Pathophysiology and consequences of Craniosynostosis:Novel approaches in genetic, neuroimaging and aesthetic studies

Chapter 1 describes the background and rationale for the studies presented in this thesis. Craniosynostosis is a rare congenital anomaly defined by premature fusion of one or more cranial sutures leading to a deformed skull shape. Craniosynostosis can have a wide range of adverse effects throughout the course of life. Our understanding of the development of the cranial vault and premature suture fusion has improved over the last decades, but many factors involved in the pathophysiology of craniosynostosis remain unknown. Therefore, the general aim of this thesis was to gain further insight into the pathophysiology and long-term consequences of craniosynostosis, for which we focused on three main objectives: 1) To investigate the genetic diagnostics yield in craniosynostosis; 2) to obtain further insight into the pathophysiology of metopic synostosis and associated disorders; 3) to establish methods for uniform assessments of phenotypical severity and aesthetic outcome for metopic and sagittal synostosis across Europe to facilitate cross-center comparison of treatment outcome in the future. The studies presented in this thesis were conducted in (subsets of) a large clinical cohort of patients with craniosynostosis in the Erasmus Medical Center, Rotterdam, the Netherlands and in a collaboration with European reference network (ERN) CRANIO. Chapter 2 describes studies on the genetic background of craniosynostosis. In Chapter 2.1, we describe the genetic diagnostic yield in a large 11-year birth cohort of craniosynostosis patients. We found pathogenic variants in 35% of patients who underwent genetic diagnostics. Diagnostic yield was significantly higher in syndromic craniosynostosis (62%) than in non-syndromic craniosynostosis (6%). We conclude that microarray and the Whole Exome Sequencing (WES) craniosynostosis panel are key to identifying pathogenic variants in craniosynostosis patients. Given the advances in genetic diagnostics, we recommend to consider extensive genetic diagnostics if no diagnosis is obtained through microarray and/or WES craniosynostosis panel. In Chapter 2.2, we present a case report and review of the literature on the co-occurrence of craniosynostosis and congenital diaphragmatic hernia. Through clinical whole exome sequencing, we identified a BCL11B missense variant in a patient with a congenital diaphragmatic hernia as well as sagittal suture craniosynostosis. After targeted sequencing of BCL11B in patients with craniosynostosis, we identified three additional BCL11B missense variants in patients with craniosynostosis. We conclude that both craniosynostosis as well as congenital diaphragmatic hernia may be associated with BCL11B pathogenic variants, although further studies are needed to establish if BCL11B variants are causative pathogenic variants for both conditions. Chapter 3 describes studies focused on radiological imaging in patients with metopic synostosis to gain insight into the pathophysiology of metopic synostosis and associated disorders. In Chapter 3.1, we present our study on white matter microstructure of major frontal lobe white matter tracts in young, unoperated metopic synostosis patients. Using automated diffusion tension imaging analyses, we found no significant differences in white matter microstructure in metopic synostosis patients compared to healthy controls. In Chapter 3.2, we found that preoperatively, total white matter volume was significantly smaller in young metopic synostosis patients compared to healthy controls. We observed an accelerated catch-up growth pattern of white matter volume. This pattern appears to be similar to white matter growth patterns described in autism. We observed no significant differences in total intracranial volume, total brain volume, total cerebrospinal fluid volume and total grey matter volume volumes in metopic synostosis patients compared to controls. In Chapter 3.3 we found that metopic synostosis patients have more shallow, wider, higher orbits with decreased interorbital distance compared to sagittal synostosis patients. We also found smaller globe height in metopic synostosis patients, although other eye dimensions (width and axial length) were similar to those in sagittal synostosis patients. In Chapter 4 studies conducted within ERN CRANIO are described. ERNs are networks of specialized health care providers, that aim to pool together disease-specific expertise and resource from across Europe to ensure equal high quality care to all patients in Europe. ERN CRANIO is the ERN for rare and complex craniofacial anomalies, including craniosynostosis, and ear, nose and throat disorders. In Chapter 4.1 we observed that ERN CRANIO’s coverage of craniosynostosis varies across European countries and should be optimized further. In Chapter 4.2 and Chapter 4.3, we developed 2D photo scores for assessing phenotypical severity and aesthetic outcome of metopic and sagittal synostosis, respectively, the two most common types of craniosynostosis. In both studies we observed substantial interrater agreement on overall assessment of phenotypical severity, but considerable interrater differences were present for specific phenotypical features, despite the fact that scoring was done by experienced expert surgeons that were also involved in defining the photo scores. Finally, Chapter 5 provides a general discussion in which the main findings from the work in this thesis are described in context of previous literature. We discuss methodological considerations, clinical implications, and suggestions for future research. To conclude, the studies in this thesis further underline the importance of genetic diagnostics in patients with craniosynostosis. Our observations in patients with metopic synostosis, suggest an intrinsic brain anomaly may be a key pathophysiological mechanism contributing to the neurodevelopmental disorders associated with metopic synostosis. The photo scores for uniform assessment of phenotypical severity of metopic and sagittal synostosis, developed in this thesis, may provide a useful clinical tool to uniformly score and compare aesthetic outcome in metopic and sagittal synostosis patients after treatment in multicenter settings.<br/

