Noncoding Regulatory Region Research Articles

Assembly and annotation of the first complete mitochondrial genome of Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

The cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is a significant economic pest with a global distribution. Despite the increasing volume of literature on mitochondrial genome sequencing, complete mitochondrial genome sequences have not been reported for P. solenopsis yet. Here, we assembled the complete mitochondrial genome of P. solenopsis using high throughput DNA sequencing technology. The genome is 14,831 bp in length and is comprised of 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes. The total length of all PCGs is 9,678 bp, accounting for 65.2% of the overall mitogenome. Among the PCGs, atp8 is the smallest gene (99 bp) and nad5 is the largest (1,593 bp). Of the 13 PCGs, seven (nad3, cox3, atp6, atp8, cox2, cox1, nad2) are encoded on the majority strand (J-strand), while six (nad1, nad6, cob, nad4L, nad4, nad5) are on the minority strand (N-strand). The analysis revealed a predominant A+T base composition, making up 90.3% of the total genome. However, the non-coding regulatory control region (CR) is missing due to the non-overlapping endpoints of the linear assembly. This assembly provides comprehensive information for investigating the evolutionary relationships between scale insects and for the precise identification of insects.

Escherichia coli non-coding regulatory regions are highly conserved.

Microbial genome sequences are rapidly accumulating, enabling large-scale studies of sequence variation. Existing studies primarily focus on coding regions to study amino acid substitution patterns in proteins. However, non-coding regulatory regions also play a distinct role in determining physiologic responses. To investigate intergenic sequence variation on a large-scale, we identified non-coding regulatory region alleles across 2350 Escherichia coli strains. This 'alleleome' consists of 117781 unique alleles for 1169 reference regulatory regions (transcribing 1975 genes) at single base-pair resolution. We find that 64% of nucleotide positions are invariant, and variant positions vary in a median of just 0.6% of strains. Additionally, non-coding alleles are sufficient to recover E. coli phylogroups. We find that core promoter elements and transcription factor binding sites are significantly conserved, especially those located upstream of essential or highly-expressed genes. However, variability in conservation of transcription factor binding sites is significant both within and across regulons. Finally, we contrast mutations acquired during adaptive laboratory evolution with wild-type variation, finding that the former preferentially alter positions that the latter conserves. Overall, this analysis elucidates the wealth of information found in E. coli non-coding sequence variation and expands pangenomic studies to non-coding regulatory regions at single-nucleotide resolution.

Novel lambdapapillomavirus in northern sea otters Enhydra lutris kenyoni, associated with oral hyperplastic nodules.

A novel papillomavirus (PV) associated with hyperplastic nodules scattered over the muco-cutaneous border of the oral cavity of a dead, wild, subadult northern sea otter Enhydra lutris kenyoni (NSO) in 2004 in Homer, Alaska, USA, was genetically characterized. Primers for the amplification of 2 large overlapping DNA fragments that contained the complete genome of the NSO PV were designed. Sanger methodology generated sequences from which new specific primers were designed for the primer-walking approach. The NSO PV genome consists of 8085 nucleotides and contains an early region composed of E6, E7, E1, and E2 open reading frames (ORFs), an E4 ORF (contained within E2) lacking an in-frame proximal ATG start codon, an unusually long (907 nucleotide) stretch lacking any ORFs, a late region that contains the capsid genes L2 and L1, and a non-coding regulatory region (ncRR). This NSO PV has been tentatively named Enhydra lutris kenyoni PV2 (ElkPV2). Pairwise and multiple sequence alignments of the complete L1 ORF nucleotides and concatenated E1-E2-L1 amino acid sequences showed that the NSO PV is a novel PV, phylogenetically most closely related to southern sea otter PV1. The carboxy end of the E6 oncoprotein does not contain the PDZ-binding motif with a strong correlation with oncogenicity, suggesting a low-risk PV, which is in agreement with histopathological findings. However, the ElkPV2 E7 oncoprotein does contain the retinoblastoma (pRb) binding domain LXCXE (LQCYE in ElkPV2), associated with oncogenicity in some high-risk PVs. Further studies on the prevalence and clinical significance of ElkPV2 infections in NSO are needed.

