Arthur R. Brothman

Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

Prognostic value of an RNA expression signature derived from cell cycle proliferation genes in patients with prostate cancer: a retrospective study

BACKGROUND Optimum management of clinically localised prostate cancer presents unique challenges because of the highly variable and often indolent natural history of the disease. To predict disease aggressiveness, clinicians combine clinical variables to create prognostic models, but the models have limited accuracy. We assessed the prognostic value of a predefined cell cycle progression (CCP) score in two cohorts of patients with prostate cancer. METHODS We measured the expression of 31 genes involved in CCP with quantitative RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tumour samples, and created a predefined score and assessed its usefulness in the prediction of disease outcome. The signature was assessed retrospectively in a cohort of patients from the USA who had undergone radical prostatectomy, and in a cohort of randomly selected men with clinically localised prostate cancer diagnosed by use of a transurethral resection of the prostate (TURP) in the UK who were managed conservatively. The primary endpoint was time to biochemical recurrence for the cohort of patients who had radical prostatectomy, and time to death from prostate cancer for the TURP cohort. FINDINGS After prostatectomy, the CCP score was useful for predicting biochemical recurrence in the univariate analysis (hazard ratio for a 1-unit change [doubling] in CCP 1·89; 95% CI 1·54-2·31; p=5·6×10(-9)) and the best multivariate analysis (1·77, 1·40-2·22; p=4·3×10(-6)). In the best predictive model (final multivariate analysis), the CCP score and prostate-specific antigen (PSA) concentration were the most important variables and were more significant than any other clinical variable. In the TURP cohort, the CCP score was the most important variable for prediction of time to death from prostate cancer in both univariate analysis (2·92, 2·38-3·57, p=6·1×10(-22)) and the final multivariate analysis (2·57, 1·93-3·43; p=8·2×10(-11)), and was stronger than all other prognostic factors, although PSA concentration also added useful information. Heterogeneity in the hazard ratio for the CCP score was not noted in any case for any clinical variables. INTERPRETATION The results of this study provide strong evidence that the CCP score is a robust prognostic marker, which, after additional validation, could have an essential role in determining the appropriate treatment for patients with prostate cancer. FUNDING Cancer Research UK, Queen Mary University of London, Orchid Appeal, US National Institutes of Health, and Koch Foundation.

Copy number variations and clinical cytogenetic diagnosis of constitutional disorders

The recent appreciation of widespread copy number variation in the genomes of healthy human beings has presented a significant challenge to clinical cytogeneticists who wish to use genome-wide array comparative genomic hybridization (CGH) assays for clinical diagnostic purposes. Clinical cytogeneticists need to differentiate between copy number variants (CNVs) that are likely to be pathogenic and CNVs that are less likely to contribute to an affected individuals clinical presentation. Unfortunately, our knowledge of the phenotypic effects of most CNVs is minimal, leading to the classification of many CNVs as genomic imbalances of unknown clinical significance. This has caused many laboratories to resist the use of higher-resolution genome-wide array CGH assays for clinical purposes. Ironically, the accumulation and annotation of such array CGH data can lead to the rapid identification of pathogenic CNVs and the definition of new genomic syndromes that, in turn, are useful for accurate clinical genetic diagnoses.

Analysis of chromosome breakpoints in neuroblastoma at sub‐kilobase resolution using fine‐tiling oligonucleotide array CGH

Understanding the genes and genetic pathways targeted by recurrent chromosomal imbalances in malignancy, along with the molecular mechanisms that generate the imbalances, are important problems in cancer biology. In this report, we demonstrate that oligonucleotide array CGH (oaCGH) analysis can routinely map chromosomal imbalance breakpoints at exon‐level resolution, including imbalances that are single copy number genomic alterations. Different tiling‐path array designs were used in this study: a whole‐genome array with a 6‐kb median probe spacing and fine‐tiling arrays for selected genomic regions with either 50‐ or 140‐bp median probe spacing. In both array formats, oligonucleotide probes were of isothermal design and were tiled through genic and inter‐genic regions. Whole‐genome oaCGH analysis of two neuroblastoma cell lines and three primary tumors led to the identification of 58 chromosomal breakpoints that generated 45 large‐scale partial chromosomal imbalances (>2 Mb). An unexpectedly high proportion (34%) of these breakpoint intervals mapped to regions containing segmental duplications. In addition, 88 smaller‐sized regions (<2 Mb) of imbalance were detected, the majority of which mapped to segmentally duplicated regions and may reflect constitutional copy number polymorphisms. The chromosomal breakpoints for 12 recurrent abnormalities exhibited in neuroblastoma tumors and cell lines, including MYCN amplicon boundaries, loss of 3p, loss of 11q, and gain of 17q, could be mapped to intervals ranging from 50 bp to 10 kb in size using high‐density fine‐tiling oligonucleotide microarrays. Fine‐tiling oaCGH analysis provides an unprecedented level of resolution, allowing detailed mapping of recurrent unbalanced chromosomal abnormalities. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat/index.html.

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.