Pathophysiology and consequences of Craniosynostosis:Novel approaches in genetic, neuroimaging and aesthetic studies

Chapter 1 describes the background and rationale for the studies presented in this thesis. Craniosynostosis is a rare congenital anomaly defined by premature fusion of one or more cranial sutures leading to a deformed skull shape. Craniosynostosis can have a wide range of adverse effects throughout the course of life. Our understanding of the development of the cranial vault and premature suture fusion has improved over the last decades, but many factors involved in the pathophysiology of craniosynostosis remain unknown. Therefore, the general aim of this thesis was to gain further insight into the pathophysiology and long-term consequences of craniosynostosis, for which we focused on three main objectives: 1) To investigate the genetic diagnostics yield in craniosynostosis; 2) to obtain further insight into the pathophysiology of metopic synostosis and associated disorders; 3) to establish methods for uniform assessments of phenotypical severity and aesthetic outcome for metopic and sagittal synostosis across Europe to facilitate cross-center comparison of treatment outcome in the future. The studies presented in this thesis were conducted in (subsets of) a large clinical cohort of patients with craniosynostosis in the Erasmus Medical Center, Rotterdam, the Netherlands and in a collaboration with European reference network (ERN) CRANIO. Chapter 2 describes studies on the genetic background of craniosynostosis. In Chapter 2.1, we describe the genetic diagnostic yield in a large 11-year birth cohort of craniosynostosis patients. We found pathogenic variants in 35% of patients who underwent genetic diagnostics. Diagnostic yield was significantly higher in syndromic craniosynostosis (62%) than in non-syndromic craniosynostosis (6%). We conclude that microarray and the Whole Exome Sequencing (WES) craniosynostosis panel are key to identifying pathogenic variants in craniosynostosis patients. Given the advances in genetic diagnostics, we recommend to consider extensive genetic diagnostics if no diagnosis is obtained through microarray and/or WES craniosynostosis panel. In Chapter 2.2, we present a case report and review of the literature on the co-occurrence of craniosynostosis and congenital diaphragmatic hernia. Through clinical whole exome sequencing, we identified a BCL11B missense variant in a patient with a congenital diaphragmatic hernia as well as sagittal suture craniosynostosis. After targeted sequencing of BCL11B in patients with craniosynostosis, we identified three additional BCL11B missense variants in patients with craniosynostosis. We conclude that both craniosynostosis as well as congenital diaphragmatic hernia may be associated with BCL11B pathogenic variants, although further studies are needed to establish if BCL11B variants are causative pathogenic variants for both conditions. Chapter 3 describes studies focused on radiological imaging in patients with metopic synostosis to gain insight into the pathophysiology of metopic synostosis and associated disorders. In Chapter 3.1, we present our study on white matter microstructure of major frontal lobe white matter tracts in young, unoperated metopic synostosis patients. Using automated diffusion tension imaging analyses, we found no significant differences in white matter microstructure in metopic synostosis patients compared to healthy controls. In Chapter 3.2, we found that preoperatively, total white matter volume was significantly smaller in young metopic synostosis patients compared to healthy controls. We observed an accelerated catch-up growth pattern of white matter volume. This pattern appears to be similar to white matter growth patterns described in autism. We observed no significant differences in total intracranial volume, total brain volume, total cerebrospinal fluid volume and total grey matter volume volumes in metopic synostosis patients compared to controls. In Chapter 3.3 we found that metopic synostosis patients have more shallow, wider, higher orbits with decreased interorbital distance compared to sagittal synostosis patients. We also found smaller globe height in metopic synostosis patients, although other eye dimensions (width and axial length) were similar to those in sagittal synostosis patients. In Chapter 4 studies conducted within ERN CRANIO are described. ERNs are networks of specialized health care providers, that aim to pool together disease-specific expertise and resource from across Europe to ensure equal high quality care to all patients in Europe. ERN CRANIO is the ERN for rare and complex craniofacial anomalies, including craniosynostosis, and ear, nose and throat disorders. In Chapter 4.1 we observed that ERN CRANIO’s coverage of craniosynostosis varies across European countries and should be optimized further. In Chapter 4.2 and Chapter 4.3, we developed 2D photo scores for assessing phenotypical severity and aesthetic outcome of metopic and sagittal synostosis, respectively, the two most common types of craniosynostosis. In both studies we observed substantial interrater agreement on overall assessment of phenotypical severity, but considerable interrater differences were present for specific phenotypical features, despite the fact that scoring was done by experienced expert surgeons that were also involved in defining the photo scores. Finally, Chapter 5 provides a general discussion in which the main findings from the work in this thesis are described in context of previous literature. We discuss methodological considerations, clinical implications, and suggestions for future research. To conclude, the studies in this thesis further underline the importance of genetic diagnostics in patients with craniosynostosis. Our observations in patients with metopic synostosis, suggest an intrinsic brain anomaly may be a key pathophysiological mechanism contributing to the neurodevelopmental disorders associated with metopic synostosis. The photo scores for uniform assessment of phenotypical severity of metopic and sagittal synostosis, developed in this thesis, may provide a useful clinical tool to uniformly score and compare aesthetic outcome in metopic and sagittal synostosis patients after treatment in multicenter settings.<br/

