Evolving applications of microarray technology in postnatal diagnosis (review)

Microarray-based cytogenetics is revealing the tremendous fluidity and complexity of the human genome, and is starting to illustrate the implications of genomic variability with respect to human health and disease. In the last few years, the robustness of array-based technologies has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner for identifying genomic defects of congenital and developmental abnormalities including developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASD) and/or multiple congenital anomalies (MCA). The implementation of this technology in these categories of disorders has been thoroughly evaluated and is now recommended as a first-line diagnostic approach for clinically suspected genetic disorders. However, clinical application of array-CGH in postnatal evaluation raises the debate of whether array-CGH will replace traditional cytogenetics in the near future and whether there is still a role for karyotyping and FISH. In this article, we therefore review the current status of array-based technology use for postnatal diagnosis and predict that it will replace standard cytogenetics as a first-line test for clinical evaluation in these population groups.     

Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements

The sequence of the mouse genome allows one to compare the conservation of synteny between the human and mouse genome and exploration of regions that might have been involved in major rearrangements during the evolution of these two species (evolutionary genome rearrangements). Recent segmental duplications (or duplicons) are paralogous DNA sequences with high sequence identity that account for about 3.5-5% of the human genome and have emerged during the past approximately 35 million years of evolution. These regions are susceptible to illegitimate recombination leading to rearrangements that result in genomic disorders or genomic mutations. A catalogue of several hundred segmental duplications potentially leading to genomic rearrangements has been reported. The authors and others have observed that some chromosome regions involved in genomic disorders are shuffled in orientation and order in the mouse genome and that regions flanked by segmental duplications are often polymorphic. We have compared the human and mouse genome sequences and demonstrate here that recent segmental duplications correlate with breaks of synteny between these two species. We also observed that nine primary regions involved in human genomic disorders show changes in the order or the orientation of mouse/human synteny segments, were often flanked by segmental duplications in the human sequence. We found that 53% of all evolutionary rearrangement breakpoints associate with segmental duplications, as compared with 18% expected in a random location of breaks along the chromosome (P<0.0001). Our data suggest that segmental duplications have participated in the recent evolution of the human genome, as driving forces for evolutionary rearrangements, chromosome structure polymorphisms and genomic disorders.     

Expression analysis of 6p22 genomic gain in retinoblastoma

To identify gene(s) targeted by 6p22 genomic gain, present in more than 50% retinoblastoma tumors, we used real-time RT-PCR to quantify the expression of seven genes in normal human retina and retinoblastoma. Six genes are located in the quantitative multiplex PCR-defined 0.6 Mb minimal region of gain at 6p22 (DEK, AOF1, TPMT, NHLRC1, KIF13A, and NUP153), and E2F3 is 2 Mb away from the minimal region of gain on 6p22. E2F3, DEK, KIF13A, and NUP153 were most frequently overexpressed in retinoblastoma with 6p genomic gain, compared with the normal adult human retina. E2F3 and DEK mRNA levels were increased in all human tumors showing 6p22 gain, as well as in mouse retinoblastoma induced by SV40 large T antigen expression in developing retina, compared with the normal controls (adult human retina and 7-day-old mouse retina, respectively). Only DEK showed statistically significant correlation of expression and genomic copy number (P = 0.019). E2F3 and DEK, but not NUP153, showed developmental regulation. E2F3 and DEK mRNA overexpression was always associated with protein overexpression, determined by immunoblotting or immunofluorescent staining of primary tumors, relative to the adjacent normal retina. E2F3 was strongly expressed in actively proliferating cells, while DEK was overexpressed in all tumor cells. Taking into account the proliferation-promoting role of E2F3, implication of E2F3 in bladder and prostate cancer, and the translocation and overexpression of DEK in leukemia, we conclude that either DEK or E2F3 (or both) are targeted by the 6p22 gain in retinoblastoma.     

