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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.

Background: ERF-related craniosynostosis are a rare, complex, premature trisutural fusion associated with a broad spectrum of clinical features and heterogeneous aetiology. Here we describe two cases with the same pathogenic variant and a detailed description of their clinical course. Case presentation: Two subjects; a boy with a BLSS requiring repeated skull expansions and his mother who had been operated once for sagittal synostosis. Both developed intracranial hypertension at some point during the course, which was for both verified by formal invasive intracranial pressure monitoring. Exome sequencing revealed a pathogenic truncating frame shift variant in the ERF gene. Conclusions: Here we describe a boy and his mother with different craniosynostosis patterns, but both with verified intracranial hypertension and heterozygosity for a truncating variant of ERF c.1201_1202delAA (p.Lys401Glufs*10). Our work provides supplementary evidence in support of previous phenotypic descriptions of ERF-related craniosynostosis, particularly late presentation, an evolving synostotic pattern and variable expressivity even among affected family members.

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: Genealogy and molecular genetic studies of a Swedish river valley population resulted in a large pedigree, showing that the hereditary hemochromatosis (HH) HFE/p.C282Y mutation is inherited with other recessive disorders such as Wilson´s disease (WND), a rare recessive disorder of copper overload. The population also contain individuals with the Swedish long QT syndrome (LQTS1) founder mutation (KCNQ1/p.Y111C) which in homozygotes causes the Jervell & Lange Nielsen syndrome (JLNS) and hearing loss (HL). Aims of the study were to test whether the Swedish long QT founder mutation originated in an ancestral HFE family and if carriers had an increased risk for hemochromatosis (HH), a treatable disorder. We also aimed to identify the pathogenic mutation causing the hearing loss disorder segregating in the pedigree. Methods: LQTS patients were asked about their ancestry and possible origin in a HH family. They were also offered a predictive testing for the HFE genotype. Church books were screened for families with hearing loss. One HH family had two members with hearing loss, who underwent molecular genetic analysis of the LQTS founder mutation, connexin 26 and thereafter exome sequencing. Another family with hearing loss in repeat generations was also analyzed for connexin 26 and underwent exome sequencing. Results: Of nine LQTS patients studied, four carried a HFE mutation (two p.C282Y, two p.H63D), none was homozygous. Three LQTS patients confirmed origin in a female founder ( b 1694, identical to AJ b 1694, a HFE pedigree member from the Fax river. Her descent of 44 HH families, included also 29 families with hearing loss (HL) suggesting JLNS. Eleven LQTS probands confirmed origin in a second founder couple (b 1614/1605) in which the woman b 1605 was identical to a HFE pedigree member from the Fjällsjö river. In her descent there were not only 64 HH, six WND families, one JLNS, but also 48 hearing loss families. Most hearing loss was non syndromic and

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

Background: Noonan syndrome (NS), a heterogeneous developmental disorder associated with variable clinical expression including short stature, congenital heart defect, unusual pectus deformity and typical facial features, is caused by activating mutations in genes involved in the RAS-MAPK signaling pathway. Case presentation: Here, we present a clinical and molecular characterization of a small family with Noonan syndrome. Comprehensive mutation analysis of NF1, PTPN11, SOS1, CBL, BRAF, RAF1, SHOC2, MAP2K2, MAP2K1, SPRED1, NRAS, HRAS and KRAS was performed using targeted next-generation sequencing. The result revealed a recurrent mutation in NRAS, c.179G > A (p.G60E), in the index patient. This mutation was inherited from the index patient's father, who also showed signs of NS. Conclusions: We describe clinical features in this family and review the literature for genotype-phenotype correlations for NS patients with mutations in NRAS. Neither of affected individuals in this family presented with juvenile myelomonocytic leukemia (JMML), which together with previously published results suggest that the risk for NS individuals with a germline NRAS mutation developing JMML is not different from the proportion seen in other NS cases. Interestingly, 50 % of NS individuals with an NRAS mutation (including our family) present with lentigines and/or Cafe-au-lait spots. This demonstrates a predisposition to hyperpigmented lesions in NRAS-positive NS individuals. In addition, the affected father in our family presented with a hearing deficit since birth, which together with lentigines are two characteristics of NS with multiple lentigines (previously LEOPARD syndrome), supporting the difficulties in diagnosing individuals with RASopathies correctly. The clinical and genetic heterogeneity observed in RASopathies is a challenge for genetic testing. However, next-generation sequencing technology, which allows screening of a large number of genes simultaneously, will facilitate

Background Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype. phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. Methods Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. Results The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype. phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. Conclusion A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.

Purpose: To describe morphological and functional changes in a single patient with multifocal Best vitelliform macular dystrophy (BVMD) and to perform a genotype/phenotype correlation. Methods: The proband with multifocal BVMD and three of her family members were examined with electrooculography (EOG), full-field electroretinography (full-field ERG), multifocal electroretinography (mfERG) and optical coherence tomography (OCT). Genomic DNA was screened for mutation in the BEST1 gene by DNA sequencing analysis. Results: The proband was observed regularly during a follow-up period of 4 years. Full-field ERG demonstrated reduced and delayed responses of both rods and cones. OCT demonstrated intra- and subretinal fluid which seemed to fluctuate with periods of stress, similar to that seen in chronic central serous chorioretinopathy. Two distinct heterozygous BEST1 mutations were identified in the proband, the recurrent p.R141H mutation and the p.P233A mutation. Heterozygous p.R141H mutations were also identified in two family members, while p.P233A was a de novo mutation. Abnormal EOG findings were observed in both the proband and in the carriers of p.R141H. Heterozygous carriers showed delayed implicit times in a- and b-waves of combined total rod and cone full-field ERG responses. Conclusions: The p.R141H mutation is frequently seen together with multifocal vitelliform retinopathy and biallelic mutations in BEST1. Our results show that carriers of the p.R141H mutation are clinically unaffected but present with abnormal EOG and full-field ERG findings. A patient with biallelic mutations of the BEST1 gene, causing multifocal BVMD with progressive, widespread functional disturbance of the retina, confirmed by full-field and mfERG is described.

