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RNA binding motif protein X-linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the genes importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities., Funding Agencies|Uppsala University, Faculty of Medicine, for psychiatric and neurological research; Uppsala University Hospital; Svenska Laekaresaellskapet (SLS); Jeansson foundations; Swedish Society for Medical Research (SSMF); Swedish Research Council [2020-04395]; Knut and Alice Wallenberg Foundation; Saevstaholm Foundation; Uppsala University

RNA binding motif protein X-linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the genes importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities., Funding Agencies|Uppsala University, Faculty of Medicine, for psychiatric and neurological research; Uppsala University Hospital; Svenska Laekaresaellskapet (SLS); Jeansson foundations; Swedish Society for Medical Research (SSMF); Swedish Research Council [2020-04395]; Knut and Alice Wallenberg Foundation; Saevstaholm Foundation; Uppsala University

The Nexilin F-Actin Binding Protein (Nexilin) encoded by NEXN is a cardiac Z-disc protein important for cardiac function and development in humans, zebrafish, and mice. Heterozygote variants in the human NEXN gene have been reported to cause dilated and hypertrophic cardiomyopathy. Homozygous variants in NEXN cause a lethal form of human fetal cardiomyopathy, only described in two patients before. In a Swedish, four-generation, non-consanguineous family comprising 42 individuals, one female had three consecutive pregnancies with intrauterine fetal deaths caused by a lethal form of dilated cardiomyopathy. Whole-exome sequencing and variant analysis revealed that the affected fetuses were homozygous for a NEXN variant (NM_144573:c.1302del;p.(Ile435Serfs*3)). Moreover, autopsy and histology staining declared that they presented with cardiomegaly and endocardial fibroelastosis. Immunohistochemistry staining for Nexilin in the affected fetuses revealed reduced antibody staining and loss of striation in the heart, supporting loss of Nexilin function. Clinical examination of seven heterozygote carriers confirmed dilated cardiomyopathy (two individuals), other cardiac findings (three individuals), or no cardiac deviations (two individuals), indicating incomplete penetrance or age-dependent expression of dilated cardiomyopathy. RNA sequencing spanning the variant in cDNA blood of heterozygote individuals revealed nonsense-mediated mRNA decay of the mutated transcripts. In the current study, we present the first natural course of the recessively inherited lethal form of human fetal cardiomyopathy caused by loss of Nexilin function. The affected family had uneventful pregnancies until week 23-24, followed by fetal death at week 24-30, characterized by cardiomegaly and endocardial fibroelastosis.

Lövskogarna var för 200 – 300 år sedan överexploaterade i södra Sverige och förekommer idag främst i mindre och fragmenterade områden. Efterfrågan på barrsågtimmer från industrierna steg och på de tidigare lövbevuxna markerna planterades istället gran och tall. Detta resulterade i att lövträden aktivt började rensas bort med herbicider som t.ex. Hormoslyr. Idag har lövträden en större betydelse i skogarna och framtiden ser mer positiv ut tack vare t.ex. certifieringar, ny teknik, biologisk mångfald, rekreation och ur en klimatsynpunkt. Den positiva trenden syns hos markägare då fler värnar om lövträden och i olika utsträckning väljer att satsa på dem. En tydlig indikator är den ökade försäljningen och en högre efterfrågan av lövplantor, som bl.a. setts hos Södra skogsplantor. Syftet med studien är att undersöka de främsta faktorerna som styr intresset hos markägare till att plantera lövträd, och hur skogsinspektorerna upplever intresse för lövträd bland markägarna. Undersökningen har utförts genom litteraturstudier, en enkätundersökning riktad till markägare samt intervjuer med markägare och skogsinspektorer. Enkätundersökningen och intervjuerna riktade sig till Södra skogsägarnas medlemmar som beställt lövplantor från Södra skogsplantor. Skogsinspektorerna som intervjuades var verksamma vid Södras verksamhetsområde i Oskarström. I litteraturstudien söktes relevant information om lövträd och lövplantor huvudsakligen i databasen Primo. Enligt resultatet från enkäten och intervjuerna var de främsta anledningarna till att markägarna valt att plantera lövplantor natur- och landskapsvård samt klimatpåverkan. Anledningen till att det är viktigt för markägarna med lövträd handlar bland annat om en förhoppning att lövinblandningen skapar en skog som är mer beständig mot storm, torka, rotröta etc. Andra faktorer som visade sig ha stor betydelse var ståndortsanpassning, miljöhänsyn, produktionsmål, rekreation och nyfikenhet. I de flesta fall planterades det triviallöv följt a, 200 – 300 years ago the broad-leaved forests were overexploited in southern Sweden and today they only exist in smaller fragmented sites (area). The demand for timber from the industries increased and the earlier broad-leaved forests were instead planted with the fast-growing tree species such as spruce and pine which led to broad-leaved trees being cleared away with the herbicide Hormoslyr. Today broad-leaved trees have a bigger importance in the Swedish forests and the future looks brighter thanks to for example the certification, new technique, biodiversity, recreation and from a climate perspective. Among landowners there is a vaguely positive trend seen where it's more common to enshrine broad-leaved trees and in different ways of extension choosing to invest in broad-leaved trees. A clear indicator is a higher sale and increased demand for broad-leaved trees in Södra nursery. The aim with the survey is to investigate the prime factors that direct the interest among landowners to plant broad-leaved trees and how inspectors experience the interests for broad-leaved trees among landowners. The survey have been conducted by a literature study, a questionnaire survey with landowners and interviews with landowners and inspectors. The survey study and the interviews were addressed to Södras members that had ordered broad-leaved plants from Södra nursery and the interviews were done with the inspectors in Södras area of operation in Oskarström. In the literature study relevant information about broad-leaved trees was searched mainly in the databases Primo and Google Scholar. According to the results of the questionnaire survey and the interviews, the main factors why landowners chose to plant broad-leaved plants were nature and landscape conservation and climate impact. The reason why broad-leaved trees were important for landowners is for example a hope that an increased amount of broad-leaved trees will create a forest that is more resistant to storms, droughts and