Detection of anti-dsDNA by ELISA using different sources of antigens

AIM: To compare different sources of DNA for use in ELISA-based assays for anti-dsDNA antibody detection in systemic lupus erythematosus (SLE) diagnosis. METHOD: Bacterial genomic DNA from Flavobacterium menignosepticum, Proteus vulgalis, Seratia marcescens, Streptococcus pyogenes and Salmonella typhimurium and genomic DNA from human blood were used as antigens for IgG anti-dsDNA detection by enzyme-linked immunosorbent assay (ELISA). Eighty-six sera were tested, 28 derived from patients with SLE, 28 from patients with other rheumatic diseases and 30 from normal human subjects. RESULTS: Genomic DNA from Flavobacterium menignosepticum and human blood had high sensitivity (75%, 82%) and specificity (91%, 91%) for anti-dsDNA detection in diagnosis of SLE. However, human genomic DNA was the most effective antigen of all antigens studied. The assay had a higher sensitivity but lower specificity than commercial ELISA (61% sensitivity and 95% specificity). There was a high level of correlation between commercial ELISA and ELISA using human genomic DNA as antigen (r=0.776, p<0.001) and they exhibited a high level of diagnostic agreement with each other (kappa=0.890, p<0.001). CONCLUSION: The genomic DNA from human blood is a potentially useful source of antigen for the detection of anti-dsDNA by ELISA. However, further studies are required to compare the performance of ELISA using this source of antigen against commercial radioimmunoassays for anti-dsDNA detection.     

Guidelines for genomic array analysis in acquired haematological neoplastic disorders

Genetic profiling is important for disease evaluation and prediction of prognosis or responsiveness to therapy in neoplasia. Microarray technologies, including array comparative genomic hybridization and single‐nucleotide polymorphism‐detecting arrays, have in recent years been introduced into the diagnostic setting for specific types of haematological malignancies and solid tumours. It can be used as a complementary test or depending on the neoplasia investigated, also as a standalone test. However, comprehensive and readable presentation of frequently identified complex genomic profiles remains challenging. To assist diagnostic laboratories, standardization and minimum criteria for clinical interpretation and reporting of acquired genomic abnormalities detected through arrays in neoplastic disorders are presented. © 2016 Wiley Periodicals, Inc.     

Human metabotropic glutamate receptor 2 gene (GRM2): chromosomal sublocalization (3p21.1-p21.2) and genomic organization

Imbalances in glutamatergic function have been implicated in the pathogenesis of neuropsychiatric disorders. Consequently, glutamate receptors genes are promising candidates in search of susceptibility genes for these disorders. In the present study, we report the chromosomal sublocalization and genomic organization of the human metabotropic glutamate receptor 2 gene (GRM2). Using monochromosomal hybrid cell lines of NIGMS Mapping Panel 2 (Coriell Cell Repository), the GRM2 gene was localized to human chromosome 3, confirming previously reported localization. In addition, using the radiation hybrid panel RH3 (Research Genetics), we sublocalized the GRM2 gene to chromosomal region 3p21.1-p21.2. The genomic organization of the GRM2 gene was established using a premade library of adaptor-ligated, human-specific genomic DNA fragments. The gene consists of 5 exons, with sizes ranging from 74 to 1,076 bp.     

Predicting chromatin architecture from models of polymer physics

We review the picture of chromatin large-scale 3D organization emerging from the analysis of Hi-C data and polymer modeling. In higher mammals, Hi-C contact maps reveal a complex higher-order organization, extending from the sub-Mb to chromosomal scales, hierarchically folded in a structure of domains-within-domains (metaTADs). The domain folding hierarchy is partially conserved throughout differentiation, and deeply correlated to epigenomic features. Rearrangements in the metaTAD topology relate to gene expression modifications: in particular, in neuronal differentiation models, topologically associated domains (TADs) tend to have coherent expression changes within architecturally conserved metaTAD niches. To identify the nature of architectural domains and their molecular determinants within a principled approach, we discuss models based on polymer physics. We show that basic concepts of interacting polymer physics explain chromatin spatial organization across chromosomal scales and cell types. The 3D structure of genomic loci can be derived with high accuracy and its molecular determinants identified by crossing information with epigenomic databases. In particular, we illustrate the case of the Sox9 locus, linked to human congenital disorders. The model in-silico predictions on the effects of genomic rearrangements are confirmed by available 5C data. That can help establishing new diagnostic tools for diseases linked to chromatin mis-folding, such as congenital disorders and cancer.     

