Mary Ellen Ahearn

Rare Missense and Synonymous Variants in UBE1 Are Associated with X-Linked Infantile Spinal Muscular Atrophy

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3-Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G-->T, p.Met539Ile; c.1639 A-->G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C-->T substitution (c.1731 C-->T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 x 10(-10), p = 0.001815). We also demonstrated that the synonymous C-->T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C-->T transitions might have the potential to affect gene expression.

Recurrent Third-Trimester Fetal Loss and Maternal Mosaicism for Long-QT Syndrome

Background—The importance of germ-line mosaicism in genetic disease is probably underestimated, even though recent studies indicate that it may be involved in 10% to 20% of apparently de novo cases of several dominantly inherited genetic diseases. Methods and Results—We describe here a case of repeated germ-line transmission of a severe form of long-QT syndrome (LQTS) from an asymptomatic mother with mosaicism for a mutation in the cardiac sodium channel, SCN5A. A male infant was diagnosed with ventricular arrhythmias and cardiac decompensation in utero at 28 weeks and with LQTS after birth, ultimately requiring cardiac transplantation for control of ventricular tachycardia. The mother had no ECG abnormalities, but her only previous pregnancy had ended in stillbirth with evidence of cardiac decompensation at 7 months’ gestation. A third pregnancy also ended in stillbirth at 7 months, again with nonimmune fetal hydrops. The surviving infant was found to have a heterozygous mutation in SCN5A (R1623Q), previously reported as a de novo mutation causing neonatal ventricular arrhythmia and LQTS. Initial studies of the mother detected no genetic abnormality, but a sensitive restriction enzyme–based assay identified a small (8% to 10%) percentage of cells harboring the mutation in her blood, skin, and buccal mucosa. Cord blood from the third fetus also harbored the mutant allele, suggesting that all 3 cases of late-term fetal distress resulted from germ-line transfer of the LQTS-associated mutation. Conclusions—Recurrent late-term fetal loss or sudden infant death can result from unsuspected parental mosaicism for LQTS-associated mutations, with important implications for genetic counseling.

X-linked infantile spinal muscular atrophy: Clinical definition and molecular mapping

Purpose: X-linked infantile spinal-muscular atrophy (XL-SMA) is a rare disorder, which presents with the clinical characteristics of hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and death in infancy. We have previously reported a single family with XL-SMA that mapped to Xp11.3-q11.2. Here we report further clinical description of XL-SMA plus an additional seven unrelated (XL-SMA) families from North America and Europe that show linkage data consistent with the same region.Methods: We first investigated linkage to the candidate disease gene region using microsatellite repeat markers. We further saturated the candidate disease gene region using polymorphic microsatellite repeat markers and single nucleotide polymorphisms in an effort to narrow the critical region. Two-point and multipoint linkage analysis was performed using the Allegro software package.Results: Linkage analysis of all XL-SMA families displayed linkage consistent with the original XL-SMA region.Conclusion: The addition of new families and new markers has narrowed the disease gene interval for a XL-SMA locus between SNP FLJ22843 near marker DXS 8080 and SNP ARHGEF9 which is near DXS7132 (Xp11.3-Xq11.1).

Dysfunctional potassium channel subunit interaction as a novel mechanism of long QT syndrome

BACKGROUND The slowly-activating delayed rectifier current IKs contributes to repolarization of the cardiac action potential, and is composed of a pore-forming α-subunit, KCNQ1, and a modulatory β-subunit, KCNE1. Mutations in either subunit can cause long QT syndrome, a potentially fatal arrhythmic disorder. How KCNE1 exerts its extensive control over the kinetics of IKs remains unresolved OBJECTIVE To evaluate the impact of a novel KCNQ1 mutation on IKs channel gating and kinetics METHODS KCNQ1 mutations were expressed in Xenopus oocytes in the presence and absence of KCNE1. Voltage clamping and MODELLER software were used to characterize and model channel function. Mutant and wt genes were cloned into FLAG, Myc and HA expression vectors to achieve differential epitope tagging, and expressed in HEK293 cells for immunohistochemical localization and surface biotinylation assay. RESULTS We identified 2 adjacent mutations, S338F and F339S, in the KCNQ1 S6 domain in unrelated probands. The novel KCNQ1 S338F mutation segregated with prolonged QT interval and torsade de pointes; the second variant, F339S, was associated with fetal bradycardia and prolonged QT interval, but no other clinical events. S338F channels expressed in Xenopus oocytes had slightly increased peak conductance relative to wild type, with a more positive activation voltage. F339S channels conducted minimal current. Unexpectedly, S338F currents were abolished by co-expression with intact WT KCNE1 or its C-terminus (aa63-129), despite normal membrane trafficking and surface co-localization of KCNQ1 S338F and wt KCNE1. Structural modeling indicated that the S338F mutation specifically alters the interaction between the S6 domain of one KCNQ1 subunit and the S4-S5 linker of another, inhibiting voltage-induced movement synergistically with KCNE1 binding. CONCLUSIONS A novel KCNQ1 mutation specifically impaired channel function in the presence of KCNE1. Our structural model shows that this mutation effectively immobilizes voltage gating by an inhibitory interaction that is additive with that of KCNE1. Our findings illuminate a previously unreported mechanism for LQTS, and validate recent theoretical models of the closed state of the KCNQ1:KCNE1 complex.

Abstract 4196: Identification and investigation of ethnic specific gene expression differences in non-cancerous breast tissue

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Disparities in breast cancer (BC) stage of presentation and survival are well documented in patients of different ethnicities. These differences are undoubtedly due to a combination of extrinsic and intrinsic factors. Our group is focused on the genetic contributions to these disparities. Recent observations by our group and others suggest not only the involvement of specific genes contributing to BC risk in African-American (AA) women, but that multiple distinct ethnic-specific gene expression patterns exist in both matched cancerous and normal breast tissue samples. We report the first results of a parallel study which is addressing an unanswered question: are there ethnic-specific differences in the transcriptome of normal breast tissue? We are completing gene expression profiling studies in normal breast FFPE samples from 25 AA and 25 Caucasian (Cauc) women with no history of breast cancer. Laser capture micro-dissection (LCM) is used to further identify and extract epithelial vs stromal cells from each FFPE slide for use in RNA expression profiling. RNA isolation and hybridization occurs in collaboration with Almac Diagnostics (Dx) using their proprietary Breast Cancer DSA array. Gene expression data analyses involves multiple statistical packages developed for DNA microarray studies, and is focusing on comparison of gene expression profiles across four study groups- AA vs Cauc samples and epithelial vs stroma cells. For baseline comparison, a subset of this study group (10 AA and 10 Cauc samples non-LCM dissected) has also been analyzed. Results from these non-LCM dissected samples displayed expression differences between AA and Cauc in 333 known genes. Applying the stringency criteria of a fold change of two or greater and a statistical p-value of 0.01, reduced the significant gene list to 61 genes. Initial pathway analyses indicate these genes are involved in cell adhesion, G-protein signaling, apoptosis and survival pathways, and cytoskeleton remodeling - all critical pathways for cell survival. These initial results are being validated in the LCM-dissected samples, and our latest results will be presented. Results from analyses of these normal samples will be compared to our previous results obtained in matched tumor-normal triple negative AA and Cauc breast cancer samples (as well as publicly available data sets). Combined analysis of tumor and normal breast expression (transcriptome) data will help to better understand the possible significance of gene expression differences in breast tissue between ethnic groups. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4196. doi:1538-7445.AM2012-4196

Abstract 5602: Identification of ethnic specific differences in breast cancer and normal breast tissue

