Exonic trinucleotide repeat expansions in ZFHX3 cause spinocerebellar ataxia type 4: A poly-glycine disease.

Autosomal-dominant ataxia with sensory and autonomic neuropathy is a highly specific combined phenotype that we described in two Swedish kindreds in 2014; its genetic cause had remained unknown. Here, we report the discovery of exonic GGC trinucleotide repeat expansions, encoding poly-glycine, in zinc finger homeobox 3 (ZFHX3) in these families. The expansions were identified in whole-genome datasets within genomic segments that all affected family members shared. Non-expanded alleles carried one or more interruptions within the repeat. We also found ZFHX3 repeat expansions in three additional families, all from the region of Skåne in southern Sweden. Individuals with expanded repeats developed balance and gait disturbances at 15 to 60 years of age and had sensory neuropathy and slow saccades. Anticipation was observed in all families and correlated with different repeat lengths determined through long-read sequencing in two family members. The most severely affected individuals had marked autonomic dysfunction, with severe orthostatism as the most disabling clinical feature. Neuropathology revealed p62-positive intracytoplasmic and intranuclear inclusions in neurons of the central and enteric nervous system, as well as alpha-synuclein positivity. ZFHX3 is located within the 16q22 locus, to which spinocerebellar ataxia type 4 (SCA4) repeatedly had been mapped; the clinical phenotype in our families corresponded well with the unique phenotype described in SCA4, and the original SCA4 kindred originated from Sweden. ZFHX3 has known functions in neuronal development and differentiation n both the central and peripheral nervous system. Our findings demonstrate that SCA4 is caused by repeat expansions in ZFHX3.

The long non-coding RNA LINC00707 interacts with Smad proteins to regulate TGFß signaling and cancer cell invasion.

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor ß (TGFß) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFß targets, yet, their mechanism of action and biological role in cancer remain poorly understood. METHODS: Whole-genome transcriptomics identified lncRNA genes regulated by TGFß. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation, chromatin isolation by RNA purification and protein screening sought mechanistic evidence. Functional validation of TGFß-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue. RESULTS: Transcriptomics of TGFß signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFß led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFß, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFß stimulation, LINC00707 dissociated from the Smad complex, which allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues. CONCLUSIONS: LINC00707 interacts with Smad proteins and limits the output of TGFß signaling, which decreases LINC00707 expression, thus favoring cancer cell invasion. Video Abstract.

Loss of Nexilin function leads to a recessive lethal fetal cardiomyopathy characterized by cardiomegaly and endocardial fibroelastosis.

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.

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

The Value of Whole-Genome Sequencing for Mitochondrial DNA Population Studies: Strategies and Criteria for Extracting High-Quality Mitogenome Haplotypes.

Whole-genome sequencing (WGS) data present a readily available resource for mitochondrial genome (mitogenome) haplotypes that can be utilized for genetics research including population studies. However, the reconstruction of the mitogenome is complicated by nuclear mitochondrial DNA (mtDNA) segments (NUMTs) that co-align with the mtDNA sequences and mimic authentic heteroplasmy. Two minimum variant detection thresholds, 5% and 10%, were assessed for the ability to produce authentic mitogenome haplotypes from a previously generated WGS dataset. Variants associated with NUMTs were detected in the mtDNA alignments for 91 of 917 (~8%) Swedish samples when the 5% frequency threshold was applied. The 413 observed NUMT variants were predominantly detected in two regions (nps 12,612-13,105 and 16,390-16,527), which were consistent with previously documented NUMTs. The number of NUMT variants was reduced by ~97% (400) using a 10% frequency threshold. Furthermore, the 5% frequency data were inconsistent with a platinum-quality mitogenome dataset with respect to observed heteroplasmy. These analyses illustrate that a 10% variant detection threshold may be necessary to ensure the generation of reliable mitogenome haplotypes from WGS data resources.

Discovery of Novel Sequences in 1,000 Swedish Genomes.

