Identical Deletion Research Articles

Population dynamics of defective viral genomes of tomato black ring virus during host-to-host transmission.

Defective viral genomes (DVGs) have been identified in vivo and in vitro for different virus species infecting humans, animals, and plants. The ability to form DVGs during the passaging of virus in one host has been demonstrated, i.e., for tomato black ring virus (TBRV). In our research, RNA-Seq data obtained after TBRV passaging through a combination of four distinct host species were analyzed. Our results indicate that the level of DVG abundance varied across host plant combinations. Deletions were the most prevalent class of DVGs, with the domination of longer species. Additionally, the conserved junction sites in the TBRV genome were identified, resulting in the generation of identical deletions in independently evolved viral lineages. In summary, our findings provide significant insights into the origin and structure of DVGs of plant viruses. The obtained results will help in understanding viral evolution and host-virus interactions.

HNRNPC haploinsufficiency affects alternative splicing of intellectual disability-associated genes and causes a neurodevelopmental disorder

Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders.

Seedless fruit in Annona squamosa L. is monogenic and conferred by INO locus deletion in multiple accessions

Key messageInheritance of the presence/absence of seeds in Annona squamosa is mediated by a single fully recessive gene and is caused by a deletion of the INNER NO OUTER (INO) locus.For some fruits, seedless varieties are desirable for consumption and processing. In the sugar apple tree (Annona squamosa L.), the seedless trait in the Thai seedless (Ts) and Brazilian seedless (Bs) accessions was associated with defective ovules and an apparent deletion of the INNER NO OUTER (INO) ovule development gene locus. Segregation analysis of F2 and backcross descendants of crosses of Bs to fertile wild-type varieties in this species with a multi-year generation time showed that seedlessness was recessive and controlled by a single locus. Comparison of whole genome sequence of a wild-type plant and a third accession, Hawaiian seedless (Hs), identified a 16 kilobase deletion including INO in this line. Ts and Bs lines were shown to have an identical deletion, indicating a common origin from a single deletion event. Analysis of microsatellite markers could not preclude the possibility that all three seedless accessions are vegetatively propagated clones. The sequence of the deletion site enabled a codominant assay for the wild-type and mutant genes allowing observation of complete cosegregation of the seedless/defective ovule phenotype with the INO deletion, showing maximal separation of less than 3.5 cM. The observed deletion is the only significant difference between the wild-type and Hs line over 587 kilobases, likely encompassing much more than 3.5 cM, showing that the deletion is the cause of seedless trait. The codominant markers and obtained progenies will be useful for introgression of the seedless trait into elite sugar apple lines and into other Annonas through interspecific crossings.

Copy Number Variant Analysis of Spinocerebellar Ataxia Genes in a Cohort of Dutch Patients With Cerebellar Ataxia.

The spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders generally caused by single nucleotide variants (SNVs) or indels in coding regions or by repeat expansions in coding and noncoding regions of SCA genes. Copy number variants (CNVs) have now also been reported for 3 genes-ITPR1, FGF14, and SPTBN2-but not all SCA genes have been screened for CNVs as the underlying cause of the disease in patients. In this study, we aim to assess the prevalence of CNVs encompassing 36 known SCA genes. A cohort of patients with cerebellar ataxia who were referred to the University Medical Center Groningen for SCA genetic diagnostics was selected for this study. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed using the Infinium Global Screening Array. Following data processing, genotyping data were uploaded into NxClinical software to perform CNV analysis per patient and to visualize identified CNVs in 36 genes with allocated SCA symbols. The clinical relevance of detected CNVs was determined using evidence from studies based on PubMed literature searches for similar CNVs and phenotypic features. Of the 338 patients with cerebellar ataxia, we identified putative clinically relevant CNV deletions in 3 patients: an identical deletion encompassing ITPR1 in 2 patients, who turned out to be related, and a deletion involving PPP2R2B in another patient. Although the CNV deletion in ITPR1 was clearly the underlying cause of SCA15 in the 2 related patients, the clinical significance of the deletion in PPP2R2B remained unknown. We showed that CNVs detectable with the limited resolution of SNP array are a very rare cause of SCA. Nevertheless, we suggest adding CNV analysis alongside SNV analysis to SCA gene diagnostics using next-generation sequencing approaches, at least for ITPR1, to improve the genetic diagnostics for patients.