Sequencing and characterization of an Echinococcus multilocularis DNA probe and its use in the polymerase chain reaction

The nucleotide sequence of the cloned Echinococcus multilocularis DNA probe pAL1 was determined in order to simplify and improve the sensitivity of a diagnostic assay through the application of the polymerase chain reaction (PCR). The insert-specific oligonucleotides BG1 and BG2 define a 2.6-kb fragment in the genomic DNA of E. multilocularis, while BG1 and BG3 define a 0.3 kb fragment. A PCR study including 14 independent E. multilocularis isolates in addition to Echinococcus granulosus. Echinococcus vogeli, Taenia spp. and other cestodes revealed that the 2.6-kb fragment was amplified from genomic DNA of all E. multilocularis isolates tested (originating from Switzerland, Alaska, Canada, France, Germany and Japan), but from genomic DNA of none of the other cestode species. PCR with BG1 and BG2 furthermore uniquely resulted in the synthesis of a 0.55-kb fragment specific for Taenia saginata and a 0.6-kb fragment specific for T. taeniaeformis. In contrast to the species specificity of the 2.6-kb BG1/BG2 product, the 0.3 kb (BG1/BG3) product demonstrated genus specificity: the 0.3-kb product was amplified from genomic DNA of all E. multilocularis, E. granulosus and E. vogeli isolates tested, but from genomic DNA of none of the other cestode species. The diagnostic sensitivity of PCR using both primer sets was determined to be 50 pg parasite DNA, suggesting the practical utility of this simple assay in demonstrating parasite DNA in specimens from a variety of sources. At the basic level, the pAL1-derived oligonucleotides may also prove useful in assessing strain variation, RFLPs or other manifestations of genetic variation in E. multilocularis.     

Assignment of the human gene encoding eukaryotic initiation factor 4E (EIF4E) to the region q21-25 on chromosome 4

We recently cloned genomic sequences containing the promoter region for the messenger RNA cap binding protein (eIF4E). As the rate-limiting step in translation, eukaryotic initiation factor 4E is important in cellular growth control. Using oligonucleotide primers specific for the promoter region in polymerase chain reactions (PCR), we amplified the human gene in a chromosome 4-specific human/rodent somatic cell panel. This panel mapped single copy genomic sequences for eIF4E in the region 4q21 to 4q25.     

Detection of Clinically Relevant Copy Number Variants with Whole‐Exome Sequencing

Copy number variation (CNV) is a common source of genetic variation that has been implicated in many genomic disorders. This has resulted in the widespread application of genomic microarrays as a first‐tier diagnostic tool for CNV detection. More recently, whole‐exome sequencing (WES) has been proven successful for the detection of clinically relevant point mutations and small insertion–deletions exome wide. We evaluate the utility of short‐read WES (SOLiD 5500xl) to detect clinically relevant CNVs in DNA from 10 patients with intellectual disability and compare these results to data from two independent high‐resolution microarrays. Eleven of the 12 clinically relevant CNVs were detected via read‐depth analysis of WES data; a heterozygous single‐exon deletion remained undetected by all algorithms evaluated. Although the detection power of WES for small CNVs currently does not match that of high‐resolution microarray platforms, we show that the majority (88%) of rare coding CNVs containing three or more exons are successfully identified by WES. These results show that the CNV detection resolution of WES is comparable to that of medium‐resolution genomic microarrays commonly used as clinical assays. The combined detection of point mutations, indels, and CNVs makes WES a very attractive first‐tier diagnostic test for genetically heterogeneous disorders.     

ImprintSeq,a novel tool to interrogate DNA methylation at human imprinted regions and diagnose multilocus imprinting disturbance

PurposeDisruptions of genomic imprinting are associated with congenital imprinting disorders (CIDs) and other disease states, including cancer. CIDs are most often associated with altered methylation at imprinted differentially methylated regions (iDMRs). In some cases, multiple iDMRs are affected causing multilocus imprinting disturbances (MLIDs). The availability of accurate, quantitative, and scalable high-throughput methods to interrogate multiple iDMRs simultaneously would enhance clinical diagnostics and research.MethodsWe report the development of a custom targeted methylation sequencing panel that covered most relevant 63 iDMRs for CIDs and the detection of MLIDs. We tested it in 70 healthy controls and 147 individuals with CIDs. We distinguished loss and gain of methylation per differentially methylated region and classified high and moderate methylation alterations.ResultsAcross a range of CIDs with a variety of molecular mechanisms, ImprintSeq performed at 98.4% sensitivity, 99.9% specificity, and 99.9% accuracy (when compared with previous diagnostic testing). ImprintSeq was highly sensitive for detecting MLIDs and enabled diagnostic criteria for MLID to be proposed. In a child with extreme MLID profile a probable genetic cause was identified.ConclusionImprintSeq provides a novel assay for clinical diagnostic and research studies of CIDs, MLIDs, and the role of disordered imprinting in human disease states.     