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Disparities in breast cancer stage of presentation and survival rates exist in patients of different ethnicities. These differences are undoubtedly a result of a combination of factors, including socio-economic, lifestyle, tumor characteristics and inherent factors, such as genetic composition. Our group is analyzing the genetic contributions to these disparities, with the goal to increase understanding of the underlying biology, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. Here we report our results of a recently completed study focusing on gene expression profiling in a multi-ethnic cohort of triple negative breast cancer patients. We analyzed breast cancer and self-matched normal tissue samples from 10 African-American (AA), 10 Hispanic (His), and 10 non-Hispanic white (Caucasian) patients from south Florida. Study samples were cut from FFPE (Formalin Fixed Paraffin-embedded tissue) blocks marked by pathology as normal vs. tumor tissue, and sent to Almac Diagnostics for RNA isolation, cDNA preparation, and hybridization of tumor/normal cDNAs to a breast cancer focused gene expression array (Breast Cancer DSA Research Tool). From the Breast Cancer DSA arrays data, a two-way ANOVA (disease state and ethnicity) was used to identify transcripts with a p-value less than 0.01. Data QC indicated that samples clustered well with respect to ethnicity and adjacent normal vs. tumor tissue. We have identified ethnic-specific expression patterns in the matched normal and tumor samples. Initial pathway analysis using MetaCore Program shows that a number of genes related to the DNA repair pathway are differentially expressed across the ethnicities. In a set of ten DNA repair/cell cycle genes, the direction of change (increased or decreased expression) was the same for all three ethnic groups, however, the level of change differed greatly between ethnic groups. Fold change in this set of ten genes ranged from -6.54 to +5.53 with all being greater than 2 fold change in at least one ethnic group. Additional pathway analysis and validation of these results is ongoing. In follow up to this study, we have initiated parallel analysis in normal tissue samples (reduction mammoplasty) samples from AA and Cau non-cancer patients. Combined analysis of tumor and normal expression data will help to better understand the possible significance of gene expression differences in breast tissue between ethnic groups. These studies have important implications for addressing BC health disparities, as well as tailored approaches to prediction, prevention and treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5602. doi:10.1158/1538-7445.AM2011-5602

Abstract B39: Results of a pilot study: Identification of ethnicspecific gene expression differences in normal breast tissue

Disparities in breast cancer stage of presentation and survival rates exist in patients of different ethnicities. These differences are undoubtedly a result of a combination of factors, including socio-economic, lifestyle, tumor characteristics and inherent factors, such as genetic composition. Our group remains focused on analyzing genetic/genomic contributions to these disparities, with the ultimate goal of increased biological understanding, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. We have previously reported ethnicspecific expression patterns in matched tumor and adjacent samples from a cohort of triple negative breast cancer (BC) samples. Here we report results from a parallel study focusing on gene expression profiling in a multi-ethnic collection of normal breast tissues. Study samples were cut from FFPE (formalin fixed paraffin-embedded tissue) blocks saved from local reduction mammoplasty cases [5 Caucasians (CAU); 7 African-American (AA); and 4 Hispanics (HIS) women with no personal or family BC history]. These were sent to Almac Diagnostics for RNA isolation, cDNA preparation, and hybridization of cDNAs to a cancer focused gene expression array (Xcel) containing 110,961 probes, representing 19,905 unique known genes. Arrays were quantile normalized and log transformed to the median of all samples. The probes were filtered to remove variation within each ethnic group to ≤ 0.5 SD, while the variation between the three groups was maintained at ≥ 0.2 SD. Samples which had Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B39.

