Kelley Kneile

OBJECTIVE: Creatine kinase (CK) levels are increased on dried blood spots in newborns related to the birthing process. As a marker for newborn screening, CK in Duchenne muscular dystrophy (DMD) results in false-positive testing. In this report, we introduce a 2-tier system using the dried blood spot to first assess CK with follow-up DMD gene testing. METHODS: A fluorometric assay based upon the enzymatic transphosphorylation of adenosine diphosphate to adenosine triphosphate was used to measure CK activity. Preliminary studies established a population-based range of CK in newborns using 30,547 deidentified anonymous dried blood spot samples. Mutation analysis used genomic DNA extracted from the dried blood spot followed by whole genome amplification with assessment of single-/multiexon deletions/duplications in the DMD gene using multiplex ligation-dependent probe amplification. RESULTS: DMD gene mutations (all exonic deletions) were found in 6 of 37,649 newborn male subjects, all of whom had CK levels>2,000U/l. In 3 newborns with CK>2,000U/l in whom DMD gene abnormalities were not found, we identified limb-girdle muscular dystrophy gene mutations affecting DYSF, SGCB, and FKRP. INTERPRETATION: A 2-tier system of analysis for newborn screening for DMD has been established. This path for newborn screening fits our health care system, minimizes false-positive testing, and uses predetermined levels of CK on dried blood spots to predict DMD gene mutations.

Limb-girdle muscular dystrophy type 2C (LGMD2C) is considered one of the severe forms of childhood-onset muscular dystrophy. The geographical distribution of founder mutations in the SGCG gene has a prominent effect on the prevalence of LGMD2C in certain populations. The aim of this study was to confirm the hypothesis that the c.787G>A (p.E263K) mutation in the SGCG gene is a founder mutation among Puerto Rican Hispanics and to characterize the associated clinical and immunohistochemical phenotype. Genotyping of six polymorphic microsatellite markers internal to (D13S232) and flanking (D13S175, D13S292, D13S787, D13S1243, D13S283) the SGCG gene was performed on four unrelated Puerto Rican patients with LGMD2C. Preserved ambulation to the second decade of life was observed in at least two subjects. Immunostaining of skeletal muscle demonstrated absence of γ-sarcoglycan in all affected subjects. Two markers, D13S232 and D13S292, were highly informative and confirmed that all four families share the haplotype of the mutant allele. Our findings confirm that the E263K missense mutation in the SGCG gene is a founder mutation in Puerto Rican Hispanics. A slowly progressive disease course with prolonged preservation of ambulation can be seen in association with this mutation, providing evidence for phenotypic variability.

Abstract BACKGROUND: Features of cancer genomics including gene expression levels can be employed to create biomarker profiles that can predict prior to therapy the diagnosis and prognosis of an individual patient. We aim to translate robust and reproducible diagnostic and prognostic profiles for pediatric solid tumors into clinical tools applicable to all tumor specimens, fresh frozen (FF) or formalin fixed (FFPE). RESULTS: We used Affymetrix GeneChip Human Exon 1.0 ST (HuEx) arrays on 24 cell lines or tumors (4 fusion-positive rhabdomyosarcoma [RMSpos], 5 fusion-negative RMS [RMSneg], 6 Ewing sarcoma family of tumors [ESFT], 5 neuroblastoma [NB], and 4 osteosarcoma [OS]) to identify a 41-feature diagnostic metagene that clearly distinguished both the original test samples and a set of validation samples. We then incorporated the 41-feature metagene into a 48-gene panel. To translate this diagnostic signature, we compared the performance of HuEx arrays with several mid-plex assay platforms including Fluidigm's quantitative reverse-transcriptase PCR (q-RT-PCR) and NanoString's nCounter™ Digital Analyzer by measuring gene expression on an 18-cell line panel (4 RMSpos, 4 RMSneg, 5 ESFT, 1 NB, and 4 OS). With q-RT-PCR, we observed r2 values of 0.703 ± 0.085 (RMSpos), 0.558 ± 0.138 (RMSneg), 0.689 ± 0.070 (ESFT), and 0.607 ± 0.121 (OS). nCounter reported r2 values of 0.672 ± 0.171 (RMSpos), 0.620 ± 0.143 (RMSneg), 0.675 ± 0.094 (ESFT), and 0.583 ± 0.129 (OS). Hierarchical clustering with all platforms was able to distinguish the RMSpos, ESFT, and NB cell lines; however, the RMSneg and OS cell lines were less clearly distinguished because some of the OS cell lines lacked high expression of genes characteristic of OS tumors. Cross-platform comparisons yielded good correlation between nCounter and Q-RT-PCR (average r2 = 0.710 ± 0.183). An 18-sample dilution series revealed that EWS-FLI1 type I or PAX3-FKHR transcripts were consistently detected by q-RT-PCR or nCounter even at 1:1000 RNA dilution. Among biological duplicates, q-RT-PCR reported an average r2 = 0.916 ± 0.039 while nCounter obtained average r2 = 0.983 ± 0.011 including raw cell lysates. In addition, we also compared Fluidigm's q-RT-PCR with Applied Biosystems’ Taqman Low-Density Arrays (TLDA) on five NB patient bone marrow samples to detect residual tumor cells. We observed an average r2 = 0.929 ± 0.119 across five NB genes and one housekeeping gene. Finally, we have also successfully profiled FF vs. FFPE tumors on HuEx and are currently applying these mid-plex technologies to paired FF and FFPE samples in order to create diagnostic profiles applicable to routine FFPE material. CONCLUSIONS: Mid-plex platform can reliably distinguish bone and soft tissue sarcoma cell lines and can be used to translate the application of diagnostic signatures. These data warrant further studies to analyze FFPE samples, to compare additional mid-plex platforms, and to test potential prognostic signatures. 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 4350. doi:10.1158/1538-7445.AM2011-4350