A large deletion in a non-coding regulatory region leads to NFKB1 haploinsufficiency in two adult siblings

Mutations in NFkB pathway genes can cause inborn errors of immunity (IEI), with NFKB1 haploinsufficiency being a significant etiology for common variable immunodeficiency (CVID). Indeed, mutations in NFKB1 are found in 4 to 5% of in European and United States CVID cohorts, respectively; CVID representing almost ¼ of IEI patients in European countries registries. This case study presents a 49-year-old patient with respiratory infections, chronic diarrhea, immune thrombocytopenia, hypogammaglobulinemia, and secondary lymphoma. Comprehensive genetic analysis, including high-throughput sequencing of 300 IEI-related genes and copy number variation analysis, identified a critical 2.6-kb deletion spanning the first untranslated exon and its upstream region. The region's importance was confirmed through genetic markers indicative of enhancers and promoters. The deletion was also found in the patient's brother, who displayed similar but milder symptoms. Functional analysis supported haploinsufficiency with reduced mRNA and protein expression in both patients. This case underscores the significance of copy number variation (CNV) analysis and targeting noncoding exons within custom gene panels, emphasizing the broader genomic approaches needed in medical genetics.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Abstract LB350: Novel mouse model to study HPV associated pathologies

Abstract Papillomaviruses are species specific, non-enveloped, double-stranded DNA viruses that have an ~8kb circular genome enclosed by a 55 nm icosahedral capsid. Human papillomaviruses (HPV) are associated with both benign and malignant epithelial lesions. High-risk HPV subtypes are the causative agents of cervical and the majority of oropharyngeal squamous cell carcinomas. Persistent HPV infection is required for HPV-associated tumorigenesis; however, cellular factors needed to support the viral persistence and HPV genes expression are not completely understood. Several transgenic mouse models, developed in last decades, helped tremendously to study HPV-induced carcinogenesis. However, the absence of HPV non-coding upstream regulatory region and late coding region, as well as targeted early genes expression driven by an artificial promoter - features that are shared by all existent HPV transgenic mice - limit our ability to investigate HPV-associated malignant transformation and the role of host factors in this process. Therefore, we generated HPV16 transgenic mouse by using CRISPR/Cas9-mediated genome editing to insert the full-length HPV16 genome into the Rosa26 locus. Two founder mice, with single HPV copy and with tandem HPV integration, were used to establish mouse colonies. Interestingly, regardless of HPV copy number, ~30% of the HPV knock-in mice exhibited early death during the first 2 weeks after birth, while survived pups were significantly smaller than the same age wild-type mice, and had severe hair loss. Intriguingly, the growth retardant and hair loss phenotype in the HPV16 knock-in mice disappeared in ~4 weeks, most likely indicating that viral genes compromise specific stages of mouse development. We evaluated the expression of HPV genes during development, in different mouse organs, and in established fibroblasts and epithelial cell cultures. Our newly developed HPV16 knock-in mouse model provides viral gene expression from natural viral promoters offering an essential basis to explore the role of cellular factors in HPV-driven pathologies. Citation Format: Xue Li, Travis Schrank, Aditi Kothari, Hina Rehmani, Jason Tasoulas, Sulgi Kim, Wendell Yarbrough, Natalia Isaeva. Novel mouse model to study HPV associated pathologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB350.

The functional verification and analysis of Fugu promoter of cardiac gene tnni1a in zebrafish.

Troponin I type 1b (Tnni1b) is thought to be a novel isoform that is expressed only in the zebrafish heart. Knocking down of tnni1b can lead to cardiac defects in zebrafish. Although both the zebrafish tnni1b and human troponin I1 (TNNI1) genes are thought to be closely associated with fatal cardiac development, the regulatory molecular mechanisms of these genes are poorly understood. Analyzing the functionally conserved sequence, especially in the noncoding regulatory region involved in gene expression, clarified these mechanisms. In this study, we isolated a 3kb fragment upstream of Fugu tnni1a that can regulate green fluorescence protein (GFP) expression in a heart-specific manner, similar to the pattern of zebrafish homologue expression. Three evolutionarily conserved regions (ECRs) in the 5'-flanking sequence of Fugu tnni1a were identified by sequence alignment. Deletion analysis led to the identification of ECR2 as a core sequence that affects the heart-specific expression function of the Fugu tnni1a promoter. Interestingly, both the Fugu tnni1a promoter and ECR2 sequence were functionally conserved in zebrafish, although they shared no sequence similarity. Together, the findings of our study provided further evidence for the important role of tnni1a homologous in cardiac development and demonstrated that two functionally conserved sequences in the zebrafish and Fugu genomes may be ECRs, despite their lack of similarity.

