannot be incorporated. Based on the assumption that deleterious variants and protective variants cluster or occur in different parts of the genomic region of interest, we propose a testing strategy for rare variants that builds on spatial cluster methodology and that guides the identification of the biological relevant segments of the region. Our approach does not require any assumption about the directions of the genetic effects. Results: In simulation studies, we assess the power of the clustering approach and compare it with existing methodology. Our simulation results suggest that the clustering approach for rare variants is well powered, even in situations that are ideal for standard methods. The efficiency of our spatial clustering approach is not affected by the presence of rare variants that have opposite effect size directions. An application to a sequencing study for non-syndromic cleft lip with or without cleft palate (NSCL/P) demonstrates its practical relevance. The proposed testing strategy is applied to a genomic region on chromosome 15q13.3 that was implicated in NSCL/P etiology in a previous genome-wide association study, and its results are compared with standard approaches. Availability: Source code and documentation for the implementation in R will be provided online. Currently, the R-implementation only supports genotype data. We currently are working on an extension for VCF files. Contact: [email protected]

Disruption of neural crest enhancer landscapes as an etiological mechanism for human neurocristopathies

The embryonic development of the human facial features is a highly complex mechanism which requires very exact spatial and temporal regulation of gene expression during neural crest (NC) development. NC cells (NCC) are a transient embryonic cell type with wide differentiation potential that contributes to the formation and morphogenesis of multiple tissues and organs, including many parts of the face. Just like any other cell type, NCC possess a characteristic set of enhancers that, by controlling the expression of specific genes, define cellular identity. Impairment of this regulation can lead to craniofacial malformations, such as orofacial cleft (OFC), which are frequently referred to as neurocristopathies and that represent a heavy burden on both the affected individuals and society. Understanding how genetic or structural disruption of enhancer activity during NC development can lead to human neurocristopathies is the central goal of this work. In the long term, the gained knowledge should serve to enable early detection and show potential therapeutic approaches. Here we investigate the pathomechanism of both syndromic (i.e. Branchiooculofacial Syndrome (BOFS)) and non-syndromic (i.e. OFC) neurocristopathies, by combining in vitro and in vivo NC developmental models with genetic engineering approaches and multiple genomic methods. First, we describe a unique patient with BOFS, who, in contrast to previously reported cases, does not present a heterozygous mutations within TFAP2A, a NC master regulator. Instead, the patient carries a de novo heterozygous 89 Mb inversion in which one of the breakpoints is located 40 kb downstream of TFAP2A. We first showed that this inversion separates TFAP2A from enhancers that are located within the same large topologically associating domain (TAD) and that are essential for TFAP2A expression in NCC. Importantly, using patient-specific human induced pluripotent stem cells (hiPSC) and a robust in vitro differentiation system towards NCC, we then showed that the inversion causes a loss of physical interactions between the inverted TFAP2A allele and its cognate enhancers, leading to TFAP2A monoallelic and haploinsufficient expression in human NCC. Overall, this first part provides a powerful approach to investigate the pathological mechanisms of structural variants predicted to disrupt 3D genome organization of gene regulatory landscapes and that, due to various reasons (i.e. limited access to relevant patient material, differences in gene dosage sensitivity between mice and humans, difficulties in recapitulating certain structural variants), cannot be properly evaluated in vivo. Second, we combined previously generated hNCC enhancer maps with OFC risk-loci identified through genome-wide association studies (GWAS) and, as a result, we revealed a highly conserved enhancer (i.e. Enh2p24.2) as a potential candidate harboring genetic variants involved in OFC. GWAS link common single nucleotide polymorphisms (SNPs) with quantitative traits and complex disorders. However, most disease-associated SNPs occur in non-coding regions of the human genome and consequently, the etiological relevance of these genetic variants cannot be easily connected to a gene. Nevertheless, accumulating evidences suggest that these disease-associated SNPs may contribute to human disease susceptibility by altering enhancers. Interestingly, SNPs associated with OFC are overrepresented in NCC enhancers. Therefore, we hypothesize that SNPs associated with OFC contribute to the etiology of the disorder by altering NCC enhancers and, consequently, the expression of relevant genes. Using Enh2p24.2 as a bait in circularized chromosome conformation capture sequencing (4C-seq) experiments, we identified two distally located genes, MYCN and DDX1, as its potential targets. Using in vitro and in vivo NCC developmental models, we then demonstrated that both genes are essential for normal facial development. While MYCN was not a surprising candidate to be involved in the etiology of OFC, the identification of DDX1 as a novel regulator of facial development might provide new insights into the molecular processes (e.g. transcription-coupled DNA repair) implicated in OFC and, potentially, other human neurocristopathies (e.g. neuroblastoma)

