Identification and functional validation of pathogenic small nucleotide variants in patients with craniosynostosis

Craniosynostosis, the premature fusion of the cranial sutures, affects ~1 in 2,000 live births. It is a heterogenous disorder with documented monogenic, polygenic, chromosomal, and environmental causes. Although many patients with a genetically determined cause harbour a variant in one of just seven genes, or have a chromosomal abnormality, there is a long tail of rarer genetic diagnoses with over 60 genes known to be recurrently mutated. Despite the recent uplift in knowledge of the genetic causes of craniosynostosis and the biogenesis of the cranial suture, there remains a suspected 10% of undiagnosed patients with a putatively pathogenic monogenic cause. In my analyses I aimed to target this subset of patients to delineate novel genetic causes of craniosynostosis. Using data generated from genome and exome sequencing I developed systematic pipelines to screen for small nucleotide variants. This approach identified 8 variants that explained the phenotype of previously undiagnosed patients (n = 114) enrolled in the UK 100,000 Genomes Project (100kGP) and highlighted additional candidate genes for further scrutiny. The candidate genes were included alongside 35 additional genes with reasonable evidence for involvement in craniosynostosis in a panel-based resequencing analysis of 617 individuals without a genetic diagnosis. Through this approach, I identified 8 diagnostic variants and 15 additional candidate variants. Drawing on the identification of pathogenic variants from the UK 100kGP, my resequencing analysis, and genes identified through other sources, I undertook experiments to explore and functionally validate the likely pathophysiological mechanisms associated with variants in three genes (PRRX1, PTCH1, and SPRY1) by using immunofluorescence, mouse models, and RNA and protein assays. Overall, this work has achieved improved diagnostic pipelines for craniosynostosis, and rare disease more widely, and has also served to delineate three new genetic causes of craniosynostosis by providing a key evidence base to support variant pathogenicity

Review of recurrently mutated genes in craniosynostosis supports expansion of diagnostic gene panels

Craniosynostosis, the premature fusion of the cranial sutures, affects ~1 in 2000 children. Although many patients with a genetically determined cause harbor a variant in one of just seven genes or have a chromosomal abnormality, over 60 genes are known to be recurrently mutated, thus comprising a long tail of rarer diagnoses. Genome sequencing for the diagnosis of rare diseases is increasingly used in clinical settings, but analysis of the data is labor intensive and involves a trade-off between achieving high sensitivity or high precision. PanelApp, a crowd-sourced disease-focused set of gene panels, was designed to enable prioritization of variants in known disease genes for a given pathology, allowing enhanced identification of true-positives. For heterogeneous disorders like craniosynostosis, these panels must be regularly updated to ensure that diagnoses are not being missed. We provide a systematic review of genetic literature on craniosynostosis over the last 5 years, including additional results from resequencing a 42-gene panel in 617 affected individuals. We identify 16 genes (representing a 25% uplift) that should be added to the list of bona fide craniosynostosis disease genes and discuss the insights that these new genes provide into pathophysiological mechanisms of craniosynostosis

BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome

Introduction: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely-related paralogue KCTD1 cause scalp-ear-nipple (SEN) syndrome. Methods: Exome sequencing was performed on a 2-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. Results: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p. (Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. Conclusion: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex

Breakfast Consumption Is Positively Associated with Usual Nutrient Intakes among Food Pantry Clients Living in Rural Communities

Background: Breakfast consumption has declined over the past 40 y and is inversely associated with obesity-related diet and health outcomes. The breakfast pattern of food pantry clients and its association with diet is unknown. Objective: The objective is to investigate the association of breakfast consumption with diet quality and usual nutrient intakes among food pantry clients (n = 472) living in rural communities. Methods: This was an observational study using cross-sectional analyses. English-speaking participants ≥18 y (or ≥19 y in Nebraska) were recruited from 24 food pantries in rural high-poverty counties in Indiana, Michigan, Missouri, Nebraska, Ohio, and South Dakota. Participants were surveyed at the pantry regarding characteristics and diet using 24-h recall. A second recall was self-completed or completed via assisted phone call within 2 wk of the pantry visit. Participants were classified as breakfast skippers when neither recall reported breakfast ≥230 kcal consumed between 04:00 and 10:00; breakfast consumers were all other participants. The Healthy Eating Index-2010 was modeled with breakfast pattern using multiple linear regression. Mean usual intake of 16 nutrients was estimated using the National Cancer Institute Method and compared across breakfast pattern groups. Usual nutrient intake was compared with the Estimated Average Requirement (EAR) or Adequate Intake (AI) to estimate the proportion of population not meeting the EAR or exceeding the AI. Results: A total of 56% of participants consumed breakfast. Compared with breakfast skippers, breakfast consumers had 10–59% significantly higher usual mean intakes of all nutrients (P ≤ 0.05), and had 12–21% lower prevalence of at-risk nutrient intakes except for vitamin D, vitamin E, and magnesium. Conclusions: Adult food pantry clients living in rural communities experienced hardships in meeting dietary recommendations. Breakfast consumption was positively associated with usual nutrient intakes in this population. This trial was registered at clinicaltrials.gov as NCT03566095

