Identification of RNA editing sites in the SNP database

The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing calls for a careful distinction between inherited SNPs and somatic DNA and RNA modifications. In order to demonstrate that some of the SNP database records are actually somatic modification, we focus on one type of these modifications, namely A-to-I RNA editing, and present evidence for hundreds of dbSNP records that are actually editing sites. We provide a list of 102 RNA editing sites previously annotated in dbSNP database as SNPs, and experimentally validate seven of these. Interestingly, we show how dbSNP can serve as a starting point to look for new editing sites. Our results, for this particular type of RNA editing, demonstrate the need for a careful analysis of SNP databases in light of the increasing recognition of the significance of somatic sequence modifications

Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: Efficient identification of known microduplications and identification of a novel microduplication in ASMT

<p>Abstract</p> <p>Background</p> <p>It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications.</p> <p>Methods</p> <p>In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region.</p> <p>Results</p> <p>MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, <it>de novo </it>duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include <it>GABRB3 </it>and <it>ATP10A </it>in one case, and <it>MKRN3</it>, <it>MAGEL2 </it>and <it>NDN </it>in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the <it>ASPA </it>gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the <it>TM4SF2 </it>gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the <it>ASMT </it>gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003).</p> <p>Conclusion</p> <p>MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new <it>de novo </it>small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in <it>TM4SF2</it> are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the <it>ASMT </it>locus indicate that further studies of the duplication of the <it>ASMT </it>gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.</p

Mutation analysis of the ferritin L-chain gene in age-related cataract

Purpose: To investigate whether acquired somatic mutations in the iron response element of the ferritin L-chain gene account for the age-related cataract. Methods: The 15 most prevalent point mutations causing hereditary hyperferritinemia cataract syndrome (HHCS) were screened in patients with age-related cataract using MALDI-TOF Mass Spectrometry. DNA samples were obtained from the lens capsules of patients following cataract surgery, and subjected to PCR amplification. Products were analyzed by a Sequenom® mass spectrometer, and classified as a mutation or wild type according to molecular weight. For a positive control, L-ferritin G32T mutation detected by direct sequencing in 3 members of an Israeli family known to be affected by HHCS was used. Results: DNA samples were isolated from the lens capsules of 90 patients, mean age 73.86, and screened for L-ferritin mutations. While the G32T mutation was detected in all 3 positive control cases, all other patients were negative for the 15 mutations. Conclusions: Somatic mutations in the iron response elements (IRE) of the L-ferritin gene are infrequent in the age-related cataract. The role of L-ferritin genetic variations in the pathogenesis of age-related cataract is yet to be explored

Transient abnormal myelopoiesis with extramedullary involvement in a down syndrome preemie leading to an unresponsive course despite chemotherapy

Introduction: Transient abnormal myelopoiesis (TAM) is a transient, clonal myeloproliferative disorder unique to Down Syndrome (DS) babies. It is characterized by increased peripheral blasts and presence of GATA1 mutation. The clinical spectrum ranges from jaundice and hepatosplenomegaly to multi-organ failure and death. Here we present a case of a premature baby with DS diagnosed to have TAM with extramedullary involvement at birth who had a fatal outcome. Case report: A 30.3-week-old female fetus with DS had leukocytosis (WBC: 187.82 K/uL) with neutrophilia (ANC 27.65 K/uL), macrocytic anemia (RBC: 2.41 m/uL, Hb 8.8 g/dL, MCV 108.3, MCH 36.5, MCHC 33.7) and thrombocytosis (platelet count 361 K/uL) at birth. Liver panels demonstrated normal bilirubin levels with elevated liver enzymes (AST = 239 U/L, ALT = 216 U/L). Results: Peripheral smear showed marked leukocytosis with increased blasts (70%), nucleated RBCs, giant platelets, and megakaryocytic elements. Flow cytometry demonstrated two populations of cells: 20% myeloblasts and 26% dim CD45 CD34- cells. GATA1 mutation was present. Based on these findings a diagnosis of TAM with extramedullary hematopoiesis was made. She received two cycles of cytarabine chemotherapy. Though her WBC levels reached a low of 18.93 K/uL, she developed multi-organ failure, eventually leading to death on day 45. Discussion: TAM is a transient condition resulting in disease resolution in around 80% of cases. Death is reported in 10% of cases. Risk factors associated with early death include prematurity, hyperleukocytosis, elevated bilirubin levels. Management of high-risk babies with chemotherapy is recommended to improve survival