Three-Dimensional Morphometric Analysis of the Craniofacial Complex in the Unaffected Relatives of Individuals with Nonsyndromic Orofacial Clefts

Numerous studies have described altered patterns of craniofacial form in the unaffected relatives of individuals with nonsyndromic oral clefts. Unfortunately, results from these studies have been highly variable and have failed to provide a reliable method for discriminating at-risk relatives from controls. In the present study, we compared craniofacial shape between a sample of unaffected relatives (33 females; 14 males) from CL/P multiplex families and an equal number of age/sex/ethnicity-matched controls. A total of 16 x,y,z facial landmark coordinates derived from 3D photogrammetry were analyzed via Euclidean Distance Matrix Analysis (EDMA), while 14 additional linear distances from direct anthropometry were analyzed via t-tests. Variables identified as significantly different (p ≤ 0.10 from EDMA; 0.05 from t-tests) were then entered into a two-group discriminant function analysis. All analyses were carried out for each sex separately. Compared to controls, female unaffected relatives demonstrated increased upper facial width, midface reduction and lateral displacement of the alar cartilage. A single discriminant function was derived (canonical correlation = 0.43; p = 0.01) which correctly classified 70% of female unaffected relatives and 73% of female controls. Male unaffected relatives demonstrated increased upper facial and cranial base width, increased lower facial height and decreased upper facial height. Again, a single discriminant function was derived (canonical correlation = 0.79; p < 0.001) which correctly classified 86% of male unaffected relatives and 93% of male controls. In both males and females, upper facial width contributed most to group discrimination. Based on the discriminant function results, unaffected relatives were classified into risk/liability classes (high risk or low risk) based on the degree of phenotypic divergence from controls. Results suggest that the craniofacial shape differences characterizing unaffected relatives are partly sex-specific and perhaps more pronounced in males. The pattern of relative-control differences observed in both sexes is in broad agreement with previous findings from both humans and animal models. Although preliminary, these results suggest that a quantitative assessment of the craniofacial phenotype may allow for the identification of at-risk individuals within CL/P multiplex families. Importantly, the identification of such individuals could lead to improvements in recurrence risk estimation and gene mapping