Novel sequences (NSs), not present in the human reference genome, are abundant and remain largely unexplored. Here, we utilize de novo assembly to study NS in 1,000 Swedish individuals first sequenced as part of the SweGen project revealing a total of 46 Mb in 61,044 distinct contigs of sequences not present in GRCh38. The contigs were aligned to recently published catalogs of Icelandic and Pan-African NSs, as well as the chimpanzee genome, revealing a great diversity of shared sequences. Analyzing the positioning of NS across the chimpanzee genome, we find that 2,807 NS align confidently within 143 chimpanzee orthologs of human genes. Aligning the whole genome sequencing data to the chimpanzee genome, we discover ancestral NS common throughout the Swedish population. The NSs were searched for repeats and repeat elements: revealing a majority of repetitive sequence (56%), and enrichment of simple repeats (28%) and satellites (15%). Lastly, we align the unmappable reads of a subset of the thousand genomes data to our collection of NS, as well as the previously published Pan-African NS: revealing that both the Swedish and Pan-African NS are widespread, and that the Swedish NSs are largely a subset of the Pan-African NS. Overall, these results highlight the importance of creating a more diverse reference genome and illustrate that significant amounts of the NS may be of ancestral origin.

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting.

Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base-pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.

Molecular characterization of a large unselected cohort of metastatic colorectal cancers in relation to primary tumor location, rare metastatic sites and prognosis.

Background: We have reported that BRAF V600E mutations and microsatellite instability-high (MSI-H) are more prevalent in a population-based cohort of metastatic colorectal cancer (mCRC) patients than has been reported from clinical trials or hospital-based patient groups. The aim was to explore if other mutations in mCRC differ in prevalence between these cohorts in relation to mismatch repair status and primary tumor location and if presence of bone or brain metastases is associated with any mutations.Material and methods: A population-based cohort of 798 mCRC patients from three regions in Scandinavia was used. Forty-four cancer related genes were investigated in a custom designed Ampliseq hotspot panel. Differences in survival were analyzed using the Kaplan-Meier estimator and the Cox regression analysis.Results: Determination of mutations was possible in 449/501 patients for 40/44 genes. Besides BRAF V600E, seen in 19% of the tumors, none of the other mutations appeared more prevalent than in trial cohorts. BRAF V600E and MSI-H, seen in 8%, were associated with poor prognosis as was right-sided primary tumor location (39%) when compared to left-sided and rectum together; however, in a multivariable regression, only the BRAF mutation retained its statistical significance. No other mutations were associated with poor prognosis. ERBB2 alterations were more common if bone metastases were present at diagnosis (17% vs. 4%, p = .011). No association was found for brain metastases. Fifty-two percent had an alteration that is treatable with an FDA-approved targeted therapy, chiefly by EGFR-inhibitor for RAS wild-type and a check-point inhibitor for MSI-H tumors.Conclusions: Right-sided tumor location, BRAF V600E mutations, but no other investigated mutation, and MSI-H are more commonly seen in an unselected cohort than is reported from clinical patient cohorts, likely because they indicate poor prognosis. Half of the patients have a tumor that is treatable with an already FDA-approved targeted drug for mCRC.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.

Invasive cervical tumors with high and low HPV titer represent molecular subgroups with different disease etiology.

Invasive cervical cancer (ICC) with very low titer of high-risk human papillomavirus (HPV) has worse clinical outcome than cases with high titer, indicating a difference in molecular etiology. Fresh-frozen ICC tumors (n = 49) were classified into high- and low-HPV-titer cases using real-time PCR-based HPV genotyping. The mutation spectra were studied using the AmpliSeq Comprehensive Cancer Panel and the expression profiles using total RNA sequencing, and the results were validated using the AmpliSeq Transcriptome assay. HPV DNA genotyping and RNA sequencing showed that 16.6% of ICC tumors contained very low levels of HPV DNA and HPV transcripts. Tumors with low HPV levels had more mutations with a high allele frequency and fewer mutations with low allele frequency relative to tumors with high HPV titer. A number of genes showed significant expression differences between HPV titer groups, including genes with somatic mutations. Gene ontology and pathway analyses implicated the enrichment of genes involved in DNA replication, cell cycle control and extracellular matrix in tumors with low HPV titer. The results indicate that in low titer tumors, HPVs act as trigger of cancer development whereas somatic mutations are clonally selected and become drivers of the tumor development process. In contrast, in tumors with high HPV titer the expression of HPV oncoproteins plays a major role in tumor development and the many low frequency somatic mutations represent passengers. This putative subdivision of invasive cervical tumors may explain the higher radiosensitivity of ICC tumors with high HPV titer and thereby have consequences for clinical management.

