Large-scale Deletions Research Articles

Mitochondrial Retinopathies.

The retina is an exquisite target for defects of oxidative phosphorylation (OXPHOS) associated with mitochondrial impairment. Retinal involvement occurs in two ways, retinal dystrophy (retinitis pigmentosa) and subacute or chronic optic atrophy, which are the most common clinical entities. Both can present as isolated or virtually exclusive conditions, or as part of more complex, frequently multisystem syndromes. In most cases, mutations of mtDNA have been found in association with mitochondrial retinopathy. The main genetic abnormalities of mtDNA include mutations associated with neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) sometimes with earlier onset and increased severity (maternally inherited Leigh syndrome, MILS), single large-scale deletions determining Kearns–Sayre syndrome (KSS, of which retinal dystrophy is a cardinal symptom), and mutations, particularly in mtDNA-encoded ND genes, associated with Leber hereditary optic neuropathy (LHON). However, mutations in nuclear genes can also cause mitochondrial retinopathy, including autosomal recessive phenocopies of LHON, and slowly progressive optic atrophy caused by dominant or, more rarely, recessive, mutations in the fusion/mitochondrial shaping protein OPA1, encoded by a nuclear gene on chromosome 3q29.

Advanced approach for comprehensive mtDNA genome testing in mitochondrial disease

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test (‘MitoGenome’) using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations.

Phenotypic spectrum and clinical course of single large-scale mitochondrial DNA deletion disease in the paediatric population: a multicentre study

BackgroundLarge-scale mitochondrial DNA deletions (LMD) are a common genetic cause of mitochondrial disease and give rise to a wide range of clinical features. Lack of longitudinal data means the natural...

Abstract 17081: End Stage Mitochondrial Cardiomyopathy And Heart Transplantation Due To Pathogenic Biallelic C1QBP Variants

Case Presentation: A 29-year-old male with LVH diagnosed in childhood was admitted with acute HF. TTE showed LVEF 5-10% and LV thrombi for which he was anticoagulated. He received inappropriate ICD shocks due to T wave oversensing, leading to cardiogenic shock requiring VA-ECMO support. Serum lactate peaked at 17 mmol/L due to cardiac and metabolic decompensation. He underwent heart transplantation (HT) on hospital day (HD) 8 and tolerated standard immunosuppression. First endomyocardial biopsy showed acute cellular rejection requiring pulse steroids. He was discharged on HD 33. Trio whole exome and mitochondrial genome sequencing revealed biallelic variants in complement component 1Q subcomponent-binding protein ( C1QBP ), due to a maternally inherited likely pathogenic variant c.612C>G (p.F204L in exon 5) and an apparently de novo deletion of 17p13.2, spanning exons 4-6 of C1QBP and exon 6 of the RPAIN gene. Mitochondrial genome sequencing of the explanted heart revealed multiple large-scale mitochondrial DNA deletions at 33% heteroplasmy. Discussion: C1QBP variants are associated with mitochondrial and multi-organ dysfunction. Only 12 patients exhibiting biallelic C1QBP variants are reported. Four died in the peripartum period due to fetal hydrops or HF; 5 exhibited early-onset cardiomyopathy (CM); 3 others had late-onset ophthalmoplegia without CM. The p.F204L variant has been reported in 1 patient with compound C1QBP p.F204L/p.C186S heterozygosity who died from hydrops fetalis and a second with p.F204L homozygosity with late-onset ophthalmoplegia and skeletal myopathy without CM. Differences in the size, heteroplasmy, and tissue distribution of mitochondrial genome secondary deletions may explain variability in disease onset and progression. We present the first patient with biallelic pathogenic C1QBP gene variants with mitochondrial CM to undergo HT and highlight the diagnosis and management of an exceptionally uncommon genetic disorder.

