Umut Aypar

Profiling Mitochondrial Proteins in Radiation-Induced Genome-Unstable Cell Lines with Persistent Oxidative Stress by Mass Spectrometry

Abstract Miller, J. H., Jin, S., Morgan, W. F., Yang, A., Wan, Y., Aypar, U., Peters, J. S. and Springer D. L. Profiling Mitochondrial Proteins in Radiation-Induced Genome-Unstable Cell Lines with Persistent Oxidative Stress by Mass Spectrometry. Radiat. Res. 169, 700–706 (2008). Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.

Genetic testing for hearing loss in the United States should include deletion/duplication analysis for the deafness/infertility locus at 15q15.3

BackgroundHearing loss is the most common birth defect and the most prevalent sensorineural disorder in developed countries. More than 50% of prelingual deafness is genetic, most often autosomal recessive and nonsyndromic, of which 50% can be attributed to the disorder DFNB1, caused by mutations in GJB2 and GJB6. Sensorineural hearing loss and male infertility (Deafness-Infertility Syndrome; DIS) is a contiguous gene deletion syndrome resulting from homozygous deletion of the CATSPER2 and STRC genes on chromosome 15q15.3. Females with DIS have only hearing loss and are fertile. Until recently this syndrome has only been described in three consanguineous families and 2 nonconsanguineous families.ResultsWe recently indentified a patient with hearing loss and macrocephaly who was found to be homozygous for this deletion. Her nonconsanguineous parents are both carriers. We examined our database of patients tested by array CGH and determined that just over 1% of our patients are heterozygous for this deletion. If this number is representative of the general population, this implies a 1% carrier frequency and prevalence of DIS of 1 in 40,000 individuals.ConclusionWe propose that DIS is a greatly under-diagnosed cause of deafness and should be considered in children with hearing loss. Likewise, current molecular genetic testing panels for hearing loss in the United States should be expanded to include deletion/duplication analysis of this region.

Impact of RNA degradation on fusion detection by RNA-seq

BackgroundRNA-seq is a well-established method for studying the transcriptome. Popular methods for library preparation in RNA-seq such as Illumina TruSeq® RNA v2 kit use a poly-A pulldown strategy. Such methods can cause loss of coverage at the 5′ end of genes, impacting the ability to detect fusions when used on degraded samples. The goal of this study was to quantify the effects RNA degradation has on fusion detection when using poly-A selected mRNA and to identify the variables involved in this process.ResultsUsing both artificially and naturally degraded samples, we found that there is a reduced ability to detect fusions as the distance of the breakpoint from the 3′ end of the gene increases. The median transcript coverage decreases exponentially as a function of the distance from the 3′ end and there is a linear relationship between the coverage decay rate and the RNA integrity number (RIN). Based on these findings we developed plots that show the probability of detecting a gene fusion (“sensitivity”) as a function of the distance of the fusion breakpoint from the 3′ end.ConclusionsThis study developed a strategy to assess the impact that RNA degradation has on the ability to detect gene fusions by RNA-seq.

Genetic and Epigenetic Changes in Chromosomally Stable and Unstable Progeny of Irradiated Cells

Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12 and CS9) and decreased Alu element methylation was observed for the other two unstable clones (115 and Fe5.0–8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0–8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes are almost as equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate.

Does parent of origin matter? Methylation studies should be performed on patients with multiple copies of the Prader–Willi/Angelman syndrome critical region

