SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline.

While it is widely thought that de novo mutations (DNMs) occur randomly, we previously showed that some DNMs are enriched because they are positively selected in the testes of aging men. These "selfish" mutations cause disorders with a shared presentation of features, including exclusive paternal origin, significant increase of the father's age, and high apparent germline mutation rate. To date, all known selfish mutations cluster within the components of the RTK-RAS-MAPK signaling pathway, a critical modulator of testicular homeostasis. Here, we demonstrate the selfish nature of the SMAD4 DNMs causing Myhre syndrome (MYHRS). By analyzing 16 informative trios, we show that MYHRS-causing DNMs originated on the paternally derived allele in all cases. We document a statistically significant epidemiological paternal age effect of 6.3 years excess for fathers of MYHRS probands. We developed an ultra-sensitive assay to quantify spontaneous MYHRS-causing SMAD4 variants in sperm and show that pathogenic variants at codon 500 are found at elevated level in sperm of most men and exhibit a strong positive correlation with donor's age, indicative of a high apparent germline mutation rate. Finally, we performed in vitro assays to validate the peculiar functional behavior of the clonally selected DNMs and explored the basis of the pathophysiology of the different SMAD4 sperm-enriched variants. Taken together, these data provide compelling evidence that SMAD4, a gene operating outside the canonical RAS-MAPK signaling pathway, is associated with selfish spermatogonial selection and raises the possibility that other genes/pathways are under positive selection in the aging human testis.

Expanding the genotypic spectrum of TXNL4A variants in Burn-McKeown syndrome.

The developmental disorder Burn-McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre-messenger RNA splicing factor TXNL4A. Most patients have a loss-of-function variant in trans with a 34-base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs *21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258-3C>G) and a type 1 Δ34 promoter deletion. We show the c.258-3C>G variant and a previously reported c.258-2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56 bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22 bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22 bp repeated motif could be relevant to BMKS aetiology. Finally, our data emphasises the need to analyse the non-coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation.

Disease modeling of core pre-mRNA splicing factor haploinsufficiency.

The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.

EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion-Almeida type.

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.

A homozygous missense variant in CHRM3 associated with familial urinary bladder disease.

CHRM3 codes for the M3 muscarinic acetylcholine receptor that is located on the surface of smooth muscle cells of the detrusor, the muscle that effects urinary voiding. Previously, we reported brothers in a family affected by a congenital prune belly-like syndrome with mydriasis due to homozygous CHRM3 frameshift variants. In this study, we describe two sisters with bladders that failed to empty completely and pupils that failed to constrict fully in response to light, who are homozygous for the missense CHRM3 variant c.352G > A; p.(Gly118Arg). Samples were not available for genotyping from their brother, who had a history of multiple urinary tract infections and underwent surgical bladder draining in the first year of life. He died at the age of 6 years. This is the first independent report of biallelic variants in CHRM3 in a family with a rare serious bladder disorder associated with mydriasis and provides important evidence of this association.

Home sleep apnea testing: comparison of manual and automated scoring across international sleep centers.

PURPOSE: To determine the agreement between the manual scoring of home sleep apnea tests (HSATs) by international sleep technologists and automated scoring systems. METHODS: Fifteen HSATs, previously recorded using a type 3 monitor, were saved in European Data Format. The studies were scored by nine experienced technologists from the sleep centers of the Sleep Apnea Global Interdisciplinary Consortium (SAGIC) using the locally available software. Each study was scored separately by human scorers using the nasal pressure (NP), flow derived from the NP signal (transformed NP), or respiratory inductive plethysmography (RIP) flow. The same procedure was followed using two automated scoring systems: Remlogic (RLG) and Noxturnal (NOX). RESULTS: The intra-class correlation coefficients (ICCs) of the apnea-hypopnea index (AHI) scoring using the NP, transformed NP, and RIP flow were 0.96 [95% CI 0.93-0.99], 0.98 [0.96-0.99], and 0.97 [0.95-0.99], respectively. Using the NP signal, the mean differences in AHI between the average of the manual scoring and the automated systems were - 0.9 ± 3.1/h (AHIRLG vs AHIMANUAL) and - 1.3 ± 2.6/h (AHINOX vs AHIMANUAL). Using the transformed NP, the mean differences in AHI were - 1.9 ± 3.3/h (AHIRLG vs AHIMANUAL) and 1.6 ± 3.0/h (AHINOX vs AHIMANUAL). Using the RIP flow, the mean differences in AHI were - 2.7 ± 4.5/h (AHIRLG vs AHIMANUAL) and 2.3 ± 3.4/h (AHINOX vs AHIMANUAL). CONCLUSIONS: There is very strong agreement in the scoring of the AHI for HSATs between the automated systems and experienced international technologists. Automated scoring of HSATs using commercially available software may be useful to standardize scoring in future endeavors involving international sleep centers.

