Aaron Theisen

Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: Is less more?

Chromosome analysis is an important component to the diagnosis of congenital anomalies, developmental delay, and mental retardation. Routine chromosome analysis identifies aneuploidy and structural rearrangements greater than 5 Mb but cannot identify abnormalities of the telomeric regions or microdeletions reliably. Molecular cytogenetic techniques were developed to overcome these limitations. High‐resolution comparative genomic hybridization (CGH)‐based microarrays (array CGH) were developed to increase the resolution of chromosomal studies and to provide a comprehensive assay by using large‐insert clones as the target for analysis. We constructed a microarray for the clinical diagnosis of medically significant and relatively common chromosomal alterations. Nine hundred six bacterial artificial chromosome (BAC) clones were chosen, the chromosomal locations of which were confirmed by fluorescence in situ hybridization (FISH). FISH‐testing showed that 7% of the clones were mismapped based on map locations obtained from two publicly available databases (58 mapped to the wrong chromosome and three mapped to a different locus on the same chromosome), 16% cross‐hybridized to other chromosomes, and 12% did not hybridize or showed poor hybridization signals under uniform FISH conditions. Thus, from a total of 906 BAC clones that were evaluated, only 589 (65%) were deemed adequate for arraying on this clinical device. The performance of this array was tested in a set of blinded experiments on a cohort of phenotypically normal individuals and on individuals with known chromosome abnormalities. The array identified deletion/duplication polymorphisms not seen by FISH in the phenotypically normal individuals and detected single copy dosage differences in all of the cases with known chromosomal abnormalities. All abnormalities detected by the array were confirmed by FISH with BACs from the appropriate loci. Our data demonstrate that the rigorous assessment of BACs and their use in array CGH is especially important when the microarray is used for clinical diagnosis. In addition, this study illustrates that when constructed carefully with proper attention to the quality of the BACs that are arrayed, array CGH is an effective and efficient tool for delineating chromosomal aberrations and an important adjunct to FISH and conventional cytogenetics.

Expanding the clinical phenotype of the 3q29 microdeletion syndrome and characterization of the reciprocal microduplication

BackgroundInterstitial deletions of 3q29 have been recently described as a microdeletion syndrome mediated by nonallelic homologous recombination between low-copy repeats resulting in an ~1.6 Mb common-sized deletion. Given the molecular mechanism causing the deletion, the reciprocal duplication is anticipated to occur with equal frequency, although only one family with this duplication has been reported.ResultsIn this study we describe 14 individuals with microdeletions of 3q29, including one family with a mildly affected mother and two affected children, identified among 14,698 individuals with idiopathic mental retardation who were analyzed by array CGH. Eleven individuals had typical 1.6-Mb deletions. Three individuals had deletions that flank, span, or partially overlap the commonly deleted region. Although the clinical presentations of individuals with typical-sized deletions varied, several features were present in multiple individuals, including mental retardation and microcephaly. We also identified 19 individuals with duplications of 3q29, five of which appear to be the reciprocal duplication product of the 3q29 microdeletion and 14 of which flank, span, or partially overlap the common deletion region. The clinical features of individuals with microduplications of 3q29 also varied with few common features. De novo and inherited abnormalities were found in both the microdeletion and microduplication cohorts illustrating the need for parental samples to fully characterize these abnormalities.ConclusionOur report demonstrates that array CGH is especially suited to identify chromosome abnormalities with unclear or variable presentations.

The discovery of microdeletion syndromes in the post-genomic era: review of the methodology and characterization of a new 1q41q42 microdeletion syndrome

Purpose: The advent of molecular cytogenetic technologies has altered the means by which new microdeletion syndromes are identified. Whereas the cytogenetic basis of microdeletion syndromes has traditionally depended on the serendipitous ascertainment of a patient with established clinical features and a chromosomal rearrangement visible by G-banding, comparative genomic hybridization using microarrays has enabled the identification of novel, recurrent imbalances in patients with mental retardation and apparently nonspecific features. Compared with the “phenotype-first” approach of traditional cytogenetics, array-based comparative genomic hybridization has enabled the detection of novel genomic disorders using a “genotype-first” approach. We report as an illustrative example the characterization of a novel microdeletion syndrome of 1q41q42.Methods: We tested more than 10,000 patients with developmental disabilities by array-based comparative genomic hybridization using our targeted microarray. High-resolution microarray analysis was performed using oligonucleotide microarrays for patients in whom deletions of 1q41q42 were identified. Fluorescence in situ hybridization was performed to confirm all 1q deletions in the patients and to exclude deletions or other chromosomal rearrangements in the parents.Results: Seven cases were found with de novo deletions of 1q41q42. The smallest region of overlap is 1.17 Mb and encompasses five genes, including DISP1, a gene involved in the sonic hedgehog signaling pathway, the deletion of which has been implicated in holoprosencephaly in mice. Although none of these patients showed frank holoprosencephaly, many had other midline defects (cleft palate, diaphragmatic hernia), seizures, and mental retardation or developmental delay. Dysmorphic features are present in all patients at varying degrees. Some patients showed more severe phenotypes and carry the clinical diagnosis of Fryns syndrome.Conclusions: This new microdeletion syndrome with its variable clinical presentation may be responsible for a proportion of Fryns syndrome patients and adds to the increasing number of new syndromes identified with array-based comparative genomic hybridization. The genotype-first approach to identifying recurrent chromosome abnormalities is contrasted with the traditional phenotype-first approach. Targeting developmental pathways in a functional approach to diagnostics may lead to the identification of additional microdeletion syndromes.