Genomic, transcriptomic, and metabolomic profiles of hiPSC-derived dopamine neurons from clinically discordant brothers with identical PRKN deletions

We previously reported on two brothers who carry identical compound heterozygous PRKN mutations yet present with significantly different Parkinson’s Disease (PD) clinical phenotypes. Juvenile cases demonstrate that PD is not necessarily an aging-associated disease. Indeed, evidence for a developmental component to PD pathogenesis is accumulating. Thus, we hypothesized that the presence of additional genetic modifiers, including genetic loci relevant to mesencephalic dopamine neuron development, could potentially contribute to the different clinical manifestations of the two brothers. We differentiated human-induced pluripotent stem cells (hiPSCs) derived from the two brothers into mesencephalic neural precursor cells and early postmitotic dopaminergic neurons and performed wholeexome sequencing and transcriptomic and metabolomic analyses. No significant differences in the expression of canonical dopamine neuron differentiation markers were observed. Yet our transcriptomic analysis revealed a significant downregulation of the expression of three neurodevelopmentally relevant cell adhesion molecules, CNTN6, CNTN4 and CHL1, in the cultures of the more severely affected brother. In addition, several HLA genes, known to play a role in neurodevelopment, were differentially regulated. The expression of EN2, a transcription factor crucial for mesencephalic dopamine neuron development, was also differentially regulated. We further identified differences in cellular processes relevant to dopamine metabolism. Lastly, wholeexome sequencing, transcriptomics and metabolomics data all revealed differences in glutathione (GSH) homeostasis, the dysregulation of which has been previously associated with PD. In summary, we identified genetic differences which could potentially, at least partially, contribute to the discordant clinical PD presentation of the two brothers.

Whole Exome Sequencing in Individuals with Idiopathic Clubfoot Reveals a Recurrent Filamin B (FLNB) Deletion.

Clubfoot, a congenital deformity that presents as a rigid, inward turning of the foot, affects approximately 1 in 1000 infants and occurs as an isolated birth defect in 80% of patients. Despite its high level of heritability, few causative genes have been identified, and mutations in known genes are only responsible for a small portion of clubfoot heritability. (1) Are any rare gene variants enriched (that is, shared) in unrelated patients with isolated clubfoot? (2) Are there other rare variants in the identified gene (Filamin B) in these patients with clubfoot? Whole-exome sequence data were generated from a discovery cohort of 183 unrelated probands with clubfoot and 2492 controls. Variants were filtered with minor allele frequency < 0.02 to identify rare variants as well as small insertions and deletions (indels) resulting in missense variants, nonsense or premature truncation, or in-frame deletions. A candidate deletion was then genotyped in another cohort of 974 unrelated patients with clubfoot (a replication cohort). Other rare variants in the candidate gene were also investigated. A segregation analysis was performed in multigenerational families of individuals with clubfoot to see if the genotypes segregate with phenotypes. Single-variant association analysis was performed using the Fisher two-tailed exact test (exact p values are presented to give an indication of the magnitude of the association). There were no recurrent variants in the known genes causing clubfoot in this study. A three-base pair in-frame codon deletion of Filamin B (FLNB) (p.E1792del, rs1470699812) was identified in 1.6% (3 of 183) of probands with clubfoot in the discovery cohort compared with 0% of controls (0 of 2492) (odds ratio infinity (inf) [95% CI 5.64 to inf]; p = 3.18 x 10-5) and 0.0016% of gnomAD controls (2 of 125,709) (OR 1.01 x 103 [95% CI 117.42 to 1.64 x 104]; p = 3.13 x 10-8). By screening a replication cohort (n = 974 patients), we found two probands with the identical FLNB deletion. In total, the deletion was identified in 0.43% (5 of 1157) of probands with clubfoot compared with 0% of controls and 0.0016% of gnomAD controls (OR 268.5 [95% CI 43.68 to 2.88 x 103]; p = 1.43 x 10-9). The recurrent FLNB p.E1792del variant segregated with clubfoot, with incomplete penetrance in two families. Affected individuals were more likely to be male and have bilateral clubfoot. Although most patients had isolated clubfoot, features consistent with Larsen syndrome, including upper extremity abnormalities such as elbow and thumb hypermobility and wide, flat thumbs, were noted in affected members of one family. We identified 19 additional rare FLNB missense variants located throughout the gene in patients with clubfoot. One of these missense variants, FLNB p.G2397D, exhibited incomplete penetrance in one family. A recurrent FLNB E1792 deletion was identified in 0.43% of 1157 isolated patients with clubfoot. Given the absence of any recurrent variants in our discovery phase (n = 183) for any of the known genes causing clubfoot, our findings support that novel and rare missense variants in FLNB in patients with clubfoot, although rare, may be among the most commonly known genetic causes of clubfoot. Patients with FLNB variants often have isolated clubfoot, but they and their family members may be at an increased risk of having additional clinical features consistent with Larsen syndrome. Identification of FLNB variants may be useful for determining clubfoot recurrence risk and comorbidities.