Genome-wide arrays: quality criteria and platforms to be used in routine diagnostics

Whole-genome analysis using genome-wide arrays, also called "genomic arrays," "microarrays," or "arrays," has become the first-tier diagnostic test for patients with developmental abnormalities and/or intellectual disabilities. In addition to constitutional anomalies, genomic arrays are also used to diagnose acquired disorders. Despite the rapid implementation of these technologies in diagnostic laboratories, external quality control schemes (such as CEQA, EMQN, UK NEQAS, and the USA QA scheme CAP) and interlaboratory comparisons show that there are huge differences in quality, interpretation, and reporting among laboratories. We offer guidance to laboratories to help assure the quality of array experiments and to standardize minimum detection resolution, and we also provide guidelines to standardize interpretation and reporting.     

The genome of human coronavirus strain 229E

The genomic RNA of human coronavirus strain 229E (HCV 229E) migrated on polyacrylamide gels as a single peak with a mol. wt. of 5.8 X 10(6). Denaturation of the genome with formaldehyde did not alter its electrophoretic mobility, which suggests that the HCV 229E genome is a single-stranded molecule. At least 30% of the genomic RNA was shown to contain covalently attached polyadenylic acid [poly(A)]sequences by binding the RNA to an oligo(dT)-cellulose column. These poly(A) tracts were shown to be about 70 nucleotides in length by measuring the resistance to digestion of HCV 229E RNA with pancreatic and T1 RNases. Finally, the genomic RNA was shown to terminate at or near the 3'-terminus on the basis of its susceptibility to polynucleotide phosphorylase.     

Development and validation of a CGH microarray for clinical cytogenetic diagnosis.

PURPOSE: We developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). METHODS: The array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. RESULTS: The CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%. Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. CONCLUSION: The array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype. Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.     

Other genomic disorders and congenital heart disease

Congenital heart disease (CHD) is the common birth defect worldwide. Despite its recognized burden on public health, the etiology in the vast majority of individuals remains unknown. Chromosomal abnormality plays an important role, frequently observed as large cytogenetically visible rearrangement or small submicroscopic structural variation in the genome. Several genomic disorders are now recognized that are increasingly responsible for CHD with variable penetrance. Single gene disorders, epigenetic alterations, and environmental etiologies are also significant contributors. Our understanding of the genetic basis of CHD has increased exponentially with the escalating use of next generation sequencing to identify ever so small submicroscopic genomic imbalances at the level of coding exons in CHD. This review focuses on genomic disorders other than 22q11.2 deletion, that are major players in the etiology of human cardiac malformations.     

Detection of copy-number variation in AUTS2 gene by targeted exonic array CGH in patients with developmental delay and autistic spectrum disorders

Small genomic rearrangements and copy-number variations (CNVs) involving a single gene have been associated recently with many neurocognitive phenotypes, including intellectual disability (ID), behavioral abnormalities, and autistic spectrum disorders (ASDs). Such small CNVs in the Autism susceptibility candidate 2 (AUTS2) gene have been shown to be associated with seizures, ID, and ASDs. We report four patients with small CNVs ranging in size between 133–319 kb that disrupt AUTS2. Two patients have duplications involving single exons, whereas two have deletions that removed multiple exons. All patients had developmental delay, whereas two patients had a diagnosis of ASDs. The CNVs were detected by an exon-targeted array CGH with dense oligonucleotide coverage in exons of genes known or hypothesized to be causative of multiple human phenotypes. Our report further shows that disruption of AUTS2 results in a variety of neurobehavioral phenotypes. More importantly, it demonstrates the utility of targeted exon array as a highly sensitive clinical diagnostic tool for the detection of small genomic rearrangements in the clinically relevant regions of the human genome.     

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders

PurposeCopy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10–20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test.MethodsWe performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting.ResultsIn total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to –5.8% per disorder).ConclusionsThis study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016     

Speech delay and autism spectrum behaviors are frequently associated with duplication of the 7q11.23 Williams-Beuren syndrome region.

