Scientific Abstract

Structural and sequence variation have been described in several members of the contactin (CNTN) and contactin associated protein (CNTNAP) gene families in association with neurodevelopmental disorders, including autism. Using array comparative genome hybridization (CGH), we identified a maternally inherited ~535 kb deletion at 3p26.3 encompassing the 5′ end of the contactin 4 gene (CNTN4) in a patient with autism. Based on this finding and previous reports implicating genomic rearrangements of CNTN4 in autism spectrum disorders (ASDs) and 3p− microdeletion syndrome, we undertook sequencing of the coding regions of the gene in a local ASD cohort in comparison with a set of controls. Unique missense variants were identified in 4/75 unrelated individuals with an ASD, as well as in 1/107 controls. All of the amino acid substitutions were nonsynonomous, occurred at evolutionarily conserved positions, and were, thus, felt likely to be deleterious. However, these data did not reach statistical significance, nor did the variants segregate with disease within all of the ASD families. Finally, there was no detectable difference in binding of two of the variants to the interacting protein PTPRG in vitro. Thusadditional, larger studies will be necessary to determine whether CNTN4 functions as an autism susceptibility locus in combination with other genetic and/or environmental factors.

Purpose

This study determined the molecular characteristics and clinical significance of amplification of the 13q31 chromosomal region in alveolar rhabdomyosarcoma (ARMS), an aggressive pediatric cancer with frequent PAX3-FOXO1 and PAX7-FOXO1 gene fusions.

Experimental Design

The 13q31 amplicon was localized in an initial panel of ARMS cases using oligonucleotide arrays. A fluorescence in situ hybridization assay for this localized region was designed, and applied to more ARMS cases to determine the frequency and distribution of amplification. Quantitative reverse transcription-PCR assays were applied to measure gene expression. The clinical significance of copy number and expression was determined with Kaplan-Meier and Cox proportional hazard models.

Results

We localized the 13q31 amplicon to a 0.15 Mb region containing the MIR17HG gene encoding the polycistronic microRNA cluster, miR-17-92. This amplicon is present in 23% of ARMS cases with a marked preference for PAX7-FOXO1-positive cases. In tumors with 13q31 amplification, there is significantly increased expression of five of six microRNA’s within the miR-17-92 cluster (miR-17, miR-19a, miR-19b, miR-20a, and miR-92). In addition, a subset of non-amplified tumors with copy number-independent overexpression of all six microRNA’s was identified. In clinical analyses, there was a significantly worse outcome associated with increased expression of the five microRNA’s described above in 13q31-amplified cases when compared to non-amplified cases. There was also an improved outcome in 13q31-amplified cases with lower expression of these microRNA’s.

Conclusions

13q31 amplification and expression of the miR-17-92 cluster provide novel markers for identifying good and poor prognostic subsets of PAX7-FOXO1-positive ARMS.

Introduction

Prior reports of dysferlinopathy suggest a clinically heterogeneous group of patients. We identified specific novel molecular and phenotypic features that help distinguish dysferlinopathies from other forms of limb-girdle muscular dystrophy (LGMD).

Methods

A detailed history, physical exam, protein and mutation analysis of genomic DNA was done in all subjects.

Results

Five of 21 confirmed DYSF gene mutations were not previously reported. A distinct “bulge” of the deltoid muscle in combination with other findings was a striking feature in all patients. Six subjects had atypical calf enlargement, and three of these exhibited a paradoxical pattern of dysferlin expression: severely reduced by direct immunfluorescence with overexpression by western blots. Six patients showed amyloid deposits in muscle that extended these findings to new domains of the dysferlin gene including the C2G domain. Correlative studies showed co-localization of amyloid with deposition of dysferlin.

Discussion

This data further serves to guide clinicians facing the expensive task of molecular characterization of patients with an LGMD phenotype.

Background

The International Neuroblastoma Pathology Classification (INPC) was the first to clearly define prognostic subgroups in ganglioneuroma (GN) and ganglioneuroblastoma (GNB).

Procedure

Histopathology and tumor resectability of 552 GN/GNB cases from the CCG (Children’s Cancer Group) and COG (Children’s Oncology Group) neuroblastoma studies were reviewed. The results were analyzed along with clinical information and biological data of the cases.

Results

According to the INPC, 300 tumors were classified into the Favorable Histology (FH) group and 252 were into the Unfavorable Histology (UH) group. Tumors in the FH group included 43 ganglioneuroma-maturing (GN-M), 198 ganglioneuroblastoma-intermixed (GNB-I), and 59 ganglioneuroblastoma-nodular, favorable subset (GNB-N-FS), and were often (91%) resected completely by single or multiple surgical procedures. Patients with the FH tumors had an excellent prognosis with no tumor-related deaths. The UH group included ganglioneuroblastoma-nodular, unfavorable subset (GNB-N-US) tumors. Patients with the UH tumors had a high incidence (53%) of distant metastasis at the time of diagnosis, and their prognosis significantly depended on clinical stage (5-year EFS: 80.1% for non-stage 4 patients; 16.7% for stage 4 patients): Complete primary tumor resection was not beneficial to those GNB-N-US patients, regardless of whether metastasis was present or not. MYCN amplification was detected in 4 tumors in the FH group and 6 tumors in the UH group. The majority (160/191, 84%) of GN-M and GNB-I tumors had a diploid pattern determined by flow cytometry.

Conclusions

Stringent application of the INPC along with clinical staging was critical for prognostic evaluation of the patients with this group of tumors.

Abstract

The ɛ4 allele of the apolipoprotein E (APOE) gene has been linked to negative outcomes among adults with traumatic brain injury (TBI) across the spectrum of severity, with preliminary evidence suggesting a similar pattern among children. This study investigated the relationship of the APOE ɛ4 allele to outcomes in children with mild TBI. Participants in this prospective, longitudinal study included 99 children with mild TBI between the ages of 8 and 15 recruited from consecutive admissions to Emergency Departments at two large children's hospitals. Outcomes were assessed acutely in the Emergency Department and at follow-ups at 2 weeks, 3 months, and 12 months post-injury. Among the 99 participants, 28 had at least one ɛ4 allele. Children with and without an ɛ4 allele did not differ demographically. Children with an ɛ4 allele were significantly more likely than those without an ɛ4 allele to have a Glasgow Coma Scale score of less than 15, but the groups did not differ on any other measures of injury severity. Those with an ɛ4 allele exhibited better performance than children without an ɛ4 allele on a test of constructional skill, but the groups did not differ on any other neuropsychological tests. Children with and without an ɛ4 allele also did not differ on measures of post-concussive symptoms. Overall, the findings suggest that the APOE ɛ4 allele is not consistently related to the outcomes of mild TBI in children.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer that is related to the skeletal muscle lineage and characterized by recurrent chromosomal translocations. Within the ARMS category, there is clinical and genetic heterogeneity, consistent with the premise that “primary” genetic events collaborate with “secondary” events to give rise to subsets with varying clinical features. Previous studies demonstrated that genomic amplification occurs frequently in ARMS. In the current study, we used oligonucleotide arrays to localize two common amplicons to the 2p24 and 12q13-q14 chromosomal regions. Based on the copy number array data, we sublocalized the minimum common regions of 2p24 and 12q13-q14 amplification to a 0.83 Mb region containing the DDX1 and MYCN genes, and a 0.55 Mb region containing 27 genes, respectively. Using fluorescent in situ hybridization assays to measure copy number of the 2p24 and 12q13-q14 regions in over 100 cases, we detected these amplicons in 13% and 12% of cases, respectively. Comparison with fusion status revealed that 2p24 amplification occurred preferentially in cases positive for PAX3-FOXO1 or PAX7-FOXO1 while 12q13-q14 amplification occurred preferentially in PAX3-FOXO1-positive cases. Expression studies demonstrated that MYCN was usually overexpressed in cases with 2p24 amplification while multiple genes were overexpressed in cases with 12q13-q14 amplification. Finally, although 2p24 amplification did not have a significant association with clinical outcome, 12q13-q14 amplification was associated with significantly worse failure-free and overall survival that was independent of gene fusion status.

X-chromosome inactivation is an epigenetic process used to regulate gene dosage in mammalian females by silencing genes on one X-chromosome. While the pattern of X-chromosome inactivation is typically random in normal females, abnormalities of the X-chromosome may result in skewing due to disadvantaged cell growth. We describe a female patient with an X;1 translocation [46,X,t(X;1)(q28;q21)] and unusual pattern of X-chromosome inactivation who demonstrates functional disomy of the Xq28 region distal to the translocation breakpoint. There was complete skewing of X-chromosome inactivation in the patient, along with the atypical findings of an active normal X chromosome and an inactive derivative X. Characterization of the translocation revealed that the patient’s Xq28 breakpoint interrupts the DKC1 gene. Molecular analysis of the breakpoint region revealed functional disomy of Xq28 genes distal to DKC1. We propose that atypical X-chromosome inactivation occurred in the patient due to a post-inactivation cell selection mechanism likely initiated by disruption of DKC1. As a result, the pattern of X-chromosome inactivation is opposite that of the expected for an X;autosome translocation. Therefore, we suggest the phenotypic abnormalities found in the patient are a result of functional disomy in the Xq28 region.

Background

Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.

Results

We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.

Conclusion

A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.