Troy J. Gliem

An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities

Purpose: Copy number variants have emerged as a major cause of human disease such as autism and intellectual disabilities. Because copy number variants are common in normal individuals, determining the functional and clinical significance of rare copy number variants in patients remains challenging. The adoption of whole-genome chromosomal microarray analysis as a first-tier diagnostic test for individuals with unexplained developmental disabilities provides a unique opportunity to obtain large copy number variant datasets generated through routine patient care.Methods: A consortium of diagnostic laboratories was established (the International Standards for Cytogenomic Arrays consortium) to share copy number variant and phenotypic data in a central, public database. We present the largest copy number variant case-control study to date comprising 15,749 International Standards for Cytogenomic Arrays cases and 10,118 published controls, focusing our initial analysis on recurrent deletions and duplications involving 14 copy number variant regions.Results: Compared with controls, 14 deletions and seven duplications were significantly overrepresented in cases, providing a clinical diagnosis as pathogenic.Conclusion: Given the rapid expansion of clinical chromosomal microarray analysis testing, very large datasets will be available to determine the functional significance of increasingly rare copy number variants. This data will provide an evidence-based guide to clinicians across many disciplines involved in the diagnosis, management, and care of these patients and their families.

Deletion 17q12 Is a Recurrent Copy Number Variant that Confers High Risk of Autism and Schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.

B9D1 is revealed as a novel Meckel syndrome (MKS) gene by targeted exon-enriched next-generation sequencing and deletion analysis

Meckel syndrome (MKS) is an embryonic lethal, autosomal recessive disorder characterized by polycystic kidney disease, central nervous system defects, polydactyly and liver fibrosis. This disorder is thought to be associated with defects in primary cilia; therefore, it is classed as a ciliopathy. To date, six genes have been commonly associated with MKS (MKS1, TMEM67, TMEM216, CEP290, CC2D2A and RPGRIP1L). However, mutation screening of these genes revealed two mutated alleles in only just over half of our MKS cohort (46 families), suggesting an even greater level of genetic heterogeneity. To explore the full genetic complexity of MKS, we performed exon-enriched next-generation sequencing of 31 ciliopathy genes in 12 MKS pedigrees using RainDance microdroplet-PCR enrichment and IlluminaGAIIx next-generation sequencing. In family M456, we detected a splice-donor site change in a novel MKS gene, B9D1. The B9D1 protein is structurally similar to MKS1 and has been shown to be of importance for ciliogenesis in Caenorhabditis elegans. Reverse transcriptase-PCR analysis of fetal RNA revealed, hemizygously, a single smaller mRNA product with a frameshifting exclusion of B9D1 exon 4. ArrayCGH showed that the second mutation was a 1.713 Mb de novo deletion completely deleting the B9D1 allele. Immunofluorescence analysis highlighted a significantly lower level of ciliated patient cells compared to controls, confirming a role for B9D1 in ciliogenesis. The fetus inherited an additional likely pathogenic novel missense change to a second MKS gene, CEP290; p.R2210C, suggesting oligogenic inheritance in this disorder.

Comprehensive validation of array comparative genomic hybridization platforms: how much is enough?

Clinical testing using various array comparative genomic hybridization platforms is being incorporated rapidly into cytogenetic testing algorithms. Comprehensive validation of these complex assays presents unique challenges and very few studies reporting the validation of commercially available array platforms have been published. Sixty-seven patients with previously defined subtelomere abnormalities, representing deletions and/or duplications of all 41 clinically relevant sites, were tested in a blinded study using the Spectral Genomics Constitutional Chip 3.0. Overall, 72 of 74 (97%) subtelomeric abnormalities were concordant with previous cytogenetic studies. However, two false-negative results were documented, and issues with mismapped and suboptimal clone performance were identified that may result in failure to detect 6q and 20q subtelomeric abnormalities. The results of this study indicate that comprehensive validation is necessary before implementation of array comparative genomic hybridization platforms into a clinical setting. Specific suggestions for validation are discussed in the context of the recently proposed American College of Medical Genetics guidelines for microarray analysis for constitutional cytogenetic abnormalities.