Eye and Orbital Anatomy in Metopic Synostosis

Background: Metopic synostosis patients have a high prevalence of orthoptic anomalies, including hyperopia, astigmatism, and amblyopia. We hypothesized altered orbital anatomy contributes to suboptimal visual outcomes by adversely affecting eye anatomy and growth from early life onward. Therefore, we aimed to investigate eye and orbital anatomy in metopic synostosis. Methods: We conducted a retrospective study in nonsyndromic metopic synostosis patients (n = 134, median age 0.43 years [IQR 0.45]) with nonsyndromic sagittal synostosis patients (n = 134, median age 0.27 years [IQR 0.23]) as controls. Primary analyses focused on eye dimensions (axial length, width, and globe height) and orbital dimensions, correcting for sex and age. Measurements were obtained from preoperative computed tomography scans. Results: Axial length and width in metopic synostosis patients did not differ from sagittal synostosis patients, but globe height was significantly smaller (P = 0.0002). Lateral wall interorbital length, lateral orbital wall length, anterior medial interorbital length, and maximal medial interorbital length were significantly smaller, and anterior vertical orbital height and maximal vertical orbital height were significantly larger (P &lt; 0.001). The central orbital axis and interorbital angle were significantly narrower, and medial-to-lateral orbital wall angle was wider (P &lt; 0.001). Conclusions: Metopic synostosis patients have more shallow, wider, and higher orbits. Eye dimensions are similar in sagittal synostosis patients, although globe height was smaller. Altered orbital and eye dimensions in metopic synostosis probably have a causal relation with an unknown order of development. How these dimensions relate to future orthoptic anomalies (eg, refractive error) needs further investigation.</p