Combining a prioritization strategy and functional studies nominates 5'UTR variants underlying inherited retinal disease.

BACKGROUND: 5' untranslated regions (5'UTRs) are essential modulators of protein translation. Predicting the impact of 5'UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5'UTRs by different whole exome sequencing (WES) kits. The selected 5'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5'UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5'UTR compared to that of their canonical isoform. Depending on the probe design, 3-20% of IRD genes have 5'UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5'UTR variants implicated in IRDs and provides a systematic approach for 5'UTR annotation and validation that is applicable to other inherited diseases.

Personalized recurrence risk assessment following the birth of a child with a pathogenic de novo mutation.

Following the diagnosis of a paediatric disorder caused by an apparently de novo mutation, a recurrence risk of 1-2% is frequently quoted due to the possibility of parental germline mosaicism; but for any specific couple, this figure is usually incorrect. We present a systematic approach to providing individualized recurrence risk. By combining locus-specific sequencing of multiple tissues to detect occult mosaicism with long-read sequencing to determine the parent-of-origin of the mutation, we show that we can stratify the majority of couples into one of seven discrete categories associated with substantially different risks to future offspring. Among 58 families with a single affected offspring (representing 59 de novo mutations in 49 genes), the recurrence risk for 35 (59%) was decreased below 0.1%, but increased owing to parental mixed mosaicism for 5 (9%)-that could be quantified in semen for paternal cases (recurrence risks of 5.6-12.1%). Implementation of this strategy offers the prospect of driving a major transformation in the practice of genetic counselling.

The impact of paternal age on new mutations and disease in the next generation.

Advanced paternal age is associated with an increased risk of fathering children with genetic disorders and other adverse reproductive consequences. However, the mechanisms underlying this phenomenon remain largely unexplored. In this review, we focus on the impact of paternal age on de novo mutations that are an important contributor to genetic disease and can be studied both indirectly through large-scale sequencing studies and directly in the tissue in which they predominantly arise-the aging testis. We discuss the recent data that have helped establish the origins and frequency of de novo mutations, and highlight experimental evidence about the close link between new mutations, parental age, and genetic disease. We then focus on a small group of rare genetic conditions, the so-called "paternal age effect" disorders that show a strong association between paternal age and disease prevalence, and discuss the underlying mechanism ("selfish selection") and implications of this process in more detail. More broadly, understanding the causes and consequences of paternal age on genetic risk has important implications both for individual couples and for public health advice given that the average age of fatherhood is steadily increasing in many developed nations.

The Role of the U5 snRNP in Genetic Disorders and Cancer.

Pre-mRNA splicing is performed by the spliceosome, a dynamic macromolecular complex consisting of five small uridine-rich ribonucleoprotein complexes (the U1, U2, U4, U5, and U6 snRNPs) and numerous auxiliary splicing factors. A plethora of human disorders are caused by genetic variants affecting the function and/or expression of splicing factors, including the core snRNP proteins. Variants in the genes encoding proteins of the U5 snRNP cause two distinct and tissue-specific human disease phenotypes - variants in PRPF6, PRPF8, and SNRP200 are associated with retinitis pigmentosa (RP), while variants in EFTUD2 and TXNL4A cause the craniofacial disorders mandibulofacial dysostosis Guion-Almeida type (MFDGA) and Burn-McKeown syndrome (BMKS), respectively. Furthermore, recurrent somatic mutations or changes in the expression levels of a number of U5 snRNP proteins (PRPF6, PRPF8, EFTUD2, DDX23, and SNRNP40) have been associated with human cancers. How and why variants in ubiquitously expressed spliceosome proteins required for pre-mRNA splicing in all human cells result in tissue-restricted disease phenotypes is not clear. Additionally, why variants in different, yet interacting, proteins making up the same core spliceosome snRNP result in completely distinct disease outcomes - RP, craniofacial defects or cancer - is unclear. In this review, we define the roles of different U5 snRNP proteins in RP, craniofacial disorders and cancer, including how disease-associated genetic variants affect pre-mRNA splicing and the proposed disease mechanisms. We then propose potential hypotheses for how U5 snRNP variants cause tissue specificity resulting in the restricted and distinct human disorders.