RAD21 encodes a key component of the cohesin complex, and variants in RAD21 have been associated with Cornelia de Lange Syndrome (CdLS). Limited information on phenotypes attributable to RAD21 variants and genotype–phenotype relationships is currently published. We gathered a series of 49 individuals from 33 families with RAD21 alterations [24 different intragenic sequence variants (2 recurrent), 7 unique microdeletions], including 24 hitherto unpublished cases. We evaluated consequences of 12 intragenic variants by protein modelling and molecular dynamic studies. Full clinical information was available for 29 individuals. Their phenotype is an attenuated CdLS phenotype compared to that caused by variants in NIPBL or SMC1A for facial morphology, limb anomalies, and especially for cognition and behavior. In the 20 individuals with limited clinical information, additional phenotypes include Mungan syndrome (in patients with biallelic variants) and holoprosencephaly, with or without CdLS characteristics. We describe several additional cases with phenotypes including sclerocornea, in which involvement of the RAD21 variant is uncertain. Variants were frequently familial, and genotype–phenotype analyses demonstrated striking interfamilial and intrafamilial variability. Careful phenotyping is essential in interpreting consequences of RAD21 variants, and protein modeling and dynamics can be helpful in determining pathogenicity. The current study should be helpful when counseling families with a RAD21 variation.

RAD21 encodes a key component of the cohesin complex, and variants in RAD21 have been associated with Cornelia de Lange Syndrome (CdLS). Limited information on phenotypes attributable to RAD21 variants and genotype–phenotype relationships is currently published. We gathered a series of 49 individuals from 33 families with RAD21 alterations [24 different intragenic sequence variants (2 recurrent), 7 unique microdeletions], including 24 hitherto unpublished cases. We evaluated consequences of 12 intragenic variants by protein modelling and molecular dynamic studies. Full clinical information was available for 29 individuals. Their phenotype is an attenuated CdLS phenotype compared to that caused by variants in NIPBL or SMC1A for facial morphology, limb anomalies, and especially for cognition and behavior. In the 20 individuals with limited clinical information, additional phenotypes include Mungan syndrome (in patients with biallelic variants) and holoprosencephaly, with or without CdLS characteristics. We describe several additional cases with phenotypes including sclerocornea, in which involvement of the RAD21 variant is uncertain. Variants were frequently familial, and genotype–phenotype analyses demonstrated striking interfamilial and intrafamilial variability. Careful phenotyping is essential in interpreting consequences of RAD21 variants, and protein modeling and dynamics can be helpful in determining pathogenicity. The current study should be helpful when counseling families with a RAD21 variation.

Arthrogryposis multiplex congenita (AMC) is a heterogeneous disorder characterized by multiple joint contractures often in association with other congenital abnormalities. Pretibial linear vertical creases are a rare finding associated with arthrogryposis, and the etiology of the specific condition is unknown. We aimed to genetically and clinically characterize a boy from a consanguineous family, presenting with AMC and pretibial vertical linear creases on the shins. Whole exome sequencing and variant analysis revealed homozygous novel missense variants of ECEL1 (c.1163T > C, p.Leu388Pro, NM_004826) and MUSK (c.2572C > T, p.Arg858Cys, NM_005592). Both variants are predicted to have deleterious effects on the protein function, with amino acid positions highly conserved among species. The variants segregated in the family, with healthy mother, father, and sister being heterozygous carriers and the index patient being homozygous for both mutations. We report on a unique patient with a novel ECEL1 homozygous mutation, expanding the phenotypic spectrum of Distal AMC Type 5D to include vertical linear skin creases. The homozygous mutation in MUSK is of unknown clinical significance. MUSK mutations have previously shown to cause congenital myasthenic syndrome, a neuromuscular disorder with defects in the neuromuscular junction.