Comparison of microarray‐based detection rates for cytogenetic abnormalities in prenatal and neonatal specimens

To compare the detection rate by microarray analysis for chromosome abnormalities in a prenatal population to that of a neonatal population referred for diagnostic testing.

Identification of a previously unrecognized microdeletion syndrome of 16q11.2q12.2

We report the identification of microdeletions of 16q11.2q12.2 by microarray‐based comparative genomic hybridization (aCGH) in two individuals. The clinical features of these two individuals include hypotonia, gastroesophageal reflux, ear anomalies, and toe deformities. Other features include developmental delay, mental retardation, hypothyroidism, and seizures. The identification of common clinical features in these two individuals and those of one other report suggests microdeletion of 16q12.1q12.2 is a rare, emerging syndrome. These results illustrate that aCGH is particularly suited to identify rare chromosome abnormalities in patients with apparently non‐syndromic idiopathic mental retardation and birth defects.

The clinical utility of enhanced subtelomeric coverage in array CGH

Telomeric chromosome abnormalities are a substantial cause of mental retardation and birth defects. Although subtelomeric fluorescence in situ hybridization (FISH) probes have been widely used to identify submicroscopic telomeric rearrangements, array‐based comparative genomic hybridization (array CGH) has emerged as a more efficient and comprehensive detection method. Due to the clinical relevance of telomeric abnormalities, it has been proposed that array CGH using panels of BAC clones that map to regularly spaced intervals along the length of each telomere could be used to characterize subtelomeric aberrations more precisely in a single experiment. We have added 1,120 FISH‐mapped BAC clones to our microarray to enhance the coverage of the 41 unique human subtelomeric regions. Contigs of clones were selected in increments of ∼0.5 Mb beginning with the most distal unique sequence for each subtelomere and extending on average ∼5.7 Mb toward the centromere. We have used this microarray to characterize 169 clinically significant subtelomeric abnormalities identified out of nearly 7,000 consecutive clinical cases analyzed by array CGH in our diagnostic laboratory. The expanded telomere coverage was sufficient to define the breakpoints of over half (56%) of the chromosome abnormalities. However, 44% of the subtelomeric aberrations extended beyond the size of this expanded coverage suggesting that many subtelomeric abnormalities are >5 Mb in size and that greater representation may be of even more value. In addition to identifying 6 cases of complex rearrangements, we have identified 42 cases of interstitial deletions that would have been missed by subtelomere FISH panels that use a single clone to the most distal unique sequence for each region. Microarrays designed to investigate regions known to be involved in chromosome abnormalities will enhance the detection of cytogenetic abnormalities at unprecedented resolution and frequency.

High-resolution array CGH defines critical regions and candidate genes for microcephaly, abnormalities of the corpus callosum, and seizure phenotypes in patients with microdeletions of 1q43q44

Microdeletions of 1q43q44 result in a recognizable clinical disorder characterized by moderate to severe intellectual disability (ID) with limited or no expressive speech, characteristic facial features, hand and foot anomalies, microcephaly (MIC), abnormalities (agenesis/hypogenesis) of the corpus callosum (ACC), and seizures (SZR). Critical regions have been proposed for some of the more prominent features of this disorder such as MIC and ACC, yet conflicting data have prevented precise determination of the causative genes. In this study, the largest of pure interstitial and terminal deletions of 1q43q44 to date, we characterized 22 individuals by high-resolution oligonucleotide microarray-based comparative genomic hybridization. We propose critical regions and candidate genes for the MIC, ACC, and SZR phenotypes associated with this microdeletion syndrome. Three cases with MIC had small overlapping or intragenic deletions of AKT3, an isoform of the protein kinase B family. The deletion of only AKT3 in two cases implicates haploinsufficiency of this gene in the MIC phenotype. Likewise, based on the smallest region of overlap among the affected individuals, we suggest a critical region for ACC that contains ZNF238, a transcriptional and chromatin regulator highly expressed in the developing and adult brain. Finally, we describe a critical region for the SZR phenotype which contains three genes (FAM36A, C1ORF199, and HNRNPU). Although ~90% of cases in this study and in the literature fit these proposed models, the existence of phenotypic variability suggests other mechanisms such as variable expressivity, incomplete penetrance, position effects, or multigenic factors could account for additional complexity in some cases.