Deletion of 15q11.2‐q13 results in either Prader–Willi syndrome (PWS) or Angelman syndrome (AS) depending on the parent of origin. Duplication of the PWS/AS critical region (PWASCR) has also been reported in association with developmental delay and autism, and it has been shown that they also show a parent‐of‐origin effect. It is generally accepted that maternal duplications are pathogenic. However, there is conflicting evidence as to the pathogenicity of paternal duplications. We have identified 35 patients with gain of the PWASCR using array comparative genomic hybridization. Methylation testing was performed to determine parent of origin of the extra copies. Of the 35 cases, 22 had a supernumerary marker chromosome 15 (SMC15), 12 had a tandem duplication, and 1 had a tandem triplication. Only one patient had a paternal duplication; this patient does not have features typical of patients with maternal duplication of the PWASCR. Three of the mothers had a tandem duplication (two were paternal and one was maternal origin). While one of the two mothers with paternal duplication was noted not to have autism, the other was noted to have learning disability and depression. Based on our data, we conclude that SMC15 are almost exclusively maternal in origin and result in an abnormal phenotype. Tandem duplications/triplications are generally of maternal origin when ascertained on the basis of abnormal phenotype; however, tandem duplications of paternal origin have also been identified. Therefore, we suggest that methylation testing be performed for cases of tandem duplications/triplications since the pathogenicity of paternal gains is uncertain.

Whole-Exome Sequencing of 10 Scientists: Evaluation of the Process and Outcomes

OBJECTIVE To understand motivations, educational needs, and concerns of individuals contemplating whole-exome sequencing (WES) and determine what amount of genetic information might be obtained by sequencing a generally healthy cohort so as to more effectively counsel future patients. PATIENTS AND METHODS From 2012 to 2014, 40 medically educated, generally healthy scientists at Mayo Clinic were invited to have WES conducted on a research basis; 26 agreed to be in a drawing from which 10 participants were selected. The study involved pre- and posttest genetic counseling and completion of 4 surveys related to the experience and outcomes. Whole-exome sequencing was conducted on DNA from blood from each person. RESULTS Most variants (76,305 per person; range, 74,505-77,387) were known benign allelic variants, variants in genes of unknown function, or variants of uncertain significance in genes of known function. The results of suspected pathogenic/pathogenic variants in Mendelian disorders and pharmacogenomic variants were disclosed. The mean number of suspected pathogenic/pathogenic variants was 2.2 per person (range, 1-4). Four pharmacogenomic genes were included for reporting; variants were found in 9 of 10 participants. CONCLUSION This study provides data that may be useful in establishing reality-based patient expectations, outlines specific points to cover during counseling, and increases confidence in the feasibility of providing adequate preparation and counseling for WES in generally healthy individuals.

Offering Prenatal Screening in the Age of Genomic Medicine: A Practical Guide

AIMS In September, 2015, Mayo Clinic convened a panel of national thought leaders on prenatal screening, medical genetics, and obstetrics and gynecology practice. RESULTS During the 2-day symposium, participants discussed the implications of the shift toward broader prenatal screening using cell-free placental DNA in maternal serum (cfDNA screening). Key topics included challenges around the pace of change in the prenatal screening market, uncertainty around reimbursement, meeting the need for patient counseling, and potential challenges in interpreting and returning cfDNA screening results. INNOVATION Here, we describe the challenges discussed and offer clinical recommendations for practices who are working to meet them. CONCLUSION As the spread of prenatal genetic screening continues, providers will increasingly need to update their practice to accommodate new screening modalities.