TSC1 intragenic deletion transmitted from a mosaic father to two siblings with cardiac rhabdomyomas: Identification of two aberrant transcripts

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by non-cancerous tumors in multiple organs including the brain, kidney, lung, heart, and skin. We encountered a Japanese family consisting of two siblings (a four-year-old boy and a one-year-old girl) with multiple cardiac rhabdomyomas conveying a high risk of TSC and apparently unaffected sibling (a two-year-old girl) and parents. Whole exome sequencing and application of Integrative Genomic Viewer revealed an identical intragenic TSC1 deletion with the breakpoints on intron 15 and exon 19 in the affected siblings, but not in the apparently unaffected sibling and parents. Subsequently, PCR-based analyses were performed using primers flanking the deletion, showing that the deletion was also present in the father and that the deletion occurred between chr9:135,777,038 (bp) and chr9:135,780,540 (bp) in association with a one bp overlap. Furthermore, RT-PCR analyses were carried out using lymphoblastoid cell lines, revealing a major in-frame insertion/deletion transcript produced by aberrant splicing using a cryptic ″ag″ splice acceptor motif at intron 15 (r.1998_2438delinsTTCATTAGGTGG) and a minor frameshift transcript generated by aberrant splicing between exon 15 and exon 20 (r.1998_2502del, p.Lys666Asnfs*15) in the affected siblings. These findings imply that the intragenic deletion producing two aberrant transcripts was generated as a somatic pathogenic variant involving the germline in the father and was transmitted to the affected siblings.

Disorders Associated With Diverse, Recurrent Deletions and Duplications at 1q21.1.

The subchromosomal region 1q21.1 is one of the hotspots in the human genome for deletions and reciprocal duplications, owing to the existence of hundreds of segmental duplications. Recurrent deletions and duplications in this region are thought to be causative in patients with variable clinical manifestations. Based on the genomic locations, deletions and duplications at the 1q21.1 locus have been associated with distinguishable syndromes: chromosome 1q21.1 deletion syndrome, chromosome 1q21.1 duplication syndrome, and thrombocytopenia-absent radius (TAR) syndrome, which is partially due to deletions at the proximal 1q21.1 region. We report here diverse, recurrent deletions and duplications at the 1q21.1 locus in 36 patients from a cohort of 5,200 individuals. Among the 36 patients, 18 patients carry 1q21.1 deletions, nine individuals have reciprocal duplications at 1q21.1, two patients share an identical short deletion, and the remaining seven possess variable sizes of duplications at the proximal 1q21.1 region. Furthermore, we provide cytogenetic characterization and detailed clinical features for each patient. Notably, duplications at the proximal 1q21.1 region have not been associated with a defined disorder in publications. However, recurrent duplications at the proximal 1q21.1 region among the seven patients strongly suggested that the variants are likely pathogenic. The common phenotypical features of those disorders are also summarized to facilitate clinical diagnoses and genetic counseling.