Familial inheritance of the 3q29 microdeletion syndrome: case report and review

Abstract Background The chromosome 3q29 microdeletion syndrome is characterized by a clinical phenotype that includes behavioral features consistent with autism and attention deficit hyperactivity disorder, mild to moderate developmental delay, language-based learning disabilities, and/or dysmorphic features. In addition, recent data suggest that adults with chromosome 3q29 microdeletions have a significantly increased risk for psychosis and neuropsychiatric phenotypes. Case presentation We report a 3-year-old male with global developmental delay, anemia, and mild dysmorphic facial features. Clinical chromosomal microarray (CMA) testing of the proband detected a heterozygous 1.21 Mb deletion at chromosome 3q29, consistent with a diagnosis of the 3q29 microdeletion syndrome. Interestingly, subsequent parental testing determined that the pathogenic deletion was inherited from his otherwise healthy mother who had a history of learning disabilities. The chromosome 3q29 microdeletion was not detected in the healthy older sibling of the proband by CMA testing, nor was it prenatally detected in a subsequent maternal pregnancy. Conclusion Our report highlights the 3q29 microdeletion syndrome as an illustrative example of the importance of a molecular diagnosis for families that harbor pathogenic copy number aberrations with variable expressivity, in particular those that also impart an increased risk for adult onset neuropsychiatric phenotypes

De Novo Interstitial Deletion of 9q in a Pediatric Patient With Global Developmental Delay

Cytogenomic microarray (CMA) methodologies, including array comparative genomic hybridization (aCGH) and single-nucleotide polymorphism-detecting arrays (SNP-array), are recommended as the first-tier test for the evaluation of imbalances associated with intellectual disability, autism, and multiple congenital anomalies. The authors report on a child with global developmental delay (GDD) and a de novo interstitial 7.0 Mb deletion of 9q21.33q22.31 detected by aCGH. The patient that the authors report here is noteworthy in that she presented with GDD and her interstitial deletion is not inclusive of the 9q22.32 locus that includes the PTCH1 gene, which is implicated in Gorlin syndrome, or basal cell nevus syndrome (BCNS), has not been previously reported among patients with a similar or smaller size of the deletion in this locus suggesting that the genomic contents in the identified deletion on 9q21.33q22.31 is critical for the phenotype

Abstract 19583: Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a pathological disorder predominantly due to mutations in sarcomeric components. Germline mutations in BRAF cause a developmental syndrome called cardio-facio-cutaneous syndrome (CFCS), in which 40% of patients also develop HCM. Since the role of the RAS/MAPK pathway in HCM is still unclear, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF T599R or Q257R mutations. In order to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM, we generated a method to purify cardiomyocytes and non-cardiomyocytes simultaneously by cell sorting based on SIRPα and CD90 expression. Purified BRAF-mutant SIRPα+/CD90- cells were &gt;95% cardiomyocytes, displayed cellular hypertrophy with pro-hypertrophic gene expression, and dysregulation of the RAS/MAPK pathway. BRAF-mutant cardiomyocytes also displayed intrinsic calcium handling defects, including increased calcium transient irregularity and increased stored calcium within the sarcoplasmic reticulum. In addition, purified BRAF-mutant SIRPα-/CD90+ cells, which were fibroblast-like, displayed activation of the RAS/MAPK pathway and exhibited a pro-fibrotic phenotype. Cross-culture studies revealed that BRAF-mutant fibroblast-like cells critically modulate cardiomyocyte hypertrophy through TGFβ paracrine signaling, as TGFβ inhibition prevented cardiomyocyte hypertrophy induced by BRAF-mutant fibroblast-like cells. Additionally, inhibition of RAS/MAPK signaling was capable of rescuing BRAF-mutant cardiomyocyte hypertrophy and cardiomyocyte-intrinsic calcium handling abnormalities. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM. As fibroblast activation has been documented previously in sarcomeric HCM, our findings suggest that cardiac fibroblasts may contribute to pathologic hypertrophy in addition to causing fibrosis in primary forms of HCM. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies