Amber N. Pursley

Clinical use of array comparative genomic hybridization (aCGH) for prenatal diagnosis in 300 cases.

To evaluate the use of array comparative genomic hybridization (aCGH) for prenatal diagnosis, including assessment of variants of uncertain significance, and the ability to detect abnormalities not detected by karyotype, and vice versa.

Detection of Clinically Relevant Exonic Copy-Number Changes by Array CGH

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy‐number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy‐number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom‐designed, exon‐targeted oligonucleotide array to detect intragenic copy‐number changes in patients with various clinical phenotypes. Hum Mutat 31:1–17, 2010.

Parental Somatic Mosaicism Is Underrecognized and Influences Recurrence Risk of Genomic Disorders

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.

Identification of chromosome abnormalities in subtelomeric regions by microarray analysis: a study of 5,380 cases.

Subtelomeric imbalances are a significant cause of congenital disorders. Screening for these abnormalities has traditionally utilized GTG‐banding analysis, fluorescence in situ hybridization (FISH) assays, and multiplex ligation‐dependent probe amplification. Microarray‐based comparative genomic hybridization (array‐CGH) is a relatively new technology that can identify microscopic and submicroscopic chromosomal imbalances. It has been proposed that an array with extended coverage at subtelomeric regions could characterize subtelomeric aberrations more efficiently in a single experiment. The targeted arrays for chromosome microarray analysis (CMA), developed by Baylor College of Medicine, have on average 12 BAC/PAC clones covering 10 Mb of each of the 41 subtelomeric regions. We screened 5,380 consecutive clinical patients using CMA. The most common reasons for referral included developmental delay (DD), and/or mental retardation (MR), dysmorphic features (DF), multiple congenital anomalies (MCA), seizure disorders (SD), and autistic, or other behavioral abnormalities. We found pathogenic rearrangements at subtelomeric regions in 236 patients (4.4%). Among these patients, 103 had a deletion, 58 had a duplication, 44 had an unbalanced translocation, and 31 had a complex rearrangement. The detection rates varied among patients with a normal karyotype analysis (2.98%), with an abnormal karyotype analysis (43.4%), and with an unavailable or no karyotype analysis (3.16%). Six patients out of 278 with a prior normal subtelomere‐FISH analysis showed an abnormality including an interstitial deletion, two terminal deletions, two interstitial duplications, and a terminal duplication. In conclusion, genomic imbalances at subtelomeric regions contribute significantly to congenital disorders. Targeted array‐CGH with extended coverage (up to 10 Mb) of subtelomeric regions will enhance the detection of subtelomeric imbalances, especially for submicroscopic imbalances.

Non-invasive prenatal testing for fetal chromosomal abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center

To review the performance of non‐invasive prenatal testing (NIPT) by low‐coverage whole‐genome sequencing of maternal plasma DNA at a single center.

Prenatal chromosomal microarray analysis in a diagnostic laboratory; experience with >1000 cases and review of the literature

To evaluate the results of prenatal chromosomal microarray analysis (CMA) on >1000 fetal samples referred for testing at our institution and to compare these data to published reports.

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner.

Insertional translocation detected using FISH confirmation of array-comparative genomic hybridization (aCGH) results.

Insertional translocations (ITs) are rare events that require at least three breaks in the chromosomes involved and thus qualify as complex chromosomal rearrangements (CCR). In the current study, we identified 40 ITs from approximately 18,000 clinical cases (1:500) using array‐comparative genomic hybridization (aCGH) in conjunction with fluorescence in situ hybridization (FISH) confirmation of the aCGH findings, and parental follow‐up studies. Both submicroscopic and microscopically visible IT events were detected. They were divided into three major categories: (1) simple intrachromosomal and interchromosomal IT resulting in pure segmental trisomy, (2) complex IT involving more than one abnormality, (3) deletion inherited from a parent with a balanced IT resulting in pure segmental monosomy. Of the cases in which follow‐up parental studies were available, over half showed inheritance from an apparently unaffected parent carrying the same unbalanced rearrangement detected in the propositi, thus decreasing the likelihood that these IT events are clinically relevant. Nevertheless, we identified six cases in which small submicroscopic events were detected involving known disease‐associated genes/genomic segments and are likely to be pathogenic. We recommend that copy number gains detected by clinical aCGH analysis should be confirmed using FISH analysis whenever possible in order to determine the physical location of the duplicated segment. We hypothesize that the increased use of aCGH in the clinic will demonstrate that IT occurs more frequently than previously considered but can identify genomic rearrangements with unclear clinical significance.

Exon deletions of the EP300 and CREBBP genes in two children with Rubinstein-Taybi syndrome detected by aCGH

We demonstrate the utility of an exon coverage microarray platform in detecting intragenic deletions: one in exons 24–27 of the EP300 gene and another in exons 27 and 28 of the CREBBP gene in two patients with Rubinstein–Taybi syndrome (RSTS). RSTS is a heterogeneous disorder in which ∼45–55% of cases result from deletion or mutations in the CREBBP gene and an unknown portion of cases result from gene changes in EP300. The first case is a 3-year-old female with an exonic deletion of the EP300 gene who has classic facial features of RSTS without the thumb and great toe anomalies, consistent with the milder skeletal phenotype that has been described in other RSTS cases with EP300 mutations. In addition, the mother of this patient also had preeclampsia during pregnancy, which has been infrequently reported. The second case is a newborn male who has the classical features of RSTS. Our results illustrate that exon-targeted array comparative genomic hybridization (aCGH) is a powerful tool for detecting clinically significant intragenic rearrangements that would be otherwise missed by aCGH platforms lacking sufficient exonic coverage or sequencing of the gene of interest.

Challenges in clinical interpretation of microduplications detected by array CGH analysis

Due to the lack of robust diagnostic methods and limited resolution of conventional microscopy, submicroscopic genomic duplication copy number variants (CNVs) have been long underascertained. The development of array CGH has enabled detection of microduplications with nearly the same sensitivity as microdeletions and thus allowing them to be routinely identified throughout the human genome. However, in contrast to microdeletions, clinical interpretation of microduplications more often presents a diagnostic dilemma, as the functional impact of these genomic alterations is not well understood. Microduplications are especially difficult to interpret when they encompass several genes or a portion of a gene. Determining their significance involves investigative teamwork between both the diagnostic laboratory and the clinician. We present the steps for interpreting the clinical significance of microduplications and representative examples of these challenging cases.