Partial LPL deletions: rare copy-number variants contributing towards severe hypertriglyceridemia

Severe hypertriglyceridemia (HTG) is a relatively common form of dyslipidemia with a complex pathophysiology and serious health complications. HTG can develop in the presence of rare genetic factors disrupting genes involved in the triglyceride (TG) metabolic pathway, including large-scale copy-number variants (CNVs). Improvements in next-generation sequencing technologies and bioinformatic analyses have better allowed assessment of CNVs as possible causes of or contributors to severe HTG. We screened targeted sequencing data of 632 patients with severe HTG and identified partial deletions of the LPL gene, encoding the central enzyme involved in the metabolism of TG-rich lipoproteins, in four individuals (0.63%). We confirmed the genomic breakpoints in each patient with Sanger sequencing. Three patients carried an identical heterozygous deletion spanning the 5' untranslated region (UTR) to LPL exon 2, and one patient carried a heterozygous deletion spanning the 5'UTR to LPL exon 1. All four heterozygous CNV carriers were determined to have multifactorial severe HTG. The predicted null nature of our identified LPL deletions may contribute to relatively higher TG levels and a more severe clinical phenotype than other forms of genetic variation associated with the disease, particularly in the polygenic state. The identification of novel CNVs in patients with severe HTG suggests that methods for CNV detection should be included in the diagnostic workup and molecular genetic evaluation of patients with high TG levels.

Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing

BackgroundClade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates.ResultsIsolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes.ConclusionsWe concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

Genetic analysis identifies potential transmission of low pathogenic avian influenza viruses between poultry farms.

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between‐farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10‐year surveillance period, we identified 35 potential cases of between‐farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between‐farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between‐farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p < 0.05). The results further suggest that between‐farm transmission was not restricted to holdings of the same poultry type and not related to the housing system. Although separate introductions from the wild bird reservoir cannot be excluded, our study indicates that between‐farm transmission occurred in seven of nine virologically analysed cases. Based on these findings, it is likely that between‐farm transmission contributes considerably to the incidence of LPAI virus infections in poultry.

NIPT - POTENTIAL TOOL FOR PGT FOLLOW UP

Objective Invasive procedures such as amniocentesis and CVS, in combination with karyotyping or molecular testing, are currently used to confirm PGT results of transferred embryos. However, some couples who have achieved a pregnancy following PGT-M are reluctant to use this confirmatory approach even though the risk of miscarriage and infection associated with fetal cell sampling is low. In this study, we investigate noninvasive prenatal testing (NIPT) as an alternative tool for PGT-M follow up. Materials and Methods NIPT of the cell free fetal DNAwas performed for pregnancies at risk for a single gene disease using circulating single molecule amplification and re-sequencing technology (cSMART) to target the known parental mutations and genome-wide SNPs. Fetal genotypes were called based on the maternal plasma mutation ratios. Results In preliminary studies of naturally conceived pregnancies undergoing testing for autosomal recessive conditions, fetal genotypes predicted by cSMART assay were concordant with invasive results in 4 of 4 cases (100%) of Wilson disease, 18 of 18 cases (100%) of PKU, 73 of 80 cases (91%) of autosomal recessive hearing loss and 98 of 100 cases (98%) of beta thalassemia. There was a strong association of low fetal DNA fraction and poor quality plasma samples with discordant NIPT results. In a further study, we performed NIPT follow up for a complicated PGT-M case for Fanconi Anemia with HLA matching. The case involved a couple who were both carriers of an identical FANCG deletion mutation. Following embryo testing by mutation analysis and linked STR analysis, 2 HLA matched and disease free (normal and carrier) embryos were transferred, resulting in a twin pregnancy. At 15 weeks gestation, cSMART analysis of the pregnancy plasma determined fetal DNA fractions of 14.2% and 6.6% for twin 1 and 2, respectively. The maternal plasma FANCG mutation ratio was measured at 46.2% (50% minus half the fetal fraction of twin 2), which was consistent with the presence of a carrier fetus (twin 1) and a normal fetus (twin 2). Additional retrospective studies of the WGA products from the transferred embryos using single molecule sequencing also confirmed the FANCG genotypes of the transferred embryos and a HLA match to the sick sibling. Conclusions In proof of concept studies for autosomal recessive conditions, we demonstrate that NIPT can be successfully used to reliably and accurately determine fetal genotypes. NIPT has clinical utility as a safe alternative to invasive testing for confirmation of PGT-M cases that result in an ongoing pregnancy.