Abstract P4-08-12: Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded (FFPE) Breast Cancer Samples and Analysis of Intrinsic Subtypes

Background: The advent of microarray technology has enabled robust, high throughput analysis of breast cancer (BC) transcriptomes. Indeed, molecular classification of BC has been revolutionized by the advent of Gene Expression Profiling (GEP). FFPE tumor samples have presented a technical challenge for GEP studies due to degradation of extracted RNA. Newer technologies have overcome this challenge, and have lead to generation of quality GEP data and thus, new insights using archived tissues. Of particular interest to our group has been application of these techniques to the study of triple negative breast cancer (TNBC). TNBC is a BC sub-type characterized by a lack of erbB2 gene amplification and estrogen and progesterone receptor expression. This clinically-defined BC sub-type carries a poor prognosis, is insensitive to hormonal or HER-2 targeted therapies, and displays different incidences among ethnic groups. A better understanding of the genetic and molecular mechanisms underlying TNBC is critical to improving clinic outcomes and developing tailorized therapies. Study Objective: We demonstrate utility of FFPE BC samples in obtaining consistent, reproducible GEP data, and apply this technology to validate the ability to identify intrinsic BC subtypes in unselected specimens, as well to identify differentially expressed genes in TNBC. Methods: RNA isolation and labelled cDNA preparation were performed from freshly cut FFPE sections. Samples were hybridized to a breast cancer focused gene expression array (Breast Cancer DSA Research Tool, Almac Diagnostics Inc). DSA chip quality was assessed on parameters selected automatically from GCOS report files per chip using MATLAB script based web application developed by Almac. Data pre-processing used the Resolver Error Model. All parameters including Raw Q, Background, Scaling Factor and all controls met quality criteria set by Affymetrix and Almac Dx SOPs. Hybridization results were assessed with Principal Component Analysis and Cluster Analysis in a Rosetta Resolver Gene Expression Data Analysis System to identify potential outliers, contamination, or intra-tumor heterogeneity. In total, 47 FFPE breast cancer samples covering a range of hormonal receptor status and sub-types were profiled, as were 28 TNBC FFPE tumor samples. Results: Cluster analysis demonstrated that the Almac Breast Cancer DSA was able to clearly separate the 47 tumor samples of the mixed subtype group into the previously described intrinsic subgroups. Moreover, the DSA array contains 167 probesets which correspond to the 40 of the PAM-50 gene set used as a subtype predictor (Parker et al 2009). Analysis of the TNBC samples using the 167 probeset (based on mean intensity for probes representing each of the 40 genes) showed 100% consistency with published results demonstrating a basal-like gene expression signature. Summary: We have shown that our study methodology used can reliably measure gene expression in FFPE BC samples, and that the Breast Cancer DSA can be used to evaluate intrinsic subtypes of BC specimens. Analysis of the TNBC cases showed complete concordance between the PAM-50 gene set and the corresponding genes in the DSA. These study results are being validated in a larger data set. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-12.

Distinct multiethnic genome-wide alterations in breast cancer using paraffin embedded samples: preliminary analysis.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #2024 Background and Rational : Ethnic-specific disparities in breast cancer (BC) stage of presentation and survival rates are well documented. To further investigate possible ethnic-specific genetic contributions to these disparities, we are completing gene expression profiling studies in a multi-ethnic cohort consisting of thirty “Triple Negative” BC patients [10 each African-American (AA), Hispanic (His) and non-Hispanic white (Cauc) women] matched for age of diagnosis and hormone receptor status. The overall study aim is an increased understanding of the biological basis of ethnic-specific BC disparities, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. Two immediate study goals are to demonstrate the utility of FFPE samples in obtaining consistent, reproducible data from gene expression arrays, and secondly, to identify differentially expressed genes between tumor and normal tissue that are common or unique among the three ethnic groups. Methods : Pathology specimens were freshly cut from FFPE blocks and marked by a pathologist as to normal vs. tumor tissue. RNA isolation, labeled cDNA preparation, and hybridization of tumor and normal cDNAs to a breast cancer focused gene expression microarray (Breast Cancer DSA Research Tool) was performed by Almac Diagnostics. Each patient was self-matched (tumor vs. normal tissue) for gene expression studies. Results : Using 36 matched tumor and normal FFPE samples from 18 patients, approximately 17516 transcripts were detected on the Breast Cancer DSA with intensity significantly greater than background. For normal and tumor tissue samples, 9399 and 10,296 transcripts respectively, were detected in all three ethnic groups. Importantly, a subset of transcripts (hundreds to one thousand) was detected in only one or two ethnic groups. Using two-way ANOVA (disease state and ethnicity), a subset of 6479 transcripts was identified with p-value less than 0.01 in the statistical test and was selected and further used in data quality control. Data QC indicated that patient samples clustered well with respect to both ethnicity and normal versus tumor tissue. Additional analytical methods included K-means 2-Dimensional clustering and Principal Component Analysis. From these analyses, we identified ethnic-specific expression patterns in the matched normal and tumor tissue samples. We are completing these studies by increasing sample size and matching for stage of diagnosis, mapping clusters of differentially-expressed genes in pathway analysis, and validation by real-time PCR. In the longer term, DNA copy number variation (CNV) and chromosomal alterations will be investigated by high density arrays. Summary: These preliminary analyses shows that high quality gene expression data can be generated from FFPE samples, and that ethnic specific gene expression differences can be detected in tumor and matched normal breast tissue samples across ethnic groups. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2024.