Copy number variants of schizophrenia susceptibility loci are associated with a spectrum of speech and developmental delays and behavior problems

Purpose: Recently, molecular cytogenetic techniques have identified novel copy number variants in individuals with schizophrenia. However, no large-scale prospective studies have been performed to characterize the broader spectrum of phenotypes associated with such copy number variants in individuals with unexplained physical and intellectual disabilities encountered in a diagnostic setting.Methods: We analyzed 38,779 individuals referred to our diagnostic laboratory for microarray testing for the presence of copy number variants encompassing 20 putative schizophrenia susceptibility loci. We also analyzed the indications for study for individuals with copy number variants overlapping those found in six individuals referred for schizophrenia.Results: After excluding larger gains or losses that encompassed additional genes outside the candidate loci (e.g., whole-arm gains/losses), we identified 1113 individuals with copy number variants encompassing schizophrenia susceptibility loci and 37 individuals with copy number variants overlapping those present in the six individuals referred to our laboratory for schizophrenia. Of these, 1035 had a copy number variant of one of six recurrent loci: 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11, and 22q11.2. The indications for study for these 1150 individuals were diverse and included developmental delay, intellectual disability, autism spectrum, and multiple congenital anomalies.Conclusion: The results from our study, the largest genotype-first analysis of schizophrenia susceptibility loci to date, suggest that the phenotypic effects of copy number variants associated with schizophrenia are pleiotropic and imply the existence of shared biologic pathways among multiple neurodevelopmental conditions.

Identification of familial and de novo microduplications of 22q11.21-q11.23 distal to the 22q11.21 microdeletion syndrome region

Deletions of the 22q11.2 region distal to the 22q11.21 microdeletion syndrome region have recently been described in individuals with mental retardation and congenital anomalies. Because these deletions are mediated by low-copy repeats (LCRs), located distal to the 22q11.21 DiGeorge/velocardiofacial microdeletion region, duplications are predicted to occur with a frequency equal to the deletion. However, few microduplications of this region have been reported. We report the identification of 18 individuals with microduplications of 22q11.21-q11.23. The duplication boundaries for all individuals are within LCRs distal to the DiGeorge/velocardiofacial microdeletion region. Clinical records for nine subjects reveal shared characteristics, but also several examples of contradicting clinical features (e.g. macrocephaly versus microcephaly and upslanting versus downslanting palpebral fissures). Of 12 cases for whom parental DNA samples were available for testing, one is de novo and 11 inherited the microduplication from a parent, three of whom reportedly have learning problems or developmental delay. The variable phenotypes and preponderance of familial cases obfuscate the clinical relevance of the molecular data and emphasize the need for careful parental assessments and clinical correlations.

Development of a high-density pericentromeric region BAC clone set for the detection and characterization of small supernumerary marker chromosomes by array CGH

Purpose: Small supernumerary marker chromosomes are centric chromosomal segments that, by definition, cannot be characterized unambiguously by conventional chromosome banding. Marker chromosomes are of particular interest in clinical cytogenetics because they are nearly 10 times more frequent in individuals with mental retardation (0.426%) than in the normal population (0.043%). However, they are often found in only a small percentage of cells, making them difficult to detect and characterize in a diagnostic setting. We designed, constructed, and employed a bacterial artificial chromosome (BAC)-based microarray to demonstrate the utility of array-based comparative genomic hybridization (array CGH) for detecting and characterizing marker chromosomes in clinical diagnostic specimens.Methods: We constructed a high-density microarray using 974 BAC clones that were mapped by fluorescence in situ hybridization and cover approximately 5 Mb of the most proximal unique sequence adjacent to the centromere on all 43 unique pericentromeric regions of the human genome (excluding the acrocentric short arms). This array was used to further characterize 20 previously identified marker chromosomes that were originally found with either conventional chromosome analysis or a targeted microarray.Results: The enhanced coverage of this pericentromeric array not only identified the chromosomal origin of each marker in 15 cases, it also distinguished between the involvement of the short arm and/or the long arm of each chromosome, defined the sizes of many of the markers, and revealed complex rearrangements or multiple markers in single individuals. However, in five cases, the markers could not be identified by this assay, most likely because of very low levels of mosaicism and/or their small size and lack of detectable euchromatin. The expanded coverage of the pericentromeric regions represented on the array was adequate to refine the breakpoints in two-thirds of all cases in which a marker chromosome was identified by this assay.Conclusions: This study demonstrates the utility of array CGH in the detection and characterization of mosaic marker chromosomes. Because approximately one-third of the markers characterized in this study involved more unique sequence than that represented on this array, additional pericentromeric coverage may be even more valuable. We anticipate that this will allow detailed characterization of small supernumerary marker chromosomes that will greatly facilitate phenotype/genotype correlations and play a valuable role in the diagnosis and medical management of both pre- and postnatal cases in which marker chromosomes have been identified.