Potential role of human papillomavirus proteins associated with the development of cancer.

Papillomaviruses are viruses with double-stranded DNA that are epitheliotropic and non-enveloped that infects cutaneous epithelial and mucosal cells in a species-specific way in several higher vertebrate species and cause cellular growth."There are around 100 different human papillomaviruses (HPVs)", as "more than 150 HPV types have been isolated and fully sequenced". We classify the probability of cancer development following viral infection with each HPV genotype into two types: "low-risk" and "high-risk." As a result, HPV diagnosis is a critical component of HPV genotype identification and characterization. Based on its activities, we may classify the HPV genome into three regions: the long control region (LCR) or the non-coding upstream regulatory region (URR), the late (L) region, and the early (E) region. Functional proteins are mostly static things that are not inflexible; they have undergone both local and global movements at various times and time ranges. The structural differences between HPV16 and 18 discovered by molecular modeling of the E6 oncoprotein were associated with their carcinogenic characteristics. Similarly, the E6 protein has two sets of C-X-X-C motifs that play significant roles in transformation, transcriptional activation, interactions, and immortalization with other proteins of cells in the host environment. Here, we review the literature regarding the protein mechanisms associated with HPV and how they cause cancer. Unless otherwise noted, it described all protein activities in terms of HPV proteins. The term "papillomaviruses" refers to groups of papillomavirus proteins that have a characteristic in common. HPV proteins can study the genetic influences on pathogenicity and the therapeutic applications of genomics. The future study provides a potential advancement in HPV infections and malignant illnesses to improve preventive and treatment strategies. Patients have been able to conquer this condition using a range of therapies and vaccines that were projected to be effective and robust enough to put an end to the ailment completely. In cancer prevention strategies, HPV vaccination is one of the most effective. It is safe, efficient, and long-lasting.

The Complete Mitochondrial Genome of Plectorhinchus Chaetodonoides (Perciformes: Haemulidae)

Plectorhinchus chaetodonoides Lacepède, 1801 is a widespread multicolored sweetlips fish found in the Indo-West Pacific Ocean where its appearance and color patterns drastically change during growth. In this study, the whole mitochondrial genome of P. chaetodonoides was sequenced which revealed it is 16,546 bp long and contains 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and one noncoding regulatory region. The GC content of the whole genome was 47.5% and 48.2%, 46.3%, 46.8%, 42.5% in the protein coding genes, tRNAs, rRNAs, and control regions, respectively. Molecular phylogenetic analysis resolved P. chaetodonoides as closely associated with Diagramma pictum and nested within a clade of Haemulidae that is allied with species from the Lutjanidae, Kyphosidae, Teraponidae, and Sciaenidae families. These results provide an essential genomic resource for future evolutionary and conservation studies of P. chaetodonoides as well as the Haemulidae family.

Placental mtDNA copy number and methylation in association with macrosomia in healthy pregnancy.