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26.

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G > A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. To our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome.

PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c.

The FADS1 and FADS2 genes in the FADS cluster encode the rate-limiting enzymes in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). Genetic variation in this region has been associated with a large number of diseases and traits many of them correlated to differences in metabolism of PUFAs. However, the causative variants leading to these associations have not been identified. Here we find that the multiallelic rs174557 located in an AluYe5 element in intron 1 of FADS1 is functional and lies within a PATZ1 binding site. The derived allele of rs174557, which is the common variant in most populations, diminishes binding of PATZ1, a transcription factor conferring allele-specific downregulation of FADS1. The PATZ1 binding site overlaps with a SP1 site. The competitive binding between the suppressive PATZ1 and the activating complex of SP1 and SREBP1c determines the enhancer activity of this region, which regulates expression of FADS1.

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.

A novel ECEL1 mutation expands the phenotype of distal arthrogryposis multiplex congenita type 5D to include pretibial vertical skin creases.

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.

A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders.

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. METHODS: We 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. RESULTS: In 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. CONCLUSIONS: Our 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.

Identification of novel genetic causes of Rett syndrome-like phenotypes.

BACKGROUND: The aim of this work was to identify new genetic causes of Rett-like phenotypes using array comparative genomic hybridisation and a whole exome sequencing approach. METHODS AND RESULTS: We studied a cohort of 19 Portuguese patients (16 girls, 3 boys) with a clinical presentation significantly overlapping Rett syndrome (RTT). Genetic analysis included filtering of the single nucleotide variants and indels with preference for de novo, homozygous/compound heterozygous, or maternally inherited X linked variants. Examination by MRI and muscle biopsies was also performed. Pathogenic genomic imbalances were found in two patients (10.5%): an 18q21.2 deletion encompassing four exons of the TCF4 gene and a mosaic UPD of chromosome 3. Variants in genes previously implicated in neurodevelopmental disorders (NDD) were identified in six patients (32%): de novo variants in EEF1A2, STXBP1 and ZNF238 were found in three patients, maternally inherited X linked variants in SLC35A2, ZFX and SHROOM4 were detected in two male patients and one homozygous variant in EIF2B2 was detected in one patient. Variants were also detected in five novel NDD candidate genes (26%): we identified de novo variants in the RHOBTB2, SMARCA1 and GABBR2 genes; a homozygous variant in EIF4G1; compound heterozygous variant in HTT. CONCLUSIONS: Network analysis reveals that these genes interact by means of protein interactions with each other and with the known RTT genes. These findings expand the phenotypical spectrum of previously known NDD genes to encompass RTT-like clinical presentations and identify new candidate genes for RTT-like phenotypes.

A novel variant in MYLK causes thoracic aortic dissections: genotypic and phenotypic description.

BACKGROUND: Mutations in MYLK cause non-syndromic familial thoracic aortic aneurysms and dissections (FTAAD). Very little is known about the phenotype of affected families. We sought to characterize the aortic disease and the presence of other vascular abnormalities in FTAAD caused by a deletion in MYLK and to compare thoracic aortic diameter and stiffness in mutation carriers and non-carriers. METHODS: We studied FTAAD in a 5-generation family that included 19 living members. Exome sequencing was performed to identify the underlying gene defect. Aortic elastic properties measured by TTE, MRI and pulse wave velocity were then compared between mutation carriers and non-carriers. RESULTS: Exome sequencing led to the identification of a 2-bp deletion in MYLK (c3272_3273del, p.Ser1091*) that led to a premature stop codon and nonsense-mediated decay. Eleven people were mutation carriers and eight people were non-carriers. Five aortic ruptures or dissections occurred in this family, with two survivors. There were no differences in aortic diameter or stiffness between carriers and non-carriers of the mutation. CONCLUSIONS: Individuals carrying this deletion in MYLK have a high risk of presenting with an acute aortic dissection or rupture. Aortic events occur over a wide range of ages and are not always preceded by obvious aortic dilatation. Aortic elastic properties do not differ between carriers and non-carriers of this mutation, rendering it uncertain whether and when carriers should undergo elective prophylactic surgery.

