The TATA-box binding protein associated factor 1 (TAF1) protein is a key unit of the transcription factor II D complex that serves a vital function during transcription initiation. Variants of TAF1 have been associated with neurodevelopmental disorders, but TAF1 ’s molecular functions remain elusive. In this study, we present a five-generation family affected with X-linked intellectual disability that co-segregated with a TAF1 c.3568C>T, p.(Arg1190Cys) variant. All affected males presented with intellectual disability and dysmorphic features, while heterozygous females were asymptomatic and had completely skewed X-chromosome inactivation. We investigated the role of TAF1 and its association to neurodevelopment by creating the first complete knockout model of the TAF1 orthologue in zebrafish. A crucial function of human TAF1 during embryogenesis can be inferred from the model, demonstrating that intact taf1 is essential for embryonic development. Transcriptome analysis of taf1 zebrafish knockout revealed enrichment for genes associated with neurodevelopmental processes. In conclusion, we propose that functional TAF1 is essential for embryonic development and specifically neurodevelopmental processes.

Background One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

Objective De novo mutations contribute significantly to severe early‐onset genetic disorders. Even if the mutation is apparently de novo, there is a recurrence risk due to parental germ line mosaicism, depending on in which gonadal generation the mutation occurred.MethodsWe demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction.ResultsIn the first family, a TCOF1 variant c.3156C>T was identified in the proband with Treacher Collins syndrome. The variant affects splicing and was determined to be of paternal origin. It was present in <1% of the paternal germ cells, suggesting a very low recurrence risk. In the second family, the couple had undergone several unsuccessful pregnancies where a de novo mutation PTPN11 c.923A>C causing Noonan syndrome was identified. The variant was present in 40% of the paternal germ cells suggesting a high recurrence risk.ConclusionsOur findings highlight a successful strategy to identify the parental origin of mutations and to investigate the recurrence risk in couples that have undergone assisted reproduction with an unknown donor or in couples with gonadal mosaicism that will undergo preimplantation genetic diagnosis.

A much-debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. Here we present SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9. The methods were assessed using the human cell line HEK293, which was first re-sequenced at 18x coverage using highly accurate (HiFi) SMRT reads to get a detailed view of all on-and off-target binding regions. We then applied SMRT-OTS and Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 highconfidence gRNA binding sites identified by both methods. Twenty-five (45%) of these sites were not reported by off-target prediction software, either because they contained four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. We further discovered that a heterozygous SNP can cause allele-specific gRNA binding. Finally, by performing a de novo genome assembly of the HiFi reads, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome. This suggests that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown. In conclusion, the amplificationfree sequencing protocols revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference. Nevertheless, it is still unknown whether in vivo off-target editing would occur at these sites.

The double differential cross section of low momentum kaons (# 0.3 GeV͞c) from p 1 C collisions at subthreshold bombarding energies has been for the first time measured by the use of the CLAMSUD magnetic spectrometer installed at the CELSIUS storage ring. Invariant cross sections extracted from the data show a source rapidity shifted below the nucleon-nucleon rapidity, in agreement with the existence of multistep processes in the K 1 production cross section. The total cross section of the inclusive reaction 12 C͑p, K 1 ͒ at 1.2 GeV was extracted and compared with recent data systematics as a function of the proton bombarding energy. [S0031-9007 (98)06266-8] PACS numbers: 25.40.Ve, 25.40.QaWhile the production of pions at subthreshold energies, i.e., at energies lower than the threshold for free nucleonnucleon (NN) collisions, has been extensively studied both in proton-nucleus [1] and in nucleus-nucleus [2] collisions, only recently have the first data on the production of K 1 from proton-nucleus collisions in the subthreshold regime been published [3].

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.

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.

X-chromosome inactivation (XCI) analyses often assist in diagnostics of X-linked traits, however accurate assessment remains challenging with current methods. We developed a novel strategy using amplification-free Cas9 enrichment and Oxford nanopore technologies sequencing called XCI-ONT, to investigate and rigorously quantify XCI in human androgen receptor gene (AR) and human X-linked retinitis pigmentosa 2 gene (RP2). XCI-ONT measures methylation over 116 CpGs in AR and 58 CpGs in RP2, and separate parental X-chromosomes without PCR bias. We show the usefulness of the XCI-ONT strategy over the PCR-based golden standard XCI technique that only investigates one or two CpGs per gene. The results highlight the limitations of using the golden standard technique when the XCI pattern is partially skewed and the advantages of XCI-ONT to rigorously quantify XCI. This study provides a universal XCI-method on DNA, which is highly valuable in clinical and research framework of X-linked traits.