Purpose: To present the clinical and electrophysiological findings in four members of a family with Best vitelliform macular dystrophy (BVMD) and angle-closure glaucoma (ACG). Methods: Four members of a family with BVMD were examined clinically, including visual acuity, slit-lamp examination, biomicroscopy, Goldmann applanation tonometry and gonioscopy. Measurements of the anterior chamber depth and axial length, visual field, optical coherence tomography, full-field electroretinography, multifocal electroretinography and electrooculography were performed. In addition molecular genetic analysis of the bestrophin-1 gene (BEST1), the microphthalmia-associated transcription factor gene (MITF) and the cone-rod homeobox gene (CRX) were performed. Results: Four family members with the c.253T>C p.Y85H mutation in the BEST1 gene and BVMD in different stages also exhibited anterior segment abnormalities such as shallow anterior chambers (two cases), and reduced axial lengths in all cases. Microphthalmos (axial length <= 20mm) was found in the index patient and in her son. Hyperopia was found in all four examined patients. Closed angles/narrow angles were observed in patients with microphthalmos. The index patient developed ACG at the age of 12 years. Her son inherited microphthalmos, severe hyperopia, and narrow angles. He is at risk of developing ACG. No pathogenic mutation of the MITF or the CRX genes was detected in the index patient. Conclusions: BVMD could be associated with anterior segment abnormalities such as shallow anterior chambers, closed/narrow anterior chamber angles and ACG. Ophthalmologists should be aware of the association between ACG and BVMD. Examination of the anterior segment, gonioscopy and intraocular pressure control are recommended in patients with BVMD.

Millions of people are daily exposed to high levels of noise. Consequently, noise-induced hearing loss (NIHL) is one of the most important occupational health hazards worldwide. In this study, we performed an association study for NIHL based on a candidate gene approach. 644 Single Nucleotide Polymorphisms (SNPs) in 53 candidate genes were analyzed in two independent NIHL sample sets, a Swedish set and part of a Polish set. Eight SNPs with promising results were selected and analysed in the remaining part of the Polish samples. One SNP in PCDH15 (rs7095441), resulted in significant associations in both sample sets while two SNPs in MYH14 (rs667907 and rs588035), resulted in significant associations in the Polish sample set and significant interactions with noise exposure level in the Swedish sample set. Calculation of odds ratios revealed a significant association of rs588035 with NIHL in the Swedish high noise exposure level group. Our studies suggest that PCDH15 and MYH14 may be NIHL susceptibility genes, but further replication in independent sample sets is mandatory.

Noonan Syndrome (NS) and Neurofibromatosis type I (NF1) belong to a group of clinically related disorders that share a common pathogenesis, dysregulation of the RAS-MAPK pathway. NS is characterized by short stature, heart defect, pectus deformity and facial dysmorphism, while skin manifestations, skeletal defects, Lisch nodules and neurofibromas are characteristic of NF1. Both disorders display considerable clinical variability. Features of NS have been observed in individuals with NF1 - a condition known as Neurofibromatosis-Noonan Syndrome (NFNS). The major gene causing NFNS is NF1. Rarely, a mutation in PTPN11 in addition to an NF1 mutation is present. We present the clinical and molecular characterization of a family displaying features of both NS and NF1, with complete absence of neurofibromas. To investigate the etiology of the phenotype, mutational analysis of NF1 was conducted, revealing a novel missense mutation in exon 24, p.L1390F, affecting the GAP-domain. Additional RAS-MAPK pathway genes were examined, but no additional mutations were identified. We confirm that NF1 mutations are involved in the etiology of NFNS. Furthermore, based on our results and previous studies we suggest that evaluation of the GAP-domain of NF1 should be prioritized in NFNS.

Noise-induced hearing loss (NIHL) is one of the most important occupational health hazards. Millions of people worldwide are exposed daily to harmful levels of noise. NIHL is a complex disease resulting from an interaction between genetic and environmental factors. Although the environmental risk factors have been studied extensively, little is known about the genetic factors. Heat-shock proteins (HSPs) are induced after exposure to severe noise. When first induced by exposure to moderate sound levels, they can protect the ear from damage from excessive noise exposure. This protection is highly variable between individuals. An association of HSP70 genes with NIHL has been described by Yang et al (2006) in a Chinese sample set of noise-exposed workers. In this study, three polymorphisms (rs1043618, rs1061581 and rs2227956) in HSP70-1, HSP70-2 and HSP70-hom, respectively, were genotyped in 206 Swedish and 238 Polish DNA samples of noise-exposed subjects and analyzed. One SNP, rs2227956 in HSP70-hom, resulted in a significant association with NIHL in both sample sets. In addition, rs1043618 and rs1061581 were significant in the Swedish sample set. Analysis of the haplotypes composed of the three SNPs revealed significant associations between NIHL and haplotype GAC in both sample sets and with haplotype CGT in the Swedish sample set. In conclusion, this study replicated the association of HSP70 genes with NIHL in a second and third independent noise-exposed sample set, hereby adding to the evidence that HSP70 genes may be NIHL susceptibility genes.