Carla Rosenberg

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.

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the β-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability

Recently, the application of array-based comparative genomic hybridization (array CGH) has improved rates of detection of chromosomal imbalances in individuals with mental retardation and dysmorphic features. Here, we describe three individuals with learning disability and a heterozygous deletion at chromosome 17q21.3, detected in each case by array CGH. FISH analysis demonstrated that the deletions occurred as de novo events in each individual and were between 500 kb and 650 kb in size. A recently described 900-kb inversion that suppresses recombination between ancestral H1 and H2 haplotypes encompasses the deletion. We show that, in each trio, the parent of origin of the deleted chromosome 17 carries at least one H2 chromosome. This region of 17q21.3 shows complex genomic architecture with well-described low-copy repeats (LCRs). The orientation of LCRs flanking the deleted segment in inversion heterozygotes is likely to facilitate the generation of this microdeletion by means of non-allelic homologous recombination.

Array-CGH detection of micro rearrangements in mentally retarded individuals: Clinical significance of imbalances present both in affected children and normal parents

Background: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. Objective and methods: Array-CGH with approximately 3500 large insert clones spaced at ∼1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. Results: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. Conclusions: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Identification of Genetic Markers for Prostatic Cancer Progression

Despite the high incidence of prostate cancer, only limited data are available on genes or chromosomes specifically involved in its initiation and progression. We have applied comparative genomic hybridization to routinely processed, paraffin-embedded, tissues at different times in prostatic tumor progression to screen the tumor genome for gains and losses. Our panel included specimens derived from 56 different patients: 23 patients with primary, prostate-confined carcinomas; 18 patients with regional lymph node metastases; and 15 patients with distant metastases. Chromosome arms that most frequently showed losses, included 13q (55%), 8p (48%), 6q (43%), 5q (32%), 16q (25%), 18q (20%), 2q (18%), 4q (18%), 10q (18%), and Y (16%). Gains were often seen of chromosome arms 8q (36%), 17q (23%), Xq (23%), 7q (21%), 3q (18%), 9q (18%), 1q (16%), Xp (16%). Furthermore, specific high-level amplifications, eg, of 1q21, 1q25, and Xq12 to q13, were found in metastatic cancers. A significant accumulation of genetic changes in distant metastases was observed, eg, loss of 10q (p = 0.03) and gain of 7q (p = 0.03) sequences. In addition, investigation of a potential biomarker identified in previous studies by our group, ie, extra copies of #7 and/or #8, revealed a high prevalence of 7pq and/or 8q gain in the distant metastases (p = 0.02). Importantly, gains were observed more frequently in tumors derived from progressors after radical prostatectomy, than in nonprogressors (mean time of follow-up, 74 months). Specifically, gain of chromosome 7pq and/or 8q sequences appeared an accurate discriminator between the progressors and nonprogressors. Multivariate analysis showed a significant correlation between progressive disease and the number of chromosomes with gains. This correlation also held true when stage (p = 0.007) or grade (p = 0.002) were taken into account. Likewise, this applied for gain of chromosome 7pq and/or 8q sequences (p = 0.03 and p = 0.005 for stage or grade, respectively). Additionally, an increase in the number of chromosomes with gains per case was related to a decrease in biochemical progression-free survival (Ptrend <0.001). More specifically, the gain of 7pq and/or 8q sequences markedly reduced the biochemical progression-free survival (p < 0.001). In conclusion, this study has, firstly, documented the spectrum of chromosomal alterations in subsequent stages of prostate cancer, a number of which had not been described previously. It allowed us to identify chromosomal regions related to advanced tumor stage, ie, loss of 10q24 and gain of 7q11.2 and/or 7q31 sequences. Secondly, gain of 7pq and/or 8q was identified as a potential genetic discriminator between progressors and nonprogressors after radical surgery.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome

Background: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. Aim: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. Results: We estimate the prevalence of the syndrome to be 1 in 16 000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729–41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10−5). Conclusion: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1→q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Array-Based Comparative Genomic Hybridization Analysis Reveals Recurrent Chromosomal Alterations and Prognostic Parameters in Primary Cutaneous Large B-Cell Lymphoma

PURPOSE To evaluate the clinical relevance of genomic aberrations in primary cutaneous large B-cell lymphoma (PCLBCL). PATIENTS AND METHODS Skin biopsy samples of 31 patients with a PCLBCL classified as either primary cutaneous follicle center lymphoma (PCFCL; n = 19) or PCLBCL, leg type (n = 12), according to the WHO-European Organisation for Research and Treatment of Cancer (EORTC) classification, were investigated using array-based comparative genomic hybridization, fluorescence in situ hybridization (FISH), and examination of promoter hypermethylation. RESULTS The most recurrent alterations in PCFCL were high-level DNA amplifications at 2p16.1 (63%) and deletion of chromosome 14q32.33 (68%). FISH analysis confirmed c-REL amplification in patients with gains at 2p16.1. In PCLBCL, leg type, most prominent aberrations were a high-level DNA amplification of 18q21.31-q21.33 (67%), including the BCL-2 and MALT1 genes as confirmed by FISH, and deletions of a small region within 9p21.3 containing the CDKN2A, CDKN2B, and NSG-x genes. Homozygous deletion of 9p21.3 was detected in five of 12 patients with PCLBCL, leg type, but in zero of 19 patients with PCFCL. Complete methylation of the promoter region of the CDKN2A gene was demonstrated in one PCLBCL, leg type, patient with hemizygous deletion, in one patient without deletion, but in zero of 19 patients with PCFCL. Seven of seven PCLBCL, leg type, patients with deletion of 9p21.3 and/or complete methylation of CDKN2A died as a result of their lymphoma. CONCLUSION Our results demonstrate prominent differences in chromosomal alterations between PCFCL and PCLBCL, leg type, that support their classification as separate entities within the WHO-EORTC scheme. Inactivation of CDKN2A by either deletion or methylation of its promoter could be an important prognostic parameter for the group of PCLBCL, leg type.

Genomic imbalances associated with müllerian aplasia

Background: Aplasia of the müllerian ducts leads to absence of the uterine corpus, uterine cervix, and upper (superior) vagina. Patients with müllerian aplasia (MA) often exhibit additional clinical features such as renal, vertebral and cardiac defects. A number of different syndromes have been associated with MA, and in most cases its aetiology remains poorly understood. Objective and methods: 14 syndromic patients with MA and 46,XX G-banded karyotype were screened for DNA copy number changes by ∼1 Mb whole genome bacterial artificial chromosome (BAC) array based comparative genomic hybridisation (CGH). The detected alterations were validated by an independent method and further mapped by high resolution oligo-arrays. Results: Submicroscopic genomic imbalances affecting the 1q21.1, 17q12, 22q11.21, and Xq21.31 chromosome regions were detected in four probands. Presence of the alterations in the normal mother of one patient suggests incomplete penetrance and/or variable expressivity. Conclusion: 4 of the 14 patients (29%) were found to have cryptic genomic alterations. The imbalances on 22q11.21 support recent findings by us and others that alterations in this chromosome region may result in impairment of müllerian duct development. The remaining imbalances indicate involvement of previously unknown chromosome regions in MA, and point specifically to LHX1 and KLHL4 as candidate genes.