The phenotype of MEGF8-related Carpenter syndrome (CRPT2) is refined through the identification of eight new patients

Carpenter syndrome (CRPTS) is a rare autosomal recessive condition caused by biallelic variants in genes that encode negative regulators of hedgehog signalling (RAB23 [CRPT1] or, more rarely, MEGF8 [CRPT2]), and is characterised by craniosynostosis, polysyndactyly, and other congenital abnormalities. We describe a further six families comprising eight individuals with MEGF8-associated CRPT2, increasing the total number of reported cases to fifteen, and refine the phenotype of CRPT2 compared to CRPT1. The core features of craniosynostosis, polysyndactyly and (in males) cryptorchidism are almost universal in both CRPT1 and CRPT2. However, laterality defects are present in nearly half of those with MEGF8-associated CRPT2, but are rare in RAB23-associated CRPT1. Craniosynostosis in CRPT2 commonly involves a single midline suture in comparison to the multi-suture craniosynostosis characteristic of CRPT1. No patient to date has carried two MEGF8 gene alterations that are both predicted to lead to complete loss-of-function, suggesting that a variable degree of residual MEGF8 activity may be essential for viability and potentially contributing to variable phenotypic severity. These data refine the phenotypic spectrum of CRPT2 in comparison to CRPT1 and more than double the number of likely pathogenic MEGF8 variants in this rare disorder

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance

Purpose Studies previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the pre-osteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function variants in PRRX1 associated with craniosynostosis. Methods Trio-based genome, exome or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. Results Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9/1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, seven additional individuals (four families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multi-suture synostosis was present in 11/17 (65% of the cases). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. Conclusion This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis

Molecular High-Grade B-Cell Lymphoma: Defining a Poor-Risk Group That Requires Different Approaches to Therapy.

PURPOSE: Biologic heterogeneity is a feature of diffuse large B-cell lymphoma (DLBCL), and the existence of a subgroup with poor prognosis and phenotypic proximity to Burkitt lymphoma is well known. Conventional cytogenetics identifies some patients with rearrangements of MYC and BCL2 and/or BCL6 (double-hit lymphomas) who are increasingly treated with more intensive chemotherapy, but a more biologically coherent and clinically useful definition of this group is required. PATIENTS AND METHODS: We defined a molecular high-grade (MHG) group by applying a gene expression-based classifier to 928 patients with DLBCL from a clinical trial that investigated the addition of bortezomib to standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy. The prognostic significance of MHG was compared with existing biomarkers. We performed targeted sequencing of 70 genes in 400 patients and explored molecular pathology using gene expression signature databases. Findings were validated in an independent data set. RESULTS: The MHG group comprised 83 patients (9%), with 75 in the cell-of-origin germinal center B-cell-like group. MYC rearranged and double-hit groups were strongly over-represented in MHG but comprised only one half of the total. Gene expression analysis revealed a proliferative phenotype with a relationship to centroblasts. Progression-free survival rate at 36 months after R-CHOP in the MHG group was 37% (95% CI, 24% to 55%) compared with 72% (95% CI, 68% to 77%) for others, and an analysis of treatment effects suggested a possible positive effect of bortezomib. Double-hit lymphomas lacking the MHG signature showed no evidence of worse outcome than other germinal center B-cell-like cases. CONCLUSION: MHG defines a biologically coherent high-grade B-cell lymphoma group with distinct molecular features and clinical outcomes that effectively doubles the size of the poor-prognosis, double-hit group. Patients with MHG may benefit from intensified chemotherapy or novel targeted therapies.Supported by Bloodwise grant number 15002: Precision Medicine for Aggressive Lymphoma. The Randomized Evaluation of Molecular-Guided Therapy for DLBCL With Bortezomib (REMoDL-B) trial was endorsed by Cancer Research UK, reference number CRUKE/10/024, and Janssen-Cillag provided funding. A.S. is partly funded by the National Institute for Health Research Oxford Biomedical Research Centre. D.R.W. acknowledges UK Medical Research Council grant MR/L01629X/1 for infrastructure support