Pediatric single large-scale mtDNA deletion syndromes: The power of patient reported outcomes

There is a limited understanding of system-level clinical outcomes and interventions associated with single large-scale mitochondrial DNA deletion syndromes (SLSMDS). Additionally, no research exists that describes patient reported outcomes (PROs) of children with SLSMDS.A global and observational registry was established to understand the multi-systemic course of SLSMDS and track clinical outcomes. The development and design of the registry is described. Demographic characteristics, history and diagnoses, and system level prevalence of problems and interventions are reported for 42 children. System level problems and interventions include information on the following body systems: audiology, cardiac, endocrine, gastrointestinal (including pancreatic and hepatobiliary system), hematological, metabolic, neurological (including autonomic, mobility, & learning), ophthalmic, psychiatric, renal, and respiratory.Results emphasize the need of patient registries and suggest that the diagnostic odyssey and burden of disease for children with SLSMDS is significant. System-level findings may help families and clinical providers with diagnosis, prognostication, and treatment. A multidisciplinary team of clinical experts with a central coordinating specialist for children with SLSMDS is recommended.

Loss of Function of OsFBX267 and OsGA20ox2 in Rice Promotes Early Maturing and Semi-Dwarfism in γ-Irradiated IWP and Genome-Edited Pusa Basmati-1.

Targeted mutagenesis is now becoming the most favored methodology to improve traits in popular rice cultivars selectively. Understanding the genetic basis of already available mutants could be the first step in designing such experiment. Improved White Ponni (IWP), a popularly grown South Indian rice variety, was subjected to γ irradiation to develop WP-22-2, an M6 line superior in semi-dwarfism, early flowering, and high yield, and it has grain qualities similar to those of IWP. The exogenous application of gibberellic acid (GA3) on WP-22-2 resulted in the elongation of shorter internodes to a level similar to IWP. The expression profiling of six genes regulating plant height showed their differential expression pattern at different time points post GA3 treatment. Furthermore, the sequencing of WP-22-2 and IWP genomes revealed several single nucleotide polymorphisms (SNPs) and large-scale deletions in WP-22-2. The conversion of functional codons to stop codons was observed in OsGA20ox2 and OsFBX267, which have been reported to have roles in regulating semi-dwarfism and early flowering, respectively. The loss of function of OsGA20ox2 and OsFBX267 in WP-22-2 resulted in reduced plant height as well as early flowering, and the same has been confirmed by editing OsGA20ox2 in the rice variety Pusa Basmati1 (PB1) using the CRISPR-Cas9 approach. The targeted editing of OsGA20ox2 in PB1 conferred shorter plant height to the edited lines compared with the wild type. Altogether, the study provides evidence on mutating OsGA20ox2 and OsFBX267 genes to develop early maturing and semi-dwarf varieties that can be released to farmers after functional characterization and field trials.

DELEAT: gene essentiality prediction and deletion design for bacterial genome reduction

BackgroundThe study of gene essentiality is fundamental to understand the basic principles of life, as well as for applications in many fields. In recent decades, dozens of sets of essential genes have been determined using different experimental and bioinformatics approaches, and this information has been useful for genome reduction of model organisms. Multiple in silico strategies have been developed to predict gene essentiality, but no optimal algorithm or set of gene features has been found yet, especially for non-model organisms with incomplete functional annotation.ResultsWe have developed DELEAT v0.1 (DELetion design by Essentiality Analysis Tool), an easy-to-use bioinformatic tool which integrates an in silico gene essentiality classifier in a pipeline allowing automatic design of large-scale deletions in any bacterial genome. The essentiality classifier consists of a novel logistic regression model based on only six gene features which are not dependent on experimental data or functional annotation. As a proof of concept, we have applied this pipeline to the determination of dispensable regions in the genome of Bartonella quintana str. Toulouse. In this already reduced genome, 35 possible deletions have been delimited, spanning 29% of the genome.ConclusionsBuilt on in silico gene essentiality predictions, we have developed an analysis pipeline which assists researchers throughout multiple stages of bacterial genome reduction projects, and created a novel classifier which is simple, fast, and universally applicable to any bacterial organism with a GenBank annotation file.

Mitochondrial DNA: Unraveling the “other” genome

The mitochondrial genome, which contains all of the hereditary information within human mitochondria, consists of 16,569 base pairs of double-stranded DNA that encode 37 genes. Pathogenic mutations of mitochondrial DNA (mtDNA) cause dysfunction of the respiratory chain and the process of oxidative phosphorylation (OXPHOS), leading to impaired adenosine triphosphate synthesis. Nuclear DNA (nDNA) mutations can affect structural subunits or assembly factors of one of the five OXPHOS complexes. Mitochondrial diseases are a heterogeneous group of disorders, ranging from mtDNA single-point mutations and large-scale deletions to mitochondrial depletion syndromes, resulting from nDNA pathogenic mutations. Manifestations of mitochondrial disease are multisystemic, and organs with substantial energy requirements are most typically affected. Mitochondrial disorders are progressive in nature, and prognosis is dependent on the organs involved and the rate and severity of disease progression. A multidisciplinary team approach is needed to monitor and manage disease sequelae.