Mucinous cystadenoma of ovary in a patient with juvenile polyposis due to 10q23 microdeletion: Expansion of phenotype

Juvenile polyposis syndrome (JPS) is a hereditary condition characterized by development of gastrointestinal polyps, and caused by mutations in SMAD4 or BMPR1A genes. Juvenile polyps can also be found in a related group of syndromes with multisystemic involvement including Cowden disease, Lhermitte–Duclos disease, Bannayan–Riley–Ruvalcaba syndrome, and Proteus‐like syndrome, all grouped as PTEN hamartoma tumor syndromes (PHTS). In all these conditions including JPS, polyps manifest in older childhood or early adulthood. Infantile juvenile polyposis (JPI) is a rare entity, presenting in the first year of life with severe gastrointestinal symptoms. Many of these patients have associated macrocephaly, hypotonia, and congenital anomalies. It was recently recognized that patients with infantile polyposis have a 10q23 microdeletion, involving both BMPR1A and PTEN genes. There is a major risk for gastrointestinal malignancies in these patients, but the risk for development of other tumors is not known. We describe a patient with a history of infantile polyposis, macrocephaly, developmental delay, hypotonia, and a 10q23 microdeletion. At age 14 she presented with bilateral mucinous cystadenoma of the ovary. This type of tumor was not previously reported in association with JPS, 10q23 microdeletion syndrome, or infantile polyposis. We believe that ovarian cystadenomas may be another neoplastic complication of infantile polyposis, and that our report widens the spectrum of the 10q23 microdeletion phenotype.

Development of a Chromosomal Microarray Test for the Detection of Abnormalities in Formalin-Fixed, Paraffin-Embedded Products of Conception Specimens

Testing the products of conception (POCs) provides information about the cause of fetal loss and helps determine the recurrence risk of future losses and chromosome abnormalities in subsequent pregnancies. Historically, the Mayo Clinic Cytogenetics Laboratory performed targeted fluorescent in situ hybridization (FISH) testing to identify aneuploidy of only certain chromosomes in formalin-fixed, paraffin-embedded (FFPE) POC samples. Chromosomal microarray studies using the Affymetrix OncoScan FFPE Assay can detect copy number changes across the genome. We validated the utility of the OncoScan assay using 25 archival FFPE POC specimens with previous FISH results (five normal, 12 trisomy, six triploidy, two monosomy). Of the five normal samples, four had no clinically relevant findings, and one sample was found to have trisomy 9, which is not detectable by the FISH test. For the 20 samples with abnormal FISH results, the OncoScan assay identified all reported abnormalities along with additional findings. A sample with trisomy 22 was also found to have trisomy 7. Another sample reported as triploidy was found to have four copies of chromosome 16. In conclusion, we verified the performance characteristics of OncoScan on FFPE POC specimens and found it acceptable for clinical use. Additional information was identified in 3 of 25 cases (12%) that would explain the pregnancy loss or provide recurrence risk for the family.

Abstract 3572: Copy number detection using genomics technologies: A comparison between aCGH and NGS

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Identification of causal DNA copy number variations (CNVs) is critical to the understanding of cancer development and progression, and is important clinically for accurate prognosis and efficient treatment. T-cell lymphomas are replete with amplifications and deletions that result in over- and under-expression of genes. Array comparative genomic hybridization (aCGH) has been the technology of choice to detect CNVs but recently, it has been proposed that mate-pair Next Generation Sequencing (mpNGS) can produce similar results. Here, we report a comparison between the two techniques. Methods: We have developed an algorithm based on extreme value theory to identify CNVs across the genome at high resolution from mate-pair NGS data. The algorithm is based on plotting the distribution of read-counts in windows across the genome, and detecting deletions and gains by finding the windows that differ significantly from the distribution of the normal part of the genome. We compared this algorithm with aCGH data to detect CNVs. We ran five cases of T-cell lymphoma with both techniques and compared the results using concordance statistics. Results: With 50kb lower cutoff for a CNV, mpNGS detected 98% of the cumulative deleted genome and 94% of the amplified/gained genome that were called by aCGH. Most of the discordant CNVs that were detected by only aCGH were in regions of repetitive areas where mapping fails. Conversely, aCGH detected 75% of the cumulative deleted genome and 74% of the amplified/gained genome that were called by mpNGS. Conclusion: A detailed comparison of these techniques showed that both have pros and cons. In general, mpNGS can detect most CNVs detected by aCGH, as long as they are more than 50KB. Additionally, mpNGS offers confirmatory data, particularly for deletions, based on detection of aberrant mate pairs that map to the rejoined ends of deleted regional. Finally, the mate-pair technology can also detect balanced translocations and can also predict fusion genes. Citation Format: George Vasmatzis, Andrew L. Feldman, Sarah H. Johnson, Erik C. Thorland, Rafael Fonseca, Esteban Braggio, Troy J. Gliem. Copy number detection using genomics technologies: A comparison between aCGH and NGS. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3572. doi:10.1158/1538-7445.AM2014-3572