A Suture-specific Photo Score for Metopic Synostosis

Head shape assessments in children with metopic synostosis are a relevant outcome measure in addition to functional measures, such as neurocognitive outcomes, behavioral outcomes, and visual functioning outcomes. However, consensus on head shape assessments in children with metopic synostosis is lacking. The aim of this study is to develop a reproducible and reliable suture-specific photo score that can be used for cross-center comparison of phenotypical severity of metopic synostosis and evaluation of esthetic outcome of treatment later in childhood. We conducted a retrospective study among nonsyndromic metopic synostosis patients aged &lt;18 years. Preoperative and postoperative photosets of patients with metopic synostosis from 6 expert centers were included. The photo score was discussed in the group of expert craniofacial plastic surgeons and pediatric neurosurgeons. Interrater reliability was determined with modified weighted Fleiss' kappa and intraclass correlation coefficients. Correlation between individual photo score items with overall phenotype was assessed using Spearman correlation analyses. The metopic synostosis photo score contained the following items: "wedging of the forehead", "hypotelorism", "temporal hollowing", "biparietal widening,"and an assessment of "overall phenotype". Items were scored on a 4-point ordinal scale ranging from normal to severe. We found moderate interrater reliability for all items, but substantial agreement for the summed scores. Correlation with overall phenotype was lowest for biparietal widening. To conclude, although agreement on individual photo score items was suboptimal, the agreement on the summed score was substantial, which indicates there is consensus on the overall severity of the metopic synostosis phenotype.</p

Copy number variation of the beta-defensin genes in Europeans: no supporting evidence for association with lung function, chronic obstructive pulmonary disease or asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02–1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72–1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed

Prognostic biomarker soluble ST2 exhibits diurnal variation in chronic heart failure patients

Aim: Soluble suppression of tumorigenicity-2 (sST2) is a strong prognostic biomarker in heart failure. The emerging understanding of circadian biology in cardiovascular disease may lead to novel applications in prognosis and diagnosis and may provide insight into mechanistic aspects of the disease–biomarker interaction. So far, it is unknown whether sST2 exhibits a diurnal rhythm. Repeated measurements of sST2 may aid in clinical decision making. The goal of this study was to investigate whether sST2 exhibits diurnal variation in patients with heart failure with reduced ejection fraction (HFrEF) and in control subjects, thereby enhancing its diagnostic and prognostic values. Methods and results: The study comprised 32 subjects: 16 HFrEF patients and 16 controls. Blood was collected at seven subsequent time points during a 24 h time period. sST2, N-terminal pro-B-type natriuretic peptide (NT-proBNP), melatonin, and cortisol were measured from serum. Peak values of sST2 clustered at daytime (modal value: 5 p.m.) in 87.6% of all subjects (81.3% of patients, P = 0.021; 93.8% of controls, P = 0.001), and minimum concentrations at night-time (modal value: 5 a.m.) in 84.4% (87.5% of patients, P = 0.004 81.3% of controls, P = 0.021). A cosinor analysis of mean normalized sST2 values revealed significant cosine shaped 24 h oscillations of patients (P = 0.026) and controls (P = 0.037). NT-proBNP in contrast did not show a diurnal rhythm, while melatonin and cortisol patterns were intact in all subjects. Conclusions: sST2 exhibits a diurnal rhythm with lower values in the morning than in the late afternoon. This new insight could lead to refinement of its diagnostic and prognostic values through specified and consistent sampling times with repeated measurements. For example, by measuring sST2 during the afternoon, when levels are at their highest, false negatives on prognosis prediction could be avoided

A Photo Score for Aesthetic Outcome in Sagittal Synostosis:An ERN CRANIO Collaboration

European Reference Network (ERN) CRANIO is focused on optimizing care for patients with rare or complex craniofacial anomalies, including craniosynostosis and/or rare ear, nose, and throat disorders. The main goal of ERN CRANIO is to collect uniform data on treatment outcomes for multicenter comparison. We aimed to develop a reproducible and reliable suture-specific photo score that can be used for cross-center comparison of phenotypical severity of sagittal synostosis and aesthetic outcome of treatment. We conducted a retrospective study among nonsyndromic sagittal synostosis patients aged &lt;19 years. We included preoperative and postoperative photo sets from 6 ERN CRANIO centers. Photo sets included bird's eye, lateral, and anterior-posterior views. The sagittal synostosis photo score was discussed in the working group, and consensus was obtained on its contents. Interrater agreement was assessed with weighted Fleiss' Kappa and intraclass correlation coefficients.The photo score consisted of frontal bossing, elongated skull, biparietal narrowness, temporal hollowing, vertex line depression, occipital bullet, and overall phenotype. Each item was scored as normal, mild, moderate, or severe. Results from 36 scaphocephaly patients scored by 20 raters showed kappa values ranging from 0.38 [95% bootstrap CI: 0.31, 0.45] for biparietal narrowness to 0.56 [95% bootstrap CI: 0.47, 0.64] for frontal bossing. Agreement was highest for the sum score of individual items [intraclass correlation coefficients agreement 0.69 [95% CI: 0.57, 0.82]. This is the first large-scale multicenter study in which experts investigated a photo score to assess the severity of sagittal synostosis phenotypical characteristics. Agreement on phenotypical characteristics was suboptimal (fair-moderate agreement) and highest for the summed score of individual photo score items (substantial agreement), indicating that although experts interpret phenotypical characteristics differently, there is consensus on overall phenotypical severity.</p

Regional Differences in Prevalence of HIV-1 Discordance in Africa and Enrollment of HIV-1 Discordant Couples into an HIV-1 Prevention Trial

Background: Most HIV-1 transmission in Africa occurs among HIV-1-discordant couples (one partner HIV-1 infected and one uninfected) who are unaware of their discordant HIV-1 serostatus. Given the high HIV-1 incidence among HIV-1 discordant couples and to assess efficacy of interventions for reducing HIV-1 transmission, HIV-1 discordant couples represent a critical target population for HIV-1 prevention interventions and prevention trials. Substantial regional differences exist in HIV-1 prevalence in Africa, but regional differences in HIV-1 discordance among African couples, has not previously been reported. Methodology/Principal Findings: The Partners in Prevention HSV-2/HIV-1 Transmission Trial (“Partners HSV-2 Study”), the first large HIV-1 prevention trial in Africa involving HIV-1 discordant couples, completed enrollment in May 2007. Partners HSV-2 Study recruitment data from 12 sites from East and Southern Africa were used to assess HIV-1 discordance among couples accessing couples HIV-1 counseling and testing, and to correlate with enrollment of HIV-1 discordant couples. HIV-1 discordance at Partners HSV-2 Study sites ranged from 8–31% of couples tested from the community. Across all study sites and, among all couples with one HIV-1 infected partner, almost half (49%) of couples were HIV-1 discordant. Site-specific monthly enrollment of HIV-1 discordant couples into the clinical trial was not directly associated with prevalence of HIV-1 discordance, but was modestly correlated with national HIV-1 counseling and testing rates and access to palliative care/basic health care (r = 0.74, p = 0.09). Conclusions/Significance: HIV-1 discordant couples are a critical target for HIV-1 prevention in Africa. In addition to community prevalence of HIV-1 discordance, national infrastructure for HIV-1 testing and healthcare delivery and effective community outreach strategies impact recruitment of HIV-1 discordant couples into HIV-1 prevention trials

Genome-wide association for milk production and lactation curve parameters in Holstein dairy cows

The aim of this study was to identify genomic regions associated with 305-day milk yield and lactation curve parameters on primiparous (n = 9,910) and multiparous (n = 11,158) Holstein cows. The SNP solutions were estimated using a weighted single-step genomic BLUP approach and imputed high-density panel (777k) genotypes. The proportion of genetic variance explained by windows of 50 consecutive SNP (with an average of 165 Kb) was calculated, and regions that accounted for more than 0.50% of the variance were used to search for candidate genes. Estimated heritabilities were 0.37, 0.34, 0.17, 0.12, 0.30 and 0.19, respectively, for 305-day milk yield, peak yield, peak time, ramp, scale and decay for primiparous cows. Genetic correlations of 305-day milk yield with peak yield, peak time, ramp, scale and decay in primiparous cows were 0.99, 0.63, 0.20, 0.97 and -0.52, respectively. The results identified three windows on BTA14 associated with 305-day milk yield and the parameters of lactation curve in primi- and multiparous cows. Previously proposed candidate genes for milk yield supported by this work include GRINA, CYHR1, FOXH1, TONSL, PPP1R16A, ARHGAP39, MAF1, OPLAH and MROH1, whereas newly identified candidate genes are MIR2308, ZNF7, ZNF34, SLURP1, MAFA and KIFC2 (BTA14). The protein lipidation biological process term, which plays a key role in controlling protein localization and function, was identified as the most important term enriched by the identified genes