Different Roles of TP53 Codon 72 Polymorphism in Type 2 Diabetes and Its Complications: Evidence from a Case-Control Study on a Chinese Han Population.

ObjectiveThe purpose of this study was to investigate the relationships between TP53 Pro72Arg (rs1042522) polymorphism and susceptibility to type 2 diabetes (T2DM) and its related complications.MethodsThe TP53 Pro72Arg polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 206 T2DM patients and 446 healthy controls. Mitochondrial DNA (mtDNA) content, mtDNA transcriptional level and large-scale mtDNA deletion were evaluated in leukocytes of T2DM patients using fluorescence-based quantitative PCR (FQ-PCR), reverse transcriptase-quantitative PCR (RT-qPCR) and long-range PCR approaches, respectively. The data of our study were processed by GraphPad Prism software (version 7.00).ResultsThe distribution of TP53 Pro72Arg differed in T2DM patients from the controls, with a moderately increased proportion of TP53 Arg72 variant carriers (Pro/Arg and Arg/Arg genotypes) (88.3% vs 81.2%, p=0.022; OR=1.089, 95% CI=1.018–1.164). T2DM patients with Arg/Arg genotype had significantly decreased prevalences of diabetic neuropathy and retinopathy compared to those without (6.5% vs 19.4%, p=0.018 and 14.8% vs 30.7%, p=0.018, respectively). T2DM patients with Arg/Arg genotype had higher mtDNA content and mtRNA expression level than those who were not Arg/Arg genotype (p<0.05 for all), and we did not observe mtDNA 4977-base pair (bp) deletion mutations in the leukocytes of T2DM patients.ConclusionThere was a significant association of the TP53 Pro72Arg polymorphism with susceptibility to T2DM, and the homozygous Arg/Arg genotype of this gene locus might be a protective factor for diabetic complications. Those results suggested that the TP53 Arg72 variant had a different association with type 2 diabetes and its complications, and it might be related to mtDNA maintenance of the TP53 Arg72 variant under hyperglycemia-induced stress.

Long-term prognosis and genetic background of cardiomyopathy in 223 pediatric mitochondrial disease patients

BackgroundCardiomyopathy is a risk factor for poor prognosis in pediatric patients with mitochondrial disease. However, other risk factors including genetic factors related to poor prognosis in mitochondrial disease has yet to be fully elucidated. Methods and resultsBetween January 2004 and September 2019, we enrolled 223 consecutive pediatric mitochondrial disease patients aged <18 years with a confirmed genetic diagnosis, including 114 with nuclear gene mutations, 89 patients with mitochondrial DNA (mtDNA) point mutations, 11 with mtDNA single large-scale deletions and 9 with chromosomal aberrations. Cardiomyopathy at baseline was observed in 46 patients (21%). Hazard ratios (HR) and 95% confidence intervals (CI) were calculated for all-cause mortality. Over a median follow-up of 36 months (12–77), there were 85 deaths (38%). The overall survival rate was significantly lower in patients with cardiomyopathy than in those without (p < 0.001, log-rank test). By multivariable analysis, left ventricular (LV) hypertrophy (HR = 4.6; 95% CI: 2.8–7.3), neonatal onset (HR = 2.9; 95% CI: 1.8–4.5) and chromosomal aberrations (HR = 2.9; 95% CI: 1.3–6.5) were independent predictors of all-cause mortality. Patients with LV hypertrophy with neonatal onset and/or chromosomal aberrations had higher mortality (100% in 21 patients) than those with LV hypertrophy alone (71% in 14 patients). ConclusionIn pediatric patients with mitochondrial disease, cardiomyopathy was common (21%) and was associated with increased mortality. LV hypertrophy, neonatal onset and chromosomal aberrations were independent predictors of all-cause mortality. Prognosis is particularly unfavorable if LV hypertrophy is combined with neonatal onset and/or chromosomal aberrations.