CNKSR2 deletions: A novel cause of X-linked intellectual disability and seizures

X-linked intellectual disability (XLID) accounts for approximately 10% of intellectual disability in males and contributes to the excess of males in the intellectually disabled population (male to female ratio 1.3–1.4 to 1) [Ropers andHamel, 2005]. Underlying causes of XLID have been extensively studied in recent years, and as a result mutations causing XLID have been described in about 106 genes [Piton et al., 2013]. Recently, Houge et al. [2012] described a maternally inherited 234 kilobase deletion on the X chromosome, which removed themajority of theCNKSR2 gene (OMIM#300724; 21,375,312 – 21,609,484, genomebuild hg19; Fig. 1E) in amalewith developmental delay, epilepsy, and microcephaly. Since CNKSR2 gene is highly expressed only in the brain [Nagase et al., 1998], Houge et al. [2012] suggested that the phenotypic effects of loss of function mutations in the CNKSR2 gene may be restricted to the brain. They concluded that the CNKSR2 gene is a novel candidate gene for nonsyndromic X-linked intellectual disability. A recent report byPiton et al. [2013]onXLID-causingmutations reassessed the implications of 106 genes in their involvement in XLID and classified them into five groups: genes with known mutations, genes with questionable involvement, those that never been replicated, those awaiting replication, and some with likely involvement. The CNKSR2 gene was included in the awaiting replication category since its association with intellectual disability has not been replicated. We report on a deletion in the CNKSR2 gene in a boy with intellectual disability and seizures, therefore replicating the findings of Houge et al. [2012]. The proband was a 7-year-old boy who presented to the Neurology Department for a second opinion regarding his medically intractable focal seizures and developmental delay. He was the first child born to a 24-year-old mother. Aside from occasional alcoholic beverages (one to two drinks at a time at most twice aweek) and smokinguntil approximately fivemonths of gestation, the pregnancy was not complicated by maternal illnesses or exposure to other teratogens. The mother did not take any medications during gestation. Birth was at term and via spontaneous vaginal delivery without complications. Birth weight was 6 pounds, 14 ounces (3118 g, 25th centile), and no postnatal complications were reported. Concerns regarding the patient’s psychomotor development arose within the first year of life, particularly regarding his speech. The patient was slow in achievingmilestones but had no regression.

Biochemical and computational analyses of two phenotypically related GALT mutations (S222N and S135L) that lead to atypical galactosemia

Galactosemia is a metabolic disorder caused by mutations in the GALT gene [1,2]. We encountered a patient heterozygous for a known pathogenic H132Q mutation and a novel S222N variant of unknown significance [3]. Reminiscent of patients with the S135L mutation, our patient had loss of GALT enzyme activity in erythrocytes but a very mild clinical phenotype [3–8]. We performed splicing experiments and computational structural analyses to investigate the role of the novel S222N variant. Alamut software data predicted loss of splicing enhancers for the S222N and S135L mutations [9,10]. A cDNA library was generated from our patient׳s RNA to investigate for splicing errors, but no change in transcript length was seen [3]. In silico structural analysis was performed to investigate enzyme stability and attempt to understand the mechanism of the atypical galactosemia phenotype. Stability results are publicly available in the GALT Protein Database 2.0 [11–14]. Animations were created to give the reader a dynamic view of the enzyme structure and mutation locations. Protein database files and python scripts are included for further investigation.

Development of an NPM1/MLF1 D-FISH Probe Set for the Detection of t(3;5)(q25;q35) Identified in Patients with Acute Myeloid Leukemia

The t(3;5)(q25;q35) NPM1/MLF1 fusion has an incidence of approximately 0.5% in acute myeloid leukemia (AML) and has an intermediate prognosis at diagnosis. We have developed a dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) assay to detect fusion of the MLF1 and NPM1 genes. A blinded investigation was performed using 25 normal bone marrow specimens and 26 bone marrow samples from patients with one or more metaphases with a t(3;5)(q21-q25;q31-q35) or a der(5)t(3;5)(q21-q25;q31-q35) previously identified by chromosome analysis. Once unblinded, the results indicate our D-FISH method identified NPM1/MLF1 fusion in 15 of the 26 fully evaluated patient samples. Excluding three samples with a single abnormal t(3;5) metaphase, 15 of 17 (88%) patient samples with a balanced t(3;5) demonstrated NPM1/MLF1 fusion, and 0 of 6 patient samples with a der(5)t(3;5) demonstrated NPM1/MLF1 fusion, suggesting only the balanced form of this 3;5 translocation as observed by karyotype is associated with NPM1/MLF1 fusion. Overall, the FISH results demonstrated five different outcomes (NPM1/MLF1 fusion, MLF1 disruption, MLF1 duplication, NPM1 deletion, and normal), indicating significant molecular heterogeneity when the 3;5 translocation is identified. The development of this sensitive D-FISH strategy for the detection of NPM1/MLF1 fusion adds to the AML FISH testing repertoire and is effective in the detection of this translocation at diagnosis as well as monitoring residual disease in AML patients.