Mutations in genes involved in the cilium-centrosome complex are called ciliopathies. Meckel-Gruber syndrome (MKS) is a ciliopathic lethal autosomal recessive syndrome characterized by genetically and clinically heterogeneous manifestations, including renal cystic dysplasia, occipital encephalocele and polydactyly. Several genes have previously been associated with MKS and MKS-like phenotypes, but there are still genes remaining to be discovered. We have used whole exome sequencing (WES) to uncover the genetics of a suspected autosomal recessive Meckel syndrome phenotype in a family with two affected fetuses. RNA studies and histopathological analysis was performed for further delineation. WES lead to identification of a homozygous nonsense mutation c.256C>T (p.Arg86*) in CEP55 (centrosomal protein of 55 kDa) in the affected fetus. The variant has previously been identified in carriers in low frequencies, and segregated in the family. CEP55 is an important centrosomal protein required for the mid-body formation at cytokinesis. Our results expand the list of centrosomal proteins implicated in human ciliopathies and provide evidence for an essential role of CEP55 during embryogenesis and development of disease.

BACKGROUND: Reduced lung function in patients with chronic obstructive pulmonary disease (COPD) is likely due to both environmental and genetic factors. We report here a targeted high-throughput DNA sequencing approach to identify new and previously known genetic variants in a set of candidate genes for COPD. METHODS: Exons in 22 genes implicated in lung development as well as 61 genes and 10 genomic regions previously associated with COPD were sequenced using individual DNA samples from 68 cases with moderate or severe COPD and 66 controls matched for age, gender and smoking. Cases and controls were selected from the Obstructive Lung Disease in Northern Sweden (OLIN) studies. RESULTS: In total, 37 genetic variants showed association with COPD (p < 0.05, uncorrected). Several variants previously discovered to be associated with COPD from genetic genome-wide analysis studies were replicated using our sample. Two high-risk variants were followed-up for functional characterization in a large eQTL mapping study of 1,111 human lung specimens. The C allele of a synonymous variant, rs8040868, predicting a p.(S45=) in the gene for cholinergic receptor nicotinic alpha 3 (CHRNA3) was associated with COPD (p = 8.8 x 10(-3)). This association remained (p = 0.003 and OR = 1.4, 95 % CI 1.1-1.7) when analysing all available cases and controls in OLIN (n = 1,534). The rs8040868 variant is in linkage disequilibrium with rs16969968 previously associated with COPD and altered expression of the CHRNA5 gene. A follow-up analysis for detection of expression quantitative trait loci revealed that rs8040868-C was found to be significantly associated with a decreased expression of the nearby gene cholinergic receptor, nicotinic, alpha 5 (CHRNA5) in lung tissue. CONCLUSION: Our data replicate previous result suggesting CHRNA5 as a candidate gene for COPD and rs8040868 as a risk variant for the development of COPD in the Swedish population.

Background: Reduced lung function in patients with chronic obstructive pulmonary disease (COPD) is likely due to both environmental and genetic factors. We report here a targeted high-throughput DNA sequencing approach to identify new and previously known genetic variants in a set of candidate genes for COPD. Methods: Exons in 22 genes implicated in lung development as well as 61 genes and 10 genomic regions previously associated with COPD were sequenced using individual DNA samples from 68 cases with moderate or severe COPD and 66 controls matched for age, gender and smoking. Cases and controls were selected from the Obstructive Lung Disease in Northern Sweden (OLIN) studies. Results: In total, 37 genetic variants showed association with COPD (p <0.05, uncorrected). Several variants previously discovered to be associated with COPD from genetic genome-wide analysis studies were replicated using our sample. Two high-risk variants were followed-up for functional characterization in a large eQTL mapping study of 1,111 human lung specimens. The C allele of a synonymous variant, rs8040868, predicting a p.(S45=) in the gene for cholinergic receptor nicotinic alpha 3 (CHRNA3) was associated with COPD (p = 8.8 x 10(-3)). This association remained (p = 0.003 and OR = 1.4, 95 % CI 1.1-1.7) when analysing all available cases and controls in OLIN (n = 1,534). The rs8040868 variant is in linkage disequilibrium with rs16969968 previously associated with COPD and altered expression of the CHRNA5 gene. A follow-up analysis for detection of expression quantitative trait loci revealed that rs8040868-C was found to be significantly associated with a decreased expression of the nearby gene cholinergic receptor, nicotinic, alpha 5 (CHRNA5) in lung tissue. Conclusion: Our data replicate previous result suggesting CHRNA5 as a candidate gene for COPD and rs8040868 as a risk variant for the development of COPD in the Swedish population.