Mitochondrial Retinopathy

To report the retinal phenotype and the associated genetic and systemic findings in patients with mitochondrial disease. Retrospective case series. Twenty-three patients with retinopathy and mitochondrial disease, including chronic progressive external ophthalmoplegia (CPEO), maternally inherited diabetes and deafness (MIDD), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-Sayre syndrome, neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome, and other systemic manifestations. Review of case notes, retinal imaging, electrophysiologic assessment, molecular genetic testing including protein modeling, and histologic analysis of muscle biopsy. Phenotypic characteristics of mitochondrial retinopathy. Genetic testing identified sporadic large-scale mitochondrial DNA deletions and variants in MT-TL1, MT-ATP6, MT-TK, MT-RNR1, or RRM2B. Based on retinal imaging, 3 phenotypes could be differentiated: type 1 with mild, focal pigmentary abnormalities; type 2 characterized by multifocal white-yellowish subretinal deposits and pigment changes limited to the posterior pole; and type 3 with widespread granular pigment alterations. Advanced type 2 and 3 retinopathy presented with chorioretinal atrophy that typically started in the peripapillary and paracentral areas with foveal sparing. Two patients exhibited a different phenotype: 1 revealed an occult retinopathy, and the patient with RRM2B-associated retinopathy showed no foveal sparing, no severe peripapillary involvement, and substantial photoreceptor atrophy before loss of the retinal pigment epithelium. Two patients with type 1 disease showed additional characteristics of mild macular telangiectasia type 2. Patients with type 1 and mild type 2 or 3 disease demonstrated good visual acuity and no symptoms associated with the retinopathy. In contrast, patients with advanced type 2 or 3 disease often reported vision problems in dim light conditions, reduced visual acuity, or both. Short-wavelength autofluorescence usually revealed a distinct pattern, and near-infrared autofluorescence may be severely reduced in type 3 disease. The retinal phenotype was key to suspecting mitochondrial disease in 11 patients, whereas 12 patients were diagnosed before retinal examination. Different types of mitochondrial retinopathy show characteristic features. Even in absence of visual symptoms, their recognition may facilitate the often challenging and delayed diagnosis of mitochondrial disease, in particular in patients with mild or nebulous multisystem disease.

Engineering large-scale chromosomal deletions by CRISPR-Cas9.

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.

Abstract 2504: Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma

Abstract Introduction: Large-scale chromosomal deletions frequently occur in the progression of head and neck squamous cell carcinoma (HNSCC). Many studies have linked these deletions with particular patient outcomes. However, their precise contributions to tumorigenesis have yet to be elucidated. Here, we introduce an in vitro model for studying large-scale chromosomal deletions to establish their role as driver or passenger events in HNSCC progression. Methods: CRISPR/Cas9 vectors coding for guide RNAs (gRNAs) that flank two of the commonly deleted regions in HNSCC tumors were transfected into primary human keratinocytes. Eighteen different combinations of gRNAs were utilized to create deletions of various sizes at two different genomic loci. Genomic DNA (gDNA) was harvested 24 hours post-transfection and analyzed via endpoint PCR and Sanger sequencing to assess for the presence of the deletions. Edited cells were further sorted via fluorescence-activated cell sorting (FACS) and the efficiency of the deletions were analyzed via performing TaqMan copy number assay qPCR on extracted gDNA. Subsequently, the invasive properties of the genome-edited cells were then investigated using organotypic cultures, a near-physiological three-dimensional model system for studying squamous epithelial biology. Results: The feasibility of performing large-scale deletions was confirmed via endpoint PCR and Sanger sequencing, in which all 18 gRNA combinations created successful deletions. We further sought to create a pure population of the cells harboring the deletions by isolating the edited cells via FACS. TaqMan copy number assay qPCR on gDNA harvested from these cells indicated a 10-45% reduction in the copy number of loci of interest. Genome-edited keratinocytes containing deletions at two of the most commonly deleted sites in HNSCC tumors were used for organotypic assays, in which the preliminary results suggested that deletion of these sites can lead to a gain of invasive behavior. Conclusion: Chromosomal deletions occur at a high frequency in HNSCC tumors. However, there is no established model to determine whether these deletions exist as driver or passenger events. Here, we demonstrate the introduction of successful large-scale deletions in a primary human cell culture system that can be further utilized to study the role of these events in HNSCC tumorigenesis. Furthermore, we show that two of the most common deletions in HNSCC may act as driver events in tumor progression. Support: This study was supported by Program in Breakthrough Biological Research, UCSF School of Dentistry OHNRG pilot grant, and an R01 grant (DE029890-01 - Defining the Mutational pathogenesis of Oral Preneoplasia). Citation Format: Ramin Farhad, Vicente Planells Palop, Aaron Tward. Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2504.

Novel variants in GUCY2D causing retinopathy and the genotype-phenotype correlation

Leber congenital amaurosis (LCA) is the most severe form of retinopathy and cone/cone-rod dystrophy (CORD) is a common form of inherited retinopathy. Variants in GUCY2D constitute the most common cause of LCA and autosomal dominant CORD (ADCORD). The purpose of this study was to reveal novel variants and document associated phenotypes of patients with GUCY2D-associated retinopathy. Fifty-two potentially pathogenic variants (PPVs), including 12 novel ones (p.Gly144_Ala164del, p.Trp154Glyfs*12, p.Leu186Pro, p.Ala207Pro, p.Ala229Asp, p.Ala353Glu, p.Trp372*, p.Arg528*, p.Arg660Pro, p.Ile682Thr, p.Trp788Cys, and c.1026 + 171_*486del), were identified in 16 families with ADCORD and 34 families with autosomal recessive LCA (ARLCA). The novel variant c.1026 + 171_*486del is a large-scale (16.3 kb) deletion involving exons 4–20 of GUCY2D, and was identified in an ARLCA family in heterozygous status mimicking a homozygous p.Trp788Cys variant. Among the detected 52 PPVs, 32 (61.5%) were missense, seven (13.5%) were splicing, six (11.5%) were nonsense, four (7.7%) were inframe indel, and three (5.8%) were frameshift deletion. The median age of examination in 27 patients with ADCORD was 21.0 years (ranges 3–54) with a median visual acuity (VA) of 0.10 (ranges 0.02–0.90). There were 48.0% of patients with macular atrophy, 86.4% with severe reduced or extinguished cone responses, 77.3% with normal or mildly reduced rod responses, and 60.9% with high myopia. Visual impairment, macular dystrophy, and cone dysfunction deteriorated with age. The median age of examination in 34 patients with ARLCA was 1.1 years (ranges 0.3–25). There were 55.9% of patients with roving nystagmus, 68.2% with VA of worse than hand motion, 59.4% with almost normal fundus, 90.6% with extinguished rod and cone responses, and 50.0% with high hyperopia. In conclusions, twelve novel PPVs in GUCY2D (including a novel large-scale deletion) were identified. Most (32/52, 61.5%) of causative GUCY2D variants were missense. Progressive development of macular atrophy, cone dysfunction, visual impairment, and myopia are four major characteristics of GUCY2D-associated ADCORD. Normal fundus, roving nystagmus, and hypermetropia in early age are common findings specific to GUCY2D-associated ARLCA. The obtained data in this study will be of value in counselling patients and designing future therapeutic approaches.

KIR and HLA class I diversity in Pakistani and Yadav populations: a high-resolution study of immunogenetic diversity in South Asians

Abstract Killer cell immunoglobulin-like receptors (KIR) modulate the activity of human NK cells by interacting with HLA class I ligands, influencing their response to pathogens, cancer and during pregnancy. Combinatorial diversity of HLA and KIR allotypes impacts NK cell functions in health and disease. South Asians are under-studied for this immunogenetic diversity, despite representing almost one quarter of the world’s population. We analyzed KIR and HLA distribution of a cohort of healthy subjects comprising 81 from Karachi in Pakistan and 71 Yadav from Tamil Nadu in Southern India. To overcome genomic region complexity, we used our high throughput NGS and bioinformatics pipeline to derive high resolution gene content and allele level KIR and HLA data. We also developed a Cas9-targeted long read method to fully sequence specific structurally divergent KIR haplotypes. We identified 110 distinct HLA class I alleles in the Karachi population and 80 in the Yadav. We observed 96% individuals to have unique HLA class I genotypes and three were shown to be homozygous across all three HLA class I genes. While overall HLA diversity is lower in the Yadav compared to the Karachi, both encode approximately two KIR ligands per HLA haplotype. We identified 125 distinct KIR alleles, including 11 characterized for the first time in this study. We identified and sequenced multiple KIR haplotypes having large-scale deletions or duplications of complete genes. Most haplotypes were observed in more than one individual, but every individual studied has a unique KIR genotype. This is the first high-resolution study of KIR and HLA class I in a South Asian population, and this will lay the foundation for further immunogenic studies of subjects from this geographic region.

Distinct Roles of the Mitochondrial DNA Helicase Twinkle in Drosophila Neurons and Muscles

Mutations in the human gene TWNK, which encodes the mitochondrial DNA (mtDNA) helicase Twinkle, frequently affect the neurolocomotor system. Patients may present accumulation of multiple large-scale mtDNA deletions in muscle fibers, cerebral cortex, basal ganglia, and cerebellum throughout lifetime, as observed in cases of autosomal dominant progressive external ophthalmoplegia (adPEO). Specially in high energy demanding tissues such as muscles and neurons, mtDNA deletions and/or depletion can affect mitochondrial function, since this genome plays a central role in ATP production by means of oxidative phosphorylation (OXPHOS). Maintaining this genome intact and at the appropriate level requires an array of proteins involved in replication, transcription, repair, and recombination. Although Twinkle may participate in recombinational repair, its roles in the replication process is best understood. We have been using Drosophila melanogaster as a model to understand the role of Twinkle in mtDNA maintenance in a tissue-specific pattern, with focus on the central nervous system and the musculature. We have shown that defective versions of Twinkle that are ubiquitously expressed cause lethality at the larval stage, but directing the expression only to neurons or to muscles does not impact development. Furthermore, our results indicate that neuron-specific, but not muscle-specific, expression of ATPase-deficient versions of Twinkle leads to a dramatic shortening of adult lifespan, which is accompanied by severe locomotor problems. The lack of phenotype upon Twinkle dysfunction in muscles, however, is unexpected considering the high OXPHOS activity and high mtDNA copy number of the Drosophila flight muscles. We also overexpressed Twinkle mutants in Drosophila S2 cells in culture to get a first insight into how mtDNA maintenance may be disturbed. We have previously shown that overexpressing wild-type (WT) Twinkle leads to a decrease in intermediates at two important replication pausing regions in the mitochondrial genome. Interestingly, our preliminary findings indicate that the overexpression of an ATPase-deficient Twinkle mutant causes severe accumulation of replication intermediates at the same pausing sites. Because in whole adult fly samples the vast majority of mitochondria (and therefore mtDNA) come from the flight musculature, it is safe to assume that the mode of mtDNA replication previously described for adult individuals is in fact the mode for muscle mtDNA. In this case, it is intriguing that mtDNA maintenance in S2 cells and in adult flight muscles is so similar, and yet a severely defective Twinkle enzyme causes a strong mtDNA depletion in the former and no detectable phenotype in the latter. Since there is no available effective treatment for adPEO and other mtDNA diseases, determining if, how and when Twinkle participates in DNA maintenance and repair inside mitochondria may provide insights into how to regulate Twinkle's functions to improve conditions of mitochondrial dysfunction.

High-Resolution Analysis Identifies High Frequency of KIR-A Haplotypes and Inhibitory Interactions of KIR With HLA Class I in Zhejiang Han.

Killer cell immunoglobulin-like receptors (KIR) interact with human leukocyte antigen (HLA) class I molecules, modulating critical NK cell functions in the maintenance of human health. Characterizing the distribution and characteristics of KIR and HLA allotype diversity across defined human populations is thus essential for understanding the multiple associations with disease, and for directing therapies. In this study of 176 Zhejiang Han individuals from Southeastern China, we describe diversity of the highly polymorphic KIR and HLA class I genes at high resolution. KIR-A haplotypes, which carry four inhibitory receptors specific for HLA-A, B or C, are known to associate with protection from infection and some cancers. We show the Chinese Southern Han from Zhejiang are characterized by a high frequency of KIR-A haplotypes and a high frequency of C1 KIR ligands. Accordingly, interactions of inhibitory KIR2DL3 with C1+HLA are more frequent in Zhejiang Han than populations outside East Asia. Zhejiang Han exhibit greater diversity of inhibitory than activating KIR, with three-domain inhibitory KIR exhibiting the greatest degree of polymorphism. As distinguished by gene copy number and allele content, 54 centromeric and 37 telomeric haplotypes were observed. We observed 6% of the population to have KIR haplotypes containing large-scale duplications or deletions that include complete genes. A unique truncated haplotype containing only KIR2DL4 in the telomeric region was also identified. An additional feature is the high frequency of HLA-B*46:01, which may have arisen due to selection pressure from infectious disease. This study will provide further insight into the role of KIR and HLA polymorphism in disease susceptibility of Zhejiang Chinese.