Fetal growth and development depend on metabolic energy from placental mitochondria. However, the impact of placental mitochondria on the occurrence of macrosomia remains unclear. We aimed to explore the association between macrosomia without gestational diabetes mellitus (non-GDM) and changes in placental mitochondrial DNA (mtDNA) copy number and methylation. Fifty-four newborns with macrosomia and 54 normal birthweight controls were enrolled in this study. Placental mtDNA copy number and mRNA expression of nuclear genes related to mitochondrial replication or ATP synthesis-related genes were measured by real-time quantitative polymerase chain reaction (qPCR). Methylation levels of the non-coding regulatory region D-loop and ATP synthesis-related genes were detected by targeted bisulfite sequencing. Newborns with macrosomia had lower placental mtDNA copy number and higher methylation rates of the CpG15 site in the D-loop region (D-CpG15) and CpG6 site in the cytochrome C oxidase III (COX3) gene (COX3-CpG6) than normal birth weight newborns. After adjusting for potential covariates (gestational age, prepregnancy BMI, and infant sex), decreased placental mtDNA copy number (adjusted odds ratio [aOR]=2.09, 95% confidence interval [CI] 1.03-4.25), elevated methylation rate of D-CpG15 (aOR=2.06, 95% CI 1.03-4.09) and COX3-CpG6 (aOR=2.13, 95% CI 1.08-4.20) remained significantly associated with a higher risk of macrosomia. Reduced mtDNA copy number and increased methylation levels of specific loci at mtDNA would increase the risk of macrosomia. However, the detailed molecular mechanism needs further identification.

Genome-wide association and functional interrogation identified a variant at 3p26.1 modulating ovarian cancer survival among Chinese women

Ovarian cancer survival varies considerably among patients, to which germline variation may also contribute in addition to mutational signatures. To identify genetic markers modulating ovarian cancer outcome, we performed a genome-wide association study in 2130 Chinese ovarian cancer patients and found a hitherto unrecognized locus at 3p26.1 to be associated with the overall survival (Pcombined = 8.90 × 10−10). Subsequent statistical fine-mapping, functional annotation, and eQTL mapping prioritized a likely casual SNP rs9311399 in the non-coding regulatory region. Mechanistically, rs9311399 altered its enhancer activity through an allele-specific transcription factor binding and a long-range interaction with the promoter of a lncRNA BHLHE40-AS1. Deletion of the rs9311399-associated enhancer resulted in expression changes in several oncogenic signaling pathway genes and a decrease in tumor growth. Thus, we have identified a novel genetic locus that is associated with ovarian cancer survival possibly through a long-range gene regulation of oncogenic pathways.

Identification of a Novel Epigenetic Mechanism of MYC Deregulation in Smoldering and Newly Diagnosed Multiple Myeloma Patients

Enhanced expression of the MYC oncogene is associated with the initiation and maintenance of many human cancers, including multiple myeloma (MM). MM is a malignancy of clonal plasma cells, in which MYC deregulation is a key event in the progression from the precursor stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to symptomatic MM. Translocation and amplification of the 8q24.21 MYC locus are known mediators of MYC deregulation at premalignant stages for some patients. However, DNA and RNA sequencing of MM patients show that cases with an intact MYC locus also exhibit MYC deregulation, indicating that additional mechanisms are involved in the deregulation of MYC in MM. Here we describe a new epigenetic mechanism of transcriptional deregulation of MYC in malignant plasma cells. We show that activation of a novel non-coding regulatory region through the binding of MM-specific transcription factors (TFs) is associated with MYC dysregulation in MM.To define the MM-specific MYC epigenetic regulation mechanisms, we performed a high-throughput CRISPR interference (CRISPRi) screen in ANBL6 MM cells that harbor no MYC genetic aberrations. We infected ANBL6 cells with a library of >111,000 sgRNAs, tiling across ~1.2 Mb of sequence around MYC and induced expression of KRAB-dCas9 to epigenetically repress putative regulatory elements. We then sequenced the distribution of sgRNAs in the population before and after 14 passages of growth. Because the expression of MYC quantitatively tunes cellular growth, sgRNAs that reduce MYC expression are less abundant at passage 14. This screen identified a ~13 kb region that significantly reduced cellular proliferation when targeted with sgRNAs. We assessed the function of each enhancer region with individual sgRNAs in different MM cell lines and detected an 89% reduction in MYC mRNA levels on average 48 hours after activating KRAB-dCas9.To further characterize the new enhancer region, we performed chromatin immunoprecipitation (ChIP)- and assay for transposase-accessible chromatin (ATAC)- sequencing on MM cell lines and malignant cells obtained from the bone marrow of 13 SMM and 8 MM patients and normal plasma cells from 3 healthy donors. We found that enhancer elements were enriched for H3K27ac and showed greater chromatin accessibility in tumor cells than normal plasma cells. Motif analysis of the enhancer region recovered putative binding sites for multiple TFs, such as IRF4 and MAF, that play key roles in MM pathogenesis. ChIP-sequencing for these enhancer-associated TFs and luciferase reporter assays targeting their binding sites confirmed the binding and involvement of IRF4 and MAF in activating enhancer elements in MM cells with intact MYC loci.MYC abnormalities are well-known secondary genetic events that trigger the progression of precursor diseases to MM. To define the genetic status of the identified enhancer elements in malignant cells, we examined whole-genome sequencing (WGS) data of 906 MM patients from the MMRF CoMMpass cohort. We found focal amplification of the enhancer region in 2.8% (n = 26) of patients. Transcriptional analysis on the same patient cohort revealed a significant increase in MYC mRNA levels in enhancer-amplified patients compared to MM cases with no MYC aberrations. These results indicate that enhancer activity is required to induce MYC expression and progression of patients with intact MYC loci.Collectively, our findings reveal a novel mechanism of MYC deregulation in malignant plasma cells: selective gain of chromatin accessibility at the enhancer elements and amplification of the enhancer region allow for binding of regulatory factors IRF4 and MAF, which increase the transcription of MYC in the absence of the known MYC chromosomal abnormalities. Our results point to the importance of non-coding regulatory elements and their associated TF networks as drivers of MM progression and suggest a new approach to identify predictive biomarkers and therapeutic targets that could improve patient outcomes in MM and other cancers. DisclosuresFulco: Bristol Myers Squibb: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice

BackgroundGenes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified.MethodsTo determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior.ResultsMice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits.ConclusionsThis work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.

Intronic Variant in CNTNAP2 Gene in a Boy With Remarkable Conduct Disorder, Minor Facial Features, Mild Intellectual Disability, and Seizures.

Introduction: Mutations in the contactin-associated protein-like 2 (CNTNAP2) gene (MIM#604569) encoding for CASPR2, a cell adhesion protein of the neurexin family, are known to be associated with autism, intellectual disability, and other neuropsychiatric disorders. A set of intronic deletions of CNTNAP2 gene has also been suggested to have a causative role in individuals with a wide phenotypic spectrum, including Pitt-Hopkins syndrome, cortical dysplasia–focal epilepsy syndrome, Tourette syndrome, language dysfunction, and abnormal behavioral manifestations.Case presentation: A 10-years-old boy was referred to the hospital with mild intellectual disability and language impairment. Moreover, the child exhibited minor facial features, epileptic seizures, and notable behavioral abnormalities including impulsivity, aggressivity, and hyperactivity suggestive of the diagnosis of disruptive, impulse-control and conduct disorder (CD). Array comparative genomic hybridization (CGH) revealed a copy number variant (CNV) deletion in the first intron of CNTNAP2 gene inherited from a healthy father.Conclusions: A comprehensive description of the phenotypic features of the child is provided, revealing a distinct and remarkable alteration of social behavior not previously reported in individuals affected by disorders related to CNTNAP2 gene disruptions. A possible causative link between the deletion of a non-coding regulatory region and the symptoms presented by the boy has been advanced.

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Possibility of BKV-Associated Nephropathy in Hospitalized Burn Patients.

Although renal failure in burn patients results from some defined reasons, there are various causes which are still unclear. BK virus is a human polyomavirus, which, in case of reactivation, can cause late-onset renal dysfunction and cystitis among immunodeficient patients such as transplant, pregnant, diabetic, and HIV patients. Regarding the related challenges, Polyomavirus BK (BKV), as a ubiquitous virus, is considered as one of the potential threats in the occurrence of Polyomavirus-associated nephropathy (PAN). Hypovolemia, occurring due to the weakness of the immune system, may be regarded as the major reason for the possibility of PAN as a risk factor in burn patients. Accordingly, this study was designed to evaluate the reactivation of BKV as a probable risk factor for renal failure or a problem in the future life of burn patients. This case-control study was conducted from October 2014 to September 2016, during which 270 patients were admitted to the burn unit. The patients were divided into two groups of case and control according to the inclusion criteria, and 20 patients were assigned to each group. The serum samples were first assessed for BKV-IgG and then were quantified by specific quantitative real-time polymerase chain reaction for BKV load. Positive samples were assessed for changes in noncoding regulatory region (NCRR) compared to Archetype strain by PCR sequencing method. Amplified sequences were analyzed for NCRR arrangement while the reactivation was assessed through these changes in NCRR. In both groups, patients were seropositive for BKV-IgG. Eight patients (40%) in the case group and two patients (10%) in the control group were found to be positive for BKV DNA with a load of ≥1000 and ≥100 copies/ml, respectively. There was a significant association between BKV DNA and kidney injury in the case group. The NCRR of DNA-positive samples had a large rearrangement compared to standard strain, but they showed relatively high similarity. Compared with other patients, burn patients are among the most susceptible ones to PAN, which can be considered as a major risk factor in the treatment of burn patients and optimizing their therapy.

Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.

We performed a comprehensive assessment of rareinherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extensionof previousfindings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

Clinical, genetic and neuropathological characterization of spinocerebellar ataxia type 37.

The autosomal dominant spinocerebellar ataxias (SCAs) consist of a highly heterogeneous group of rare movement disorders characterized by progressive cerebellar ataxia variably associated with ophthalmoplegia, pyramidal and extrapyramidal signs, dementia, pigmentary retinopathy, seizures, lower motor neuron signs, or peripheral neuropathy. Over 41 different SCA subtypes have been described evidencing the high clinical and genetic heterogeneity. We previously reported a novel spinocerebellar ataxia type subtype, SCA37, linked to an 11-Mb genomic region on 1p32, in a large Spanish ataxia pedigree characterized by ataxia and a pure cerebellar syndrome distinctively presenting with early-altered vertical eye movements. Here we demonstrate the segregation of an unstable intronic ATTTC pentanucleotide repeat mutation within the 1p32 5' non-coding regulatory region of the gene encoding the reelin adaptor protein DAB1, implicated in neuronal migration, as the causative genetic defect of the disease in four Spanish SCA37 families. We describe the clinical-genetic correlation and the first SCA37 neuropathological findings caused by dysregulation of cerebellar DAB1 expression. Post-mortem neuropathology of two patients with SCA37 revealed severe loss of Purkinje cells with abundant astrogliosis, empty baskets, occasional axonal spheroids, and hypertrophic fibres by phosphorylated neurofilament immunostaining in the cerebellar cortex. The remaining cerebellar Purkinje neurons showed loss of calbindin immunoreactivity, aberrant dendrite arborization, nuclear pathology including lobulation, irregularity, and hyperchromatism, and multiple ubiquitinated perisomatic granules immunostained for DAB1. A subpopulation of Purkinje cells was found ectopically mispositioned within the cerebellar cortex. No significant neuropathological alterations were identified in other brain regions in agreement with a pure cerebellar syndrome. Importantly, we found that the ATTTC repeat mutation dysregulated DAB1 expression and induced an RNA switch resulting in the upregulation of reelin-DAB1 and PI3K/AKT signalling in the SCA37 cerebellum. This study reveals the unstable ATTTC repeat mutation within the DAB1 gene as the underlying genetic cause and provides evidence of reelin-DAB1 signalling dysregulation in the spinocerebellar ataxia type 37.

Progress on pathogenesis of progressive multifocal leukoence-phalopathy

Progressive multifocal leukoencephalopathy (PML) is a rare and lethal central nervous demyelinating disease caused by JC polyomavirus (JCV), particularly in patients with impaired immune system. The variation of JCV plays an important role in the pathogenesis of PML, including the recombination of non-coding regulatory region (NCCR), which is closely related to binding sites of transcription factors and affect the level of gene transcription. Nucleotide mutations in VP1 region determine the antigenicity and receptor specificity of JCV, play an important role in cell adsorption, immune-mediation and pathogenicity. In addition, immune cells are also involved in the pathogenesis of PML. T lymphocytes can recognize virus antigens, clear JCV, which are directly related to the prognosis of PML. B lymphocytes can serve as latent sites of JCV, and participate in viral transmission, replication, and coordination of the expression of transcription factors. This paper summarizes the roles of JCV variation and immune cells in pathogenesis of PML.

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.