MuSK: a new target for lethal fetal akinesia deformation sequence (FADS).

BACKGROUND: Fetal akinesia deformation sequence syndrome (FADS, OMIM 208150) is characterised by decreased fetal movement (fetal akinesia) as well as intrauterine growth restriction, arthrogryposis, and developmental anomalies (eg, cystic hygroma, pulmonary hypoplasia, cleft palate, and cryptorchidism). Mutations in components of the acetylcholine receptor (AChR) pathway have previously been associated with FADS. METHODS AND RESULTS: We report on a family with recurrent fetal loss, where the parents had five affected fetuses/children with FADS and one healthy child. The fetuses displayed no fetal movements from the gestational age of 17 weeks, extended knee joints, flexed hips and elbows, and clenched hands. Whole exome sequencing of one affected fetus and the parents was performed. A novel homozygous frameshift mutation was identified in muscle, skeletal receptor tyrosine kinase (MuSK), c.40dupA, which segregated with FADS in the family. Haplotype analysis revealed a conserved haplotype block suggesting a founder mutation. MuSK (muscle-specific tyrosine kinase receptor), a component of the AChR pathway, is a main regulator of neuromuscular junction formation and maintenance. Missense mutations in MuSK have previously been reported to cause congenital myasthenic syndrome (CMS) associated with AChR deficiency. CONCLUSIONS: To our knowledge, this is the first report showing that a mutation in MuSK is associated with FADS. The results support previous findings that CMS and/or FADS are caused by complete or severe functional disruption of components located in the AChR pathway. We propose that whereas milder mutations of MuSK will cause a CMS phenotype, a complete loss is lethal and will cause FADS.

Transcription factor ZBED6 affects gene expression, proliferation, and cell death in pancreatic beta cells.

We have investigated whether the recently discovered transcription factor, zinc finger BED domain-containing protein 6 (ZBED6), is expressed in insulin-producing cells and, if so, to what extent it affects beta cell function. ZBED6 was translated from a ZC3H11A transcript in which the ZBED6-containing intron was retained. ZBED6 was present in mouse ßTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. We propose that ZBED6 supports proliferation and survival of beta cells, possibly at the expense of specialized beta cell function-i.e., insulin production-because (i) the nuclear ZBED6 were the predominant forms in rapidly proliferating ßTC-6 cells, but not in human islet cells; (ii) down-regulation of ZBED6 in ßTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell-cycle arrest, increased expression of beta cell-specific genes, and higher rates of apoptosis; (iii) silencing of ZBED6 in the human PANC-1 duct cell line reduced proliferation rates; and (iv) ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis, and apoptosis. Furthermore, it is possible that beta cells, by switching from full length to a truncated form of ZBED6, can decide the subcellular localization of ZBED6, thereby achieving differential ZBED6-mediated transcriptional regulation.

Mutation in CEP63 co-segregating with developmental dyslexia in a Swedish family.

Developmental dyslexia is the most common learning disorder in children. Problems in reading and writing are likely due to a complex interaction of genetic and environmental factors, resulting in reduced power of studies of the genetic factors underlying developmental dyslexia. Our approach in the current study was to perform exome sequencing of affected and unaffected individuals within an extended pedigree with a familial form of developmental dyslexia. We identified a two-base mutation, causing a p.R229L amino acid substitution in the centrosomal protein 63 kDa (CEP63), co-segregating with developmental dyslexia in this pedigree. This mutation is novel, and predicted to be highly damaging for the function of the protein. 3D modelling suggested a distinct conformational change caused by the mutation. CEP63 is localised to the centrosome in eukaryotic cells and is required for maintaining normal centriole duplication and control of cell cycle progression. We found that a common polymorphism in the CEP63 gene had a significant association with brain white matter volume. The brain regions were partly overlapping with the previously reported region influenced by polymorphisms in the dyslexia susceptibility genes DYX1C1 and KIAA0319. We hypothesise that CEP63 is particularly important for brain development and might control the proliferation and migration of cells when those two events need to be highly coordinated.