Gene Expression Profiling of Formalin-Fixed, Pariffin-Embedded (FFPE) Tissues from Triple-Negative (TN) Breast Cancer (BC) Patients.

Background : FFPE tumor samples present a technical challenge for gene expression profiling studies. Newer technologies are resulting in quality data and new insights from these archived tissues. TNBC is a sub-type of BC characterized by a lack of erbB2 gene amplification and estrogen and progesterone receptor expression. This clinically-defined BC sub-type carries a poor prognosis, is insensitive to hormonal or HER-2 targeted therapeutic agents, and has different incidence among ethnic groups. A better understanding of the genetic and molecular mechanisms underlying TNBC is critical to improving clinic outcomes and developing individualized therapies. Study Objective : We demonstrate the utility of FFPE samples in obtaining consistent, reproducible data from gene expression arrays, and apply this technology to the identification of differentially expressed genes between TNBC and normal breast tissue that are common or unique among selected ethnic groups. Methods : RNA isolation and labeled cDNA preparation from freshly cut FFPE blocks (marked by a pathologist as to normal vs. tumor tissue) was performed using the NuGEN™ WT-Ovation™ FFPE RNA Amplification System . Hybridization of tumor and normal cDNAs occurred to a breast cancer focused gene expression array ( Breast Cancer DSA Research Tool, Almac Diagnostics Inc). Each patient9 sample served as it9s own control (tumor vs. normal). In total, 75 FFPE samples were profiled. The quality of each DSA chip was assessed on parameters selected automatically from GCOS report files per chip using MATLAB script based web application developed by Almac Dx . Data pre-processing used the Resolver Error Model. All parameters including Raw Q, Background, Scaling Factor and all controls met quality criteria set by Affymetrix and Almac Dx SOPs. Hybridization results were assessed using Principal Component Analysis and Clustering Analysis in Rosetta Resolver Gene Expression Data Analysis System 7.1 to identify potential outliers, contamination, or intra-tumor heterogeneity. Results : A Sign Agreement Matrix of FFPE and fresh frozen tissue samples during validation of the Breast Cancer DSA demonstrated that 98% of the probesets showed the same direction of fold change [ p Spearman (FC)=0.84]. The FFPE samples had an average present call ∼ 43%, and more than 90% of the FFPE samples had present call rates greater than 25%. QC analysis demonstrated that the Almac Breast Cancer DSA was able to clearly and consistently separate tumor samples from normal in FFPE tissues and to identify samples of quality or integrity issues. Application of this methodology to analyses of differentially expressed transcripts between cancer and normal tissue samples across ethnicities in the TNBC samples, detected 1350 differentially expressed genes in the African-American group, 1220 genes in the Caucasian group and 1226 genes in the White Hispanic group. We also observed certain subtle ethnic-specific expression patterns across these three ethnic groups. Summary : The above-described methodology can be used to reliably measure gene expression in FFPE breast samples. Our study results are being validated in a larger data set. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6125.