Clinical and molecular investigation of rare congenital defects of the palate

Cleft palate (CP) affects around 1/1500 live births and, along with cleft lip, is one of the most common forms of birth defect. The studies presented here focus on unusual defects of the palate, especially to understand better the rarely reported but surprisingly common condition called submucous cleft palate (SMCP). The frequency and consequences of SMCP from a surgical perspective were first investigated based on the caseload of the North Thames Cleft Service at Great Ormond Street Hospital and St Andrew's Centre, Broomfield Hospital, Mid Essex Hospitals Trust. It was previously reported that up to 80% of individuals with unrepaired SMCP experience speech difficulties as a consequence of velopharyngeal insufficiency (VPI). Attempted repair of the palatal defect can sometimes give poor results, so controversies still exist about the correct choice of surgical technique to use. Over 23 years, 222 patients at The North Thames Cleft Service underwent operations to manage SMCP. Nearly half of them (42.8%) were diagnosed with 22q11.2 deletion syndrome (22q11.2 DS). The first operation was palate repair, with an exception of one case, followed by a second surgical intervention required in approximately half of the patients. A third procedure to manage VPI was carried out in 6% of patients. To better understand the histological anatomy of the palatal muscles in cleft patients, biopsies were taken from levator veli palatini (LVP) and/or palatopharyngeus (PP) muscles during surgical correction of CP. Muscles were compared from patients with SMCP to those with overt CP and also to controls. The controls consisted of descending PP muscle fibres from healthy children who underwent a tonsillectomy operation for obstructive sleep apnoea or recurrent chronic tonsillitis. Fifty-seven biopsy samples were available from children between 10 months to 9 years of age. Individual biopsy samples were also available from patients with achondroplasia, Apert, Cornelia de Lange and Kabuki syndromes. The study showed a prevalence of fast fibres in both muscles in all CP types. However, in both SMCP LVP and SMCP 22q11.2 DS LVP, this trend was reversed in favour of slow fibres. Single cases with syndromes did not reveal any obvious differences compared to more common cleft types. Mutations in TBX22 are a frequent genetic cause of cleft palate and SMCP. The functional role of the encoded TBX22 transcription factor was investigated in a mouse model with SMCP. Cell lineage-specific fluorescence activated cell sorting of a conditional allele of Tbx22, was used to look at the RNA-Seq transcriptome in developing palatal shelves, with a view to identify downstream target genes. Eleven up regulated genes reached statistical significance after multiple testing correction in cranial mesoderm (CM) derived cells when comparing Tbx22null/Y and WT samples (Cspg4, Foxp2, Reln, Bmpr1b, Adgrb3, Sox6, Zim1, Scarna13, Fat1, Notch3, Peg3). Eleven genes were down regulated in the same comparison (Nr2f2, Lars2, Ahr, Aplnr, Emcn, Npnt, Apln, Ccr2, Tll1, Snord34, Snord99). Comparing Tbx22null/Y and WT in cranial neural crest (CNC) derived cells, only Cxcl14 was up regulated, while Tbx22 was down regulated. Osteoclast differentiation, calcium signalling, focal adhesion, Wnt signalling and cell adhesion molecule pathways were the most enriched pathways in functional annotation of significantly differentially expressed genes analysis. Finally, a family with an unusual velopharyngeal anatomy was investigated in order to determine the likely genetic cause. This involved the implementation of genetic technologies in an autosomal dominant multigeneration Egyptian family with 8 affected individuals who presented with absent uvula, short posterior border of the soft palate and abnormal pillars of the fauces. Using a combination of cytogenetic, linkage analysis and exome sequencing, followed by more detailed segregation and functional analysis, a dominantly acting missense mutation in the activation domain of FOXF2 was revealed. This variant was found to co-segregate with a copy number variant of unknown significance that could not at this stage be causally distinguished from the point mutation

Genetic Approaches for the Diagnosis and Treatment of Congenital Tooth Agenesis

Congenital tooth agenesis is the most common developmental anomaly in man. More severe forms of tooth agenesis (> 5 missing teeth) demand lengthy and expensive treatment approaches such as bone augmentation surgeries and placement of multiple implants. Tooth agenesis is caused by mutations in genes responsible for early tooth development; and ever since it had been shown that timely injections of functional recombinant gene products can overcome the corresponding, mutation-based developmental disorder, such new therapeutic strategies for the prevention of tooth agenesis should be attempted. In this research project I have pursued two objectives: 1.) Basic research into the molecular genetics and therapeutics of the tooth agenesis gene PAX9. Since PAX9 is an intra-cellular transcription factor which cannot be replaced directly, suitable downstream targets for therapy have to be identified by comparing wild type and Pax9 deficient tooth organs. 2.) Clinically oriented research into the molecular diagnostics of human tooth agenesis. We use candidate gene sequencing in large numbers of people with tooth agenesis to identify the majority of human tooth agenesis genes and to determine the molecular cause of tooth agenesis in individuals. In the first study I identify the genes and pathways that are affected by Pax9 deficiency using microarray and q-PCR technology, and find that the Fgf, Shh and Wnt pathways are more affected than Bmp4 which had previously been considered the main target of Pax9 in tooth development. The next study shows that it is possible to apply therapeutic approaches to unravel the complexity of molecular signaling within the developing craniofacial complex. Using small molecule Wnt therapies we are able to rescue palatal clefting in Pax9-deficient mice. Our third study presents a clinical aspect of human molecular genetics where we establish that tooth agenesis does not predispose women to ovarian cancer, as had been previously suggested. The last study shows that mutations in WNT10A, but not in WNT10B or WNT6, are highly prevalent in populations with tooth agenesis. We also suggest that there must be some kind of heterozygous advantage to retaining mutations in Wnt10a. However, that advantage has not been identified

MURINE TRANSGENE INSERTIONAL MUTATION INVOLVING RUNX1T1 AND GM11823 GENES AND THEIR CONTRIBUTION TO CLEFT PALATE AND RIB ANOMALIES

