Chromosomal microarray analysis (CMA) is standard of care, first-tier clinical testing for detection of genomic copy number variation among patients with developmental disabilities. Although diagnostic yield is higher than traditional cytogenetic testing, management impact has not been well studied. We surveyed genetic services providers regarding CMA ordering practices and perceptions about reimbursement. Lack of insurance coverage because of perceived lack of clinical utility was cited among the most frequent reasons why CMA was not ordered when warranted. We compiled a list of genomic regions where haploinsufficiency or triplosensitivity cause genetic conditions with documented management recommendations, estimating that at least 146 conditions potentially diagnosable by CMA testing have published literature supporting specific clinical management implications. Comparison with an existing clinical CMA database to determine the proportion of cases involving these regions showed that CMA diagnoses associated with such recommendations are found in approximately 7% of all cases (n = 28,526). We conclude that CMA impacts clinical management at a rate similar to other genetic tests for which insurance coverage is more readily approved. The information presented here can be used to address barriers that continue to contribute to inequities in patient access and care in regard to CMA testing.
Copy number variation (CNV) is a common form of structural variation detected in human genomes, occurring as both constitutional and somatic events. Cytogenetic techniques like chromosomal microarray (CMA) are widely used in analyzing CNVs. However, CMA techniques cannot resolve the full nature of these structural variations (i.e. the orientation and location of associated breakpoint junctions) and must be combined with other cytogenetic techniques, such as karyotyping or FISH, to do so. This makes the development of a next-generation sequencing (NGS) approach capable of resolving both CNVs and breakpoint junctions desirable. Mate-pair sequencing (MPseq) is a NGS technology designed to find large structural rearrangements across the entire genome. Here we present an algorithm capable of performing copy number analysis from mate-pair sequencing data. The algorithm uses a step-wise procedure involving normalization, segmentation, and classification of the sequencing data. The segmentation technique combines both read depth and discordant mate-pair reads to increase the sensitivity and resolution of CNV calls. The method is particularly suited to MPseq, which is designed to detect breakpoint junctions at high resolution. This allows for the classification step to accurately calculate copy number levels at the relatively low read depth of MPseq. Here we compare results for a series of hematological cancer samples that were tested with CMA and MPseq. We demonstrate comparable sensitivity to the state-of-the-art CMA technology, with the benefit of improved breakpoint resolution. The algorithm provides a powerful analytical tool for the analysis of MPseq results in cancer.
EIF2AK3 is a type I transmembrane protein that functions as an endoplasmic reticulum (ER) stress sensor to regulate global protein synthesis. Rare mutations in EIF2AK3 cause Wolcott-Rallison syndrome (OMIM 226980), an autosomal recessive disorder characterized by diabetes, epiphyseal dysplasia, osteoporosis, and growth retardation. To investigate the role of common genetic variation in EIF2AK3 as a determinant of bone mineral density (BMD) and osteoporosis, we sequenced all exons and flanking regions and then genotyped 6 potentially functional single nucleotide polymorphisms (SNPs) in this gene in 997 Amish subjects for association analysis, with attempted replication in 887 Mexican Americans. We found that the minor allele of a nonsynonymous SNP rs13045 had borderline associations with decreased forearm BMD in both discovery and replication cohorts (unadjusted P = 0.036 and β = −0.007 for the Amish; unadjusted P = 0.031 and β = −0.008 for Mexican Americans). A meta-analysis indicated this association achieved statistical significance in the combined sample (unadjusted P = 0.003; Bonferroni corrected P = 0.009). Rs13045 and three other potentially functional SNPs, a promoter SNP (rs6547787) and two nonsynonymous SNPs (rs867529 and rs1805165), formed two haplotypes (a low-BMD associated haplotype, denoted haplotype B (minor allele frequency (MAF) = 0.311) and a common haplotype A (MAF = 0.676)). There were no differences in mRNA expression from lymphoblastoid cell lines between the two haplotypes. However, after treating lymphoblastoid cell lines with thapsigargin to induce ER stress, cell lines with haplotype B showed increased sensitivity to ER stress (P = 0.014) compared to cell lines with haplotype A. Taken together, our results suggest that common nonsynonymous sequence variants in EIF2AK3 have a modest effect on ER stress response and may contribute to the risk for low BMD through this mechanism.
Objective: Acute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints. Method:We evaluated whether an NGS-based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML.Results: Using 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML-specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML-fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML-specific structural rearrangements in 10/88 (11%) of cases analyzed. Conclusion:Utilization of MPseq represents a precise, molecular-based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies. K E Y W O R D Sacute myeloid leukemia, molecular cytogenetics, MPseq
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