Activating Mutations in CSF1R and Additional Receptor Tyrosine Kinases in Sporadic and Familial Histiocytic Neoplasms

Genomic analyses of histiocytic neoplasms (including Langerhans Cell Histiocytosis (LCH) and the non-LCH Erdheim-Chester Disease (ECD)) have revolutionized our understanding of these disorders as clonal hematopoietic malignancies driven by MAPK signaling and led to FDA approval of vemurafenib for BRAFV600E-mutant ECD. Despite these advances, several questions about the pathogenesis of the histiocytoses remain unanswered. For example, the cell-of-origin of the histiocytoses is not definitively known. In addition, histiocytoses represent a spectrum of diseases, and genetic alterations across histiocytosis subtypes have not been comprehensively evaluated. Finally, although the histiocytoses most commonly occur as sporadic, non-hereditary disorders, familial clustering has been well documented and occurs most often in monozygotic twins. This has been taken to suggest a hereditary component of the disease, but germline genetic causes for histiocytoses are not known. Here we performed comprehensive genomic analyses of 218 patients with all subsets of histiocytoses, including monozygotic twins with histiocytosis. In so doing, we uncover a novel series of activating receptor tyrosine kinase (RTK) alterations in the histiocytoses, including the first example in any disease of recurrent, activating mutations in CSF1R, the RTK required for monocyte/macrophage development.We initially performed whole exome sequencing (WES) of skin lesions, blood, and fingernails from identical (monozygotic, dichorionic), one-year-old twins with systemic juvenile xanthogranuloma (JXG). This identified an identical in-frame deletion in CSF1R (CSF1RY546_K551del) in the skin lesions of both children. Recent data from murine models suggest that CSF1R-expressing yolk sac derived precursors of tissue-resident macrophages may be a cell-of-origin of the histiocytoses (Mass, et al. Nature 2017). Consistent with this hypothesis, the identical CSF1R mutation was shared across the histiocytosis lesions in both twins but was absent from blood or fingernails.We next sought to determine if similar mutations in CSF1R might exist in sporadic histiocytosis cases. We therefore sequenced 92 ECD (42%), 58 LCH (27%), 50 JXG (23%), 12 RDD (5%), and 6 histiocytic sarcoma (HS) (3%) lesions using WES, targeted DNA sequencing with a 585-gene panel, and targeted RNA-sequencing for fusions in 74 genes. This identified recurrent mutations in BRAFV600E, MAP2K1, N/KRAS, and ARAF, as well as BRAF, NTRK1, and ALK fusions as previously described in ECD and LCH (Fig. A-C). Interestingly, CSF1R mutations were also found in 8 cases, most commonly as CSF1RY546_K551del, and were predominantly in JXG (10%; n=5/50). Consistent with the recurrent nature of this mutation, expression of CSF1RY546_K551del, but not CSF1RWT, conferred robust cytokine-independent growth to cell lines normally dependent on cytokines (Ba/F3 and 32D cells; Fig. D). We also identified individuals with mutations in CSF3R, KIT, ALK, MET, JAK3, and CRAF, as well as a RET fusion. These studies additionally identified important differences in the spectrum of kinase alterations across histiocytoses subtypes. For example, the BRAFV600E mutation was the most common kinase alteration in LCH and ECD but was not identified in JXG or RDD. Furthermore, BRAF fusions were predominantly seen in LCH and JXG. Meanwhile, NTRK1 and ALK fusions were mainly identified in JXG and ECD, respectively.From a therapeutic perspective, CSF1R activating mutations sensitized cells to inhibition with the CSF1R-specific, small-molecule kinase inhibitors pexidartinib and BLZ945. In addition, in the course of this study, a patient bearing ALK-rearranged ECD required therapy, and we were able to evaluate response to the ALK inhibitor crizotinib. Crizotinib-treatment resulted in profound and sustained therapeutic improvements in this patient (Fig. E).Overall, the above data demonstrate the occurrence of activating CSF1R and other RTK alterations in patients with histiocytic neoplasms, many of which have direct therapeutic importance (such as the first demonstration of ALK inhibitor efficacy in ALK+ histiocytosis). In addition, the discovery of somatically acquired CSF1R activating mutations in identical twins with histiocytosis provides the first human evidence that tissue-resident macrophages may serve as a cell-of-origin of the histiocytoses. DisclosuresArcila:Invivoscribe, Inc.: Consultancy, Honoraria.

Neurological and psychological status of multiple sclerosis patients after a year in program of personalized psychological interventions

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.

Maternally inherited 133kb deletion of 14q32 causing Kagami–Ogata syndrome

We present a case of a newborn female with multiple anomalies demonstrating that the causes of imprinting disorders rely not only on the parent-of-origin of the chromosomal aberrations, but also the scope of genes contained in the imprinted region. The newborn female presented with prenatal polyhydramnios, neonatal respiratory distress, distal contractures, abdominal hernia, bell-shaped thorax, and abnormal ribs. The neonate required mechanical ventilation due to apnea, underwent surgery for laryngomalacia, and showed development delay by age 11 months. Chromosomal microarray analysis identified a single copy number loss in chromosome region 14q32.2q32.31, containing genes that are differentially expressed based on parent-of-origin. Microarray analysis also confirmed the identical deletion in the patient's mother, who was reported to be normal. Additional molecular analyses determined the exact size and breakpoints of the deletion as well as methylation states in both the patient and her mother. The maternally transmitted deletion was responsible for Kagami-Ogata syndrome in the patient.

Disruption of INOS, a Gene Encoding myo-Inositol Phosphate Synthase, Causes Male Sterility in Drosophila melanogaster.

Inositol is a precursor for the phospholipid membrane component phosphatidylinositol (PI), involved in signal transduction pathways, endoplasmic reticulum stress, and osmoregulation. Alterations of inositol metabolism have been implicated in human reproductive issues, the therapeutic effects of drugs used to treat epilepsy and bipolar disorder, spinal cord defects, and diseases including diabetes and Alzheimer’s. The sole known inositol synthetic enzyme is myo-inositol synthase (MIPS), and the homolog in Drosophilia melanogaster is encoded by the Inos gene. Three identical deletion strains (inosΔDF/CyO) were constructed, confirmed by PCR and sequencing, and homozygotes (inosΔDF/inosΔDF) were shown to lack the transcript encoding the MIPS enzyme. Without inositol, homozygous inosΔDF deletion fertilized eggs develop only to the first-instar larval stage. When transferred as pupae to food without inositol, however, inosΔDF homozygotes die significantly sooner than wild-type flies. Even with dietary inositol the homozygous inosΔDF males are sterile. An inos allele, with a P-element inserted into the first intron, fails to complement this male sterile phenotype. An additional copy of the Inos gene inserted into another chromosome rescues all the phenotypes. These genetic and phenotypic analyses establish D. melanogaster as an excellent model organism in which to examine the role of inositol synthesis in development and reproduction.

Evolutionary history of two pollen self-incompatibility factors reveals alternate routes to self-compatibility within Solanum.

Self-incompatibility (SI) prevents self-fertilization and reduces inbreeding. While SI is common in plants, transitions to self-compatibility (SC) occur frequently. Little is known about the genetic changes and evolutionary steps underlying these shifts. In the Solanaceae, SI is gametophytic, with specificity determined by S-RNases in the pistil and S-locus F-box proteins (SLFs) in pollen. We examined the role of two pollen factors, Cullin1 (CUL1) and SLF-23, in SI → SC transitions in wild tomato species from the Arcanum species group (Solanum arcanum, S. neorickii, and S. chmielewskii). Pollen compatibility was assessed on tester lines that reject pollen lacking functional SLF-23 or CUL1. Complementation tests, gene sequencing, and phylogenetic analyses were used to characterize both functional and nonfunctional alleles. We found evidence for multiple independent SI → SC transitions. In S. arcanum and S. chmielewskii, SC is caused by loss of pistil S-RNase activity, while in S. neorickii SC is associated with expression of a functional SLF-23 that recognizes the S9 type S-RNase expressed in its pistils. Interestingly, we found identical deletion mutations in CUL1 exon 7 of S. chmielewskii as previously seen in S. habrochaites. Mating system transitions in the Arcanum group have occurred via both pistil loss-of-function and pollen gain-of-function SC mutations. Mutations common to S. chmielewskii and S. habrochaites must have arisen in a common ancestor, possibly to the entire tomato clade, then became fixed in different lineages after loss of pistil-side SI function.