Objective: Cleft Palate (CP) is a common birth defect in humans occurring in 6.35/10,000 live births and it has been repeatedly shown that animal models are useful in dissecting molecular etiologies of CP. The OVE1328 mouse line develops CP as a consequence of transgene (Tg) insertion mutagenesis. Preliminary data shows Tg complex integration at chromosome 4 band A2. The goal of this work was to characterize the mutation in the OVE1328 transgenic mouse line. Methods: Genotyping microarrays and RNA-seq were used to identify the Tg insertion site. The insertion site was further validated by conventional PCR. Histology and skeletal staining were used to phenotype CP (OVE1328 (Tg/Tg)) and Wt embryos. Results: In OVE1328 embryos the transgene disrupts Runx1t1 gene at intron 12 (13,876,840 bp). The integration is associated with a deletion mutation of part of intron 12 and whole exons 13 and 14 of Runx1t1. Runx1t1 (also known as Cbfa2t1h, Eto, Mtg8) is mainly studied for its role in acute myelogenous leukemia as a fusion gene in humans and gut development in mice. The Tg insertion disruption extends to the Gm11823 gene which encodes a long non coding RNA. Little is known regarding the normal function and expression of Gm11823. The Tg integrates at intron 2 (13,949,713 bp) of Gm11823 and induces the ectopic expression of an altered message. Homozygous disruption of both genes is associated with CP (100% of OVE1328 (Tg/Tg) embryos) and rib anomalies (supernumerary ribs, 86% of OVE1328 (Tg/Tg) embryos. Furthermore, OVE1328 (Tg/Tg) embryos are ~13% smaller by weight than Wt and OVE1328 (Tg/+) littermates indicting growth delay. Supported by NIH/NIDCR DE015180.Doctor of Philosoph

Rare Variant Association Testing in Case-Parent Trio Data

The overall objective of this work is to develop a workflow for processing genome sequencing data from case-parent trios for rare variant analysis in the programming language R, and to provide publicly available software to allow others to perform rare variant analysis in case-parent trios themselves. In this thesis, we provide background on three different methods for rare variant detection designed for case-parent trios, detailing the analytical choices made in each method. We then illustrate our workflow by applying these methods to whole genome sequencing data from case-parent trios collected by the Gabriella Miller Kids First Initiative. All offspring in this dataset are affected by cleft-lip, with or without cleft palate (CL/P). We analyze both common and rare single nucleotide variants (SNVs) from the 8q24 region on chromosome 8q, a region that has been identified as containing a potential susceptibility locus for CL/P

Birth defects surveillance : a manual for programme managers

Second edition.Congenital anomalies, also known as birth defects, are structural or functional abnormalities, including metabolic disorders, which are present at birth. Congenital anomalies are a diverse group of disorders of prenatal origin, which can be caused by single-gene defects, chromosomal disorders, multifactorial inheritance, environmental teratogens or micronutrient malnutrition.This manual is intended to serve as a tool for the development, implementation and ongoing improvement of a congenital anomalies surveillance programme, particularly for countries with limited resources. The focus of the manual is on population-based and hospital-based surveillance programmes. Some countries might not find it feasible to begin with the development of a population-based programme. Therefore, the manual covers the methodology needed for the development of both population-based and hospital-based surveillance programmes. Further, although many births in predominantly low- and middle-income countries (LMICs) occur outside of hospitals, some countries with limited resources might choose to start with a hospital-based surveillance programme and expand it later into one that is population-based. Any country wishing to expand its current hospital-based programme into a population-based programme, or to begin the initial development of a population-based system, should find this manual helpful in reaching its goal.This manual provides selected examples of congenital anomalies (see Appendix A). These anomalies are severe enough that many would probably be captured during the first few days following birth. While a number of the anomalies listed are external and easily identified by physical exam, others are internal and typically require more advanced diagnostic evaluations such as imaging. However, because of their severity and frequency, all these selected conditions have significant public health impact, and for some there is a potential for primary prevention. Nevertheless, these are just suggestions; countries might choose to monitor a subset of these conditions or add other congenital anomalies to meet their needs.WHO thanks the United States Centers for Disease Control and Prevention, especially the National Center on Birth Defects and Developmental Disabilities, for providing financial support for the publication of this manual as part of the cooperative agreement 5 E11 DP002196, Global prevention of noncommunicable diseases and promotion of health. Supported in part by contract from Task Force for Global Health to the International Center on Birth Defects (ICBD) of the ICBDSR. We gratefully acknowledge and thank the United States Agency for International Development for providing financial support for this work.Suggested citation. Birth defects surveillance: a manual for programme managers, second edition. Geneva: World Health Organization; 2020. Licence: CC BY-NC-SA 3.0 IGO.9789240015395 (\u200eelectronic version)\u200e9789240015401 (\u200eprint version)\u200eBirth-Defects-Surveillance-A-Manual-for-Programme-Managers-2020Manual-P.pdfAcknowledgements -- Financial support -- Abbreviations -- Objectives of the manual -- 1. Surveillance of congenital anomalies -- 2. Planning activities and tools -- 3. Approaches to surveillance -- 4. Dianosing congenital anomalies -- 5. Congenital infectious syndromes -- 6. Coding and diagnosis -- 7. Primer on data quality in birth defects surveillance.2020cooperative agreement 5 E11 DP002196891