Ulcer metastasis? Anatomical locations of recurrence for patients in diabetic foot remission.

BACKGROUND: The "cancer analogy" is powerful for communicating risk to and organizing care for patients with diabetic foot syndrome. One potentially underappreciated similarity between cancer and foot ulcers is that both can recur at anatomical locations distinct from the primary occurrence, albeit with different physiological mechanisms. Few studies have characterized the location of diabetic foot ulcer recurrence, and these have been limited by considering only the first recurrent wound following a recent-healed wound. We therefore characterized the anatomical locations at which diabetic foot ulcers are likely to recur considering multiple wounds during follow-up and the locations of all prior wounds documented in the participant's history. METHODS: We completed a secondary analysis of existing data from a 129 participant multi-center study of participants in diabetic foot remission. The primary outcome was plantar foot ulceration, and each participant was followed for 34 weeks or until withdrawing consent, allowing characterization of all wounds occurring. We stratified the anatomical locations of wounds prior to the trial by the following outcome categories during the trial: no recurrence, recurrence to the same anatomical location, recurrence to a different anatomical location on the same foot, and recurrence to the contralateral foot. RESULTS: A large percentage (48%) of wounds recurred to the contralateral foot, and the proportion of subsequent foot ulcer to the contralateral limb was largely unaffected by the anatomical location of foot ulcer prior to the study. Only 17% of prior diabetic foot ulcers were followed by recurrence to the same anatomical location. Rates of recurrence remained high during treatment of a wound (0.41 foot ulcer/ulcer-year). Participants had documented wounds to 2.2 distinct anatomical locations on average, and more than 60% of participants had wounds to more than one plantar location by the end of the study. CONCLUSIONS: Given the significant morbidity, mortality, and resource utilization associated with foot ulcer recidivism, quality and evidenced-based preventive care is essential. Our results better characterize the burden of recurrence and to what anatomy recurrence is most likely. These insights may benefit providers and patients alike for the provision of high-quality preventive care thereby resulting in reduced morbidity, mortality, and cost. TRIAL REGISTRATION: The study providing the data for this secondary analysis was registered on ClinicalTrials.gov (NCT02647346) on January 6, 2016. The study was retrospectively registered.

Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells.

The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, branch point to 3' splice site (BPS-3'SS) distance and splice site strengths, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.

Controlling the extrudate swell in melt extrusion additive manufacturing of 3D scaffolds: a designed experiment.

Tissue engineering using three-dimensional porous scaffolds has shown promise for the restoration of normal function in injured and diseased tissues and organs. Rigorous control over scaffold architecture in melt extrusion additive manufacturing is highly restricted mainly due to pronounced variations in the deposited strand diameter upon any variations in process conditions and polymer viscoelasticity. We have designed an I-optimal, split-plot experiment to study the extrudate swell in melt extrusion additive manufacturing and to control the scaffold architecture. The designed experiment was used to generate data to relate three responses (swell, density, and modulus) to a set of controllable factors (plotting needle diameter, temperature, pressure, and the dispensing speed). The fitted regression relationships were used to optimize the three responses simultaneously. The swell response was constrained to be close to 1 while maximizing the modulus and minimizing the density. Constraining the extrudate swell to 1 generates design-driven scaffolds, with strand diameters equal to the plotting needle diameter, and allows a greater control over scaffold pore size. Hence, the modulus of the scaffolds can be fully controlled by adjusting the in-plane distance between the deposited strands. To the extent of the model's validity, we can eliminate the effect of extrudate swell in designing these scaffolds, while targeting a range of porosity and modulus appropriate for bone tissue engineering. The result of this optimization was a predicted modulus of 14 MPa and a predicted density of 0.29 g/cm3 (porosity ≈ 75%) using polycaprolactone as scaffold material. These predicted responses corresponded to factor levels of 0.6 µm for the plotting needle diameter, plotting pressure of 2.5 bar, melt temperature of 113.5 °C, and dispensing speed of 2 mm/s. The validation scaffold enabled us to quantify the percentage difference for the predictions, which was 9.5% for the extrudate swell, 19% for the density, and 29% for the modulus.