Prenatal Diagnosis of a 20 Mb Terminal Fetal 4q-Deletion in the Cytoband q33q35.2 Inherited from the Mother

AbstractThe authors present a case of prenatally diagnosed fetal terminal 4q deletion in the cytoband 4q33q35.2, inherited from the mother. The index pregnancy was referred at 28+2 weeks of gestation for polyhdramnios. Prior to performing the scan, distinctive facial features were noted in the mother. Target scan confirmed polyhdramnios, and revealed fetal retrognathia. The fetal karyotype reported a terminal 4q33&gt;ter deletion, which was further delineated by chromosomal microarray analysis. Subsequently, maternal karyotype revealed identical deletion. Subtle fetal ultrasound abnormalities should receive meticulous assessment, and evaluation using the currently available wide range of cytogenetic and molecular gene tests.

Two unrelated children with overlapping 6q25.3 deletions, motor speech disorders, and language delays

Interstitial and terminal 6q25 deletions are associated with developmental delays, hypotonia, eye pathologies, craniofacial dysmorphologies, and structural brain anomalies. In most cases, speech and language deficits are not described in detail. We report on a case (Patient 1, age 7 years) with a de novo 6q25.3-qter deletion, 11.1 Mb long and encompassing 108 genes, and a case (Patient 2, age 5 years) with an inherited interstitial 6q25.3 deletion, located within Patient 1's deletion region and 403 kb long, the smallest 6q25 deletion reported to date. Both children have hypotonia, motor speech disorders, and expressive language delays. Patient 1's speech was characterized by childhood apraxia of speech (CAS) and dysarthria. Other findings include developmental delay, ataxic cerebral palsy, optic nerve dysplagia, and atypical brain morphologies regarding the corpus callosum and gyration patterns, a clinical profile that closely matches a previously reported case with a nearly identical deletion. Patient 2 had speech characterized by CAS and typical nonverbal processing abilities. His father, a carrier, had typical speech and language but showed difficulties with complex motor speech and hand motor tasks, similar to other adults with residual signs of CAS. The small deletion in this family contains the IGF2R-AIRN-SLC22A2-SLC22A3 gene cluster, which is associated with imprinting and maternal-specific expression of Igf2R, Slc22a2, and Slc22a3 in mice, whereas imprinting in humans is a polymorphic trait. The shared phenotypes in the two patients might be associated with the deletion of the gene cluster.

The 30 kb deletion in the APOBEC3 cluster decreases APOBEC3A and APOBEC3B expression and creates a transcriptionally active hybrid gene but does not associate with breast cancer in the European population.

APOBEC3B, in addition to other members of the APOBEC3 gene family, has recently been intensively studied due to its identification as a gene whose activation in cancer is responsible for a specific pattern of massively occurring somatic mutations. It was recently shown that a common large deletion in the APOBEC3 cluster (the APOBEC3B deletion) may increase the risk of breast cancer. However, conflicting evidence regarding this association was also reported. In the first step of our study, using different approaches, including an in-house designed multiplex ligation-dependent probe amplification assay, we analyzed the structure of the deletion and showed that although the breakpoints are located in highly homologous regions, which may generate recurrent occurrence of similar but not identical deletions, there is no sign of deletion heterogeneity. This knowledge allowed us to distinguish transcripts of all affected genes, including the highly homologous canonical APOBEC3A and APOBEC3B, and the hybrid APOBEC3A/APOBEC3B gene. We unambiguously confirmed the presence of the hybrid transcript and showed that the APOBEC3B deletion negatively correlates with APOBEC3A and APOBEC3B expression and positively correlates with APOBEC3A/APOBEC3B expression, whose mRNA level is >10-fold and >1500-fold lower than the level of APOBEC3A and APOBEC3B, respectively. In the next step, we performed a large-scale association study in three different cohorts (2972 cases and 3682 controls) and showed no association of the deletion with breast cancer, familial breast cancer or ovarian cancer. Further, we conducted a meta-analysis that confirmed the lack of the association of the deletion with breast cancer in non-Asian populations.