PURPOSE: Williams-Beuren syndrome is among the most well-characterized microdeletion syndromes, caused by recurrent de novo microdeletions at 7q11.23 mediated by nonallelic homologous recombination between low copy repeats flanking this critical region. However, the clinical phenotype associated with reciprocal microduplication of this genomic region is less well described. We investigated the molecular, clinical, neurodevelopmental, and behavioral features of seven patients with dup(7)(q11.23), including two children who inherited the microduplication from one of their parents, to more fully characterize this emerging microduplication syndrome. METHODS: Patients were identified by array-based comparative genomic hybridization. Clinical examinations were performed on seven affected probands, and detailed cognitive and behavioral evaluations were carried out on four of the affected probands. RESULTS: Our findings confirm initial reports of speech delay seen in patients with dup(7)(q11.23) and further delineate and expand the phenotypic spectrum of this condition to include communication, social interactions, and repetitive interests that are often observed in individuals diagnosed with autism spectrum disorders. CONCLUSIONS: Array-based comparative genomic hybridization is a powerful means of detecting genomic imbalances and identifying molecular etiologies in the clinic setting, including genomic disorders such as Williams-Beuren syndrome and dup(7)(q11.23). We propose that dup(7)(q11.23) syndrome may be as frequent as Williams-Beuren syndrome and a previously unrecognized cause of language delay and behavioral abnormalities. Indeed, these individuals may first be referred for evaluation of autism, even if they do not ultimately meet diagnostic criteria for an autism spectrum disorder.     

Emerging themes and new challenges in defining the role of structural variation in human disease

The widespread use of array-comparative genomic hybridization (array-CGH) for the detection of copy number variants (CNVs) in both research and clinical laboratories has created a renaissance in the field of molecular cytogenetics, revealing that the human genome contains both a wealth of structural polymorphism and many novel genomic disorders. A new generation of experimental platforms enable structural variants to be identified with increasing resolution, and will require the development of more sophisticated methods to assess the pathogenic significance of novel structural variants if these technologies are to be of clinical utility. Indeed, we are now entering an era in which technologies to detect CNVs have advanced much faster than our understanding of the consequences of these variants on human phenotypes, and I argue that over the last few years the problem has now become one of interpretation rather than identification. This problem is made more complex by the realization that many genomic disorders show highly variable penetrance, blurring the boundary of how to define benign vs. pathogenic variants. I discuss insights from recent research which shed light on potential mechanisms that may underlie this phenomenon, and possible methods to determine the genetic elements that are responsible for the associated phenotype. Furthermore, there is now a growing appreciation that the underlying chromosomal architecture which catalyses many genomic disorders is polymorphic within the general population, and I discuss potential mechanisms by which inversion polymorphisms might create predispositions to genomic disorders.     

Whole exome and genome sequencing in mendelian disorders: a diagnostic and health economic analysis

Whole genome sequencing (WGS) improves Mendelian disorder diagnosis over whole exome sequencing (WES); however, additional diagnostic yields and costs remain undefined. We investigated differences between diagnostic and cost outcomes of WGS and WES in a cohort with suspected Mendelian disorders. WGS was performed in 38 WES-negative families derived from a 64 family Mendelian cohort that previously underwent WES. For new WGS diagnoses, contemporary WES reanalysis determined whether variants were diagnosable by original WES or unique to WGS. Diagnostic rates were estimated for WES and WGS to simulate outcomes if both had been applied to the 64 families. Diagnostic costs were calculated for various genomic testing scenarios. WGS diagnosed 34% (13/38) of WES-negative families. However, contemporary WES reanalysis on average 2 years later would have diagnosed 18% (7/38 families) resulting in a WGS-specific diagnostic yield of 19% (6/31 remaining families). In WES-negative families, the incremental cost per additional diagnosis using WGS following WES reanalysis was AU$36,710 (£19,407;US$23,727) and WGS alone was AU$41,916 (£22,159;US$27,093) compared to WES-reanalysis. When we simulated the use of WGS alone as an initial genomic test, the incremental cost for each additional diagnosis was AU$29,708 (£15,705;US$19,201) whereas contemporary WES followed by WGS was AU$36,710 (£19,407;US$23,727) compared to contemporary WES. Our findings confirm that WGS is the optimal genomic test choice for maximal diagnosis in Mendelian disorders. However, accepting a small reduction in diagnostic yield, WES with subsequent reanalysis confers the lowest costs. Whether WES or WGS is utilised will depend on clinical scenario and local resourcing and availability.Subject terms: Genetics research, Genetic testing, Medical genomics