Molecular characterization of the Yp11.2 region deletion in the Chinese Han population

The Y chromosome is male-specific and is important for spermatogenesis and male fertility. However, the Y chromosome is poorly characterized due to massive palindromes and inverted repeats, which increase the likelihood of genomic rearrangements, resulting in short tandem repeats on the Y chromosome or long fragment deletions. The present study reports a large-scale (2.573~2.648 Mb) deletion in the Yp11.2 region in a Chinese population based on the analysis of 34 selected Y-specific sequence-tagged sites and subsequent sequencing of the breakpoint junctions on the Y chromosome from 5,068,482–5,142,391 bp to 7,715,462–7,716,695 bp. The results of sequence analysis indicated that the deleted region included part or all of the following five genes: PCDH11Y, TSPY, AMELY, TBL1Y, and RKY. These genes are associated with spermatogenesis or amelogenesis and various other processes; however, specific physiological functions and molecular mechanisms of these genes remain unclear. Notably, individuals with this deletion pattern did not have an obvious pathological phenotype but manifested some degree of amelogenesis imperfecta.

Mitochondrial genome stability in human: understanding the role of DNA repair pathways.

Mitochondria are semiautonomous organelles in eukaryotic cells and possess their own genome that replicates independently. Mitochondria play a major role in oxidative phosphorylation due to which its genome is frequently exposed to oxidative stress. Factors including ionizing radiation, radiomimetic drugs and replication fork stalling can also result in different types of mutations in mitochondrial DNA (mtDNA) leading to genome fragility. Mitochondria from myopathies, dystonia, cancer patient samples show frequent mtDNA mutations such as point mutations, insertions and large-scale deletions that could account for mitochondria-associated disease pathogenesis. The mechanism by which such mutations arise following exposure to various DNA-damaging agents is not well understood. One of the well-studied repair pathways in mitochondria is base excision repair. Other repair pathways such as mismatch repair, homologous recombination and microhomology-mediated end joining have also been reported. Interestingly, nucleotide excision repair and classical nonhomologous DNA end joining are not detected in mitochondria. In this review, we summarize the potential causes of mitochondrial genome fragility, their implications as well as various DNA repair pathways that operate in mitochondria.

Extra-Nuclear Functions of the Transcription Factor Grainyhead-Like 3 in the Endothelium-Interaction with Endothelial Nitric Oxide Synthase.

We previously demonstrated that the transcription factor Grainyhead-like 3 (GRHL3) has essential functions in endothelial cells by inhibiting apoptosis and promoting migration as well as activation of endothelial nitric oxide synthase (eNOS). We now show that a large portion of the protein is localized to myo-endothelial projections of murine arteries suggesting extra-nuclear functions. Therefore, we generated various deletion mutants to identify the nuclear localization signal (NLS) of GRHL3 and assessed potential extra-nuclear functions. Several large-scale deletion mutants were incapable of activating a GRHL3-dependent reporter construct, which could either be due to deficiencies in transcriptional activation or to impaired nuclear import. One of these mutants encompassed a predicted bipartite NLS whose deletion led to the retention of GRHL3 outside the nucleus. Interestingly, this mutant retained functions of the full-length protein as it could still inhibit pathways inducing endothelial cell apoptosis. As apoptosis protection by GRHL3 depends on NO-production, we examined whether GRHL3 could interact with eNOS and showed a direct interaction, which was enhanced with the extra-nuclear GRHL3 variant. The observation that endogenous GRHL3 also interacts with eNOS in intact murine arteries corroborated these findings and substantiated the notion that GRHL3 has important extra-nuclear functions in the endothelium.