Exploring childhood apraxia of speech : speech and language profiles in 5-year-olds with suspected apraxia of speech or cleft palate

Introduction and aims: Childhood apraxia of speech (CAS) is a speech sound disorder (SSD) lacking a quantifiable measure discriminating all cases of CAS from other SSDs. This project aimed at exploring CAS using different perspectives when examining speech and language difficulties commonly seen in 5-year-old children with suspected CAS or children with repaired cleft palate (CP±L). Children with CP±L were added to broaden and differentiate the knowledge base on CAS and to search for factors explaining unfavorable speech outcome in this group. Material and methods: In study I, a questionnaire was constructed and used, anonymously surveying Swedish SLPs (n=178) knowledge and praxis about CAS features and assessment. Findings were compared to earlier survey findings from English contexts. Study II examined articulation proficiency and orofacial function of children with CP±L (n=52) based on SLP examination and parental interview. For measurement of intelligibility, both parent reports and SLP ratings were compared. Study III included children with CP±L and disordered speech (n=19) and children with suspected CAS (n=15). Phonetic transcription and CAS diagnostics were based on audio-recordings of single word naming. The diagnosis was built on judgement of presence or absence of speech features using a checklist constructed for English speakers. The cross-linguistic applicability of the operationalized features and checklist was tested. In study IV language competence of children with CAS and CP±L was directly assessed, and parental ratings of everyday life communication were added, and results compared. Results: Swedish SLP’s views on typical speech characteristic of CAS, surveyed in study I, corresponded in large with reports of SLP’s from English-speaking contexts. The top seven characteristics were inconsistent speech production, sequencing difficulties, oro-motor deficits, vowel errors, voicing errors, consonant cluster deletion and prosodic disturbance. In study II, 37% of children with CP±L were found to have orofacial dysfunction; however, this was not an explanatory factor for speech outcome for these children. A distinct CAS profile, found in study III, included the five features: phonemic speech inconsistency for consonants and vowels plus vowel error, voicing error, difficulty achieving initial articulatory configurations or transitionary movement gestures and stress errors. In study IV, expressive language disorder was found in 67% of children with CAS. Receptive language ability was significantly better than expressive language in all children with CAS. No such difference was observed in the group of children without CAS (non-CAS SSD). Parent ratings of communication skills reflected an increased burden on communication in everyday life when difficulties within both speech and language domains were present. Conclusions: Despite relevant theoretical and/or clinical knowledge about CAS, Swedish SLPs reported a need for further education. Swedish-speaking 5-year-olds with CAS shared a distinct speech profile including five features, with prosodic impairment almost exclusively seen in children with CAS. Findings supported cross-linguistic applicability of CAS speech feature operationalization between English and Swedish speakers. In children with CP±L and SSD, a heightened cooccurrence of CAS, compared to clinical prevalence, should be anticipated. Expressive language ability in children with CAS was worse than receptive language ability. Poor articulation proficiency in children with CP±L did not correlate with orofacial dysfunction. Parental ratings of communication abilities in everyday life added ecological validity and confirmed validity of the clinical assessment procedures