Technical Standards and Guidelines for Fragile X: The First of a Series of Disease-Specific Supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics

Preface: The Quality Assurance subcommittee of the ACMG Laboratory Practice committee has the mission of maintaining high technical standards for the performance and interpretation of genetic tests. In part, this is accomplished by the publication of the document “Standards and Guidelines for Clinical Genetics Laboratories,” which was published in its second edition in 1999 and is now maintained online (see http://www.faseb.org/genetics/acmg/index.html). This subcommittee also reviews the outcome of national proficiency testing in the genetics area and may choose to focus on specific diseases or methodologies in response to those results. Accordingly, the subcommittee selected fragile X syndrome to be the first topic in a new series of supplemental sections, recognizing that it is one of the most frequently ordered genetic tests and that it has many alternative methods with different strengths and weaknesses. This document follows the outline format of the general Standards and Guidelines. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and the methods of analysis.

RARE TRISOMY MOSAICISM DIAGNOSED IN AMNIOCYTES, INVOLVING AN AUTOSOME OTHER THAN CHROMOSOMES 13, 18, 20, AND 21: KARYOTYPE/PHENOTYPE CORRELATIONS

In order to determine the significance of trisomy mosaicism of an autosome other than chromosomes 13, 18, 20, and 21, 151 such cases diagnosed prenatally through amniocentesis were reviewed. These rare trisomy mosaicism cases include 54 from 17 cytogenetic laboratories, 34 from a previous North American mosaicism survey, and 63 from published reports. All were cases of true mosaicism with information available on pregnancy outcome, and with no evidence of biased ascertainment. There were 11 cases of 46/47,+2; 2 of 46/47,+3; 2 of 46/47,+4; 5 of 46/47,+5; 3 of 46/47,+6; 8 of 46/47,+7; 14 of 46/47,+8; 25 of 46/47,+9; 2 of 46/47,+11; 23 of 46/47,+12; 5 of 46/47,+14; 11 of 46/47,+15; 21 of 46/47,+16; 7 of 46/47,+17; 1 of 46/47,+19; and 11 of 46/47,+22. As to the risk of an abnormal outcome, the data showed a very high risk (>60 per cent) for 46/47,+2, 46/47,+16, and 46/47,+22; a high risk (40–59 per cent) for 46/47,+5, 46/47,+9, 46/47,+14, and 46/47,+15; a moderately high risk (20–39 per cent) for 46/47,+12; a moderate risk (up to 19 per cent) for 46/47,+7 and 46/47,+8; a low risk for 46/47,+17; and an undetermined risk, due to lack of cases, for the remaining autosomal trisomy mosaics. Most cases were evaluated at birth or at termination, so subtle abnormalities may have escaped detection and developmental retardation was not evaluated at all. Comparison of the phenotypes of prenatally diagnosed abnormal cases and postnatally diagnosed cases with the same diagnosis showed considerable concordance. Since the majority of anomalies noted are prenatally detectable with ultrasound, an ultrasound examination should be performed in all prenatally diagnosed cases. In cytogenetic confirmation studies, the data showed much higher confirmation rates in cases with abnormal outcomes than in cases with normal outcomes [81 per cent vs. 55 per cent for fibroblasts (from skin, fetal tissue, and/or cord); 88 per cent vs. 46 per cent for placental cells; 22 per cent vs. 10 per cent for blood cells]. The confirmation rate reached 85 per cent when both fibroblasts and placental tissues were studied in the same case (with trisomic cells found in one or the other, or both). Therefore, one must emphasize that both fibroblasts and placental tissues should be studied. Except for 46/47,+8 and 46/47,+9, PUBS is of limited value for prenatal diagnosis of rare trisomy mosaicism. DNA studies for UPD are suggested for certain chromosomes with established imprinting effects, such as chromosomes 7, 11, 14, and 15, and perhaps for chromosomes 2 and 16, where imprinting effects are likely.

Her-2/neu expression in osteosarcoma increases risk of lung metastasis and can be associated with gene amplification.

The purpose of this study was to investigate whether Her-2/ neu expression at diagnosis of osteosarcoma could provide biologic and prognostic information that predicts the risk of pulmonary metastases and outcome. Human epidermal growth factor (Her-2/ neu) expression in 25 initial pretreatment osteosarcoma biopsies and 12 posttreatment pulmonary metastatic osteosarcoma resection specimens was assessed by standard immunohistochemical techniques on formalin-fixed paraffin-embedded tissue. As a screening analysis to determine if gene amplification may be a mechanism for increased Her-2/ neu expression, FISH analysis was conducted on seven Her-2/ neu immunostain-positive samples and five Her-2/ neu immunostain-negative samples. Cytoplasmic Her-2/ neu reactivity was identified in 11/25 (44%) of primary tumors and in 7/12 (58%) resection specimens from pulmonary metastases. Cytoplasmic Her-2/ neu expression was associated with shorter overall metastasis-free survival. Her-2/ neu gene amplification was identified by FISH analysis in six of the seven immunostain-positive samples but was also identified in two of the five immunostain-negative samples. Her-2/ neu expression in patients with osteosarcoma is associated with an increased risk of metastasis and may define a subset of patients with a more aggressive tumor phenotype. Her-2/ neu gene amplification may provide a mechanism for Her-2/ neu overexpression in certain cases of osteosarcoma. Whether Her-2/ neu expression influences outcome needs to be examined further in a prospective fashion. The hope is that Her-2/ neu expression will identify patients who may benefit from the addition of directed biologic therapy.

Chromosomal clues to the development of prostate tumors.

Cytogenetic, molecular cytogenetic, and molecular studies of prostate cancer have revealed an enormous amount of data regarding chromosomal loci that are aberrant in prostate tumors.