Constitutional DNA copy number changes in ICSI children.

BACKGROUND: Over the last three decades, technological developments facilitating assisted reproductive techniques (ART) have revolutionized the treatment of subfertile couples, including men suffering from severe oligospermia or azoospermia. In parallel with the advent of these technologies, there is a great concern about the biological safety of ART. This concern is supported by the clinical observation that the frequency of congenital malformations is slightly elevated among ART-conceived children. METHODS: In this explorative study, we have used tiling-resolution BAC array-mediated comparative genomic hybridization to investigate the incidence of de novo genomic copy number changes in a group of 12 ICSI children, compared with a control group of 30 naturally conceived children. RESULTS: In 6 of the 12 ICSI children, we found 10 apparently de novo 'same direction genomic copy number changes' [i.e. simultaneous copy number gain (or loss) with respect to both biological parents], notably losses. In statistically significant contrast, similar observations were encountered only six times in the control group in 5 of the 30 children. However, our study group was small, so a larger group is needed to confirm these findings. CONCLUSIONS: Loci at which we found de novo alterations are known from the human genome database to be prone to large DNA segment copy number changes. As discussed, various molecular mechanisms, including the consequences of delayed male meiotic synapsis and replication fork stalling at early embryonic cell cycles, might trigger these copy number changes.

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

Search for genomic imbalances in a cohort of 24 Cornelia de Lange patients negative for mutations in the NIPBL and SMC1L1 genes.

Cornelia de Lange syndrome (CdLS) is a rare, multiple congenital anomaly/mental retardation syndrome characterized by varied clinical signs including facial dysmorphism, pre- and post-natal growth defects, small hands and malformations of the upper limbs. Established genetic causes include mutations in the NIPBL (50-60%), SMC1L1 and SMC3 (5%) genes. To detect chromosomal rearrangements pointing to novel positional candidate CdLS genes, we used array-CGH to analyze a subgroup of 24 CdLS patients negative for mutations in the NIPBL and SMC1L1 genes. We identified three carriers of DNA copy number alterations, including a de novo 15q26.2-qter 8-Mb deletion, and two inherited 13q14.2-q14.3 1-Mb deletion and 13q21.32-q21.33 1.5-Mb duplication, not reported among copy number variants. The clinical presentation of all three patients matched the diagnostic criteria for CdLS, and the phenotype of the patient with the 15qter deletion is compared to that of both CdLS and 15qter microdeletion patients.

High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression.

Gross cytogenetic anomalies are traditionally being used as diagnostic, prognostic and therapeutic markers in the clinical management of cancer, including childhood acute lymphoblastic leukemia (ALL). Recently, it has become increasingly clear that genetic lesions driving tumorigenesis frequently occur at the submicroscopic level and, consequently, escape standard cytogenetic observations. Therefore, we profiled the genomes of 40 childhood ALLs at high resolution. We detected multiple de novo genetic lesions, including gross aneuploidies and segmental gains and losses, some of which were subtle and affected single genes. Many of these lesions involved recurrent (partially) overlapping deletions and duplications, containing various established leukemia-associated genes, such as ETV6, RUNX1 and MLL. Importantly, the most frequently affected genes were those controlling G1/S cell cycle progression (e.g. CDKN2A, CDKN1B and RB1), followed by genes associated with B-cell development. The latter group includes microdeletions of the B-lineage transcription factors PAX5, EBF, E2-2 and IKZF1 (Ikaros), as well as genes with other established roles in B-cell development, that is RAG1 and RAG2, FYN, PBEF1 or CBP/PAG. The fact that we frequently encountered multiple lesions affecting genes involved in cell cycle regulation and B-cell differentiation strongly suggests that both these processes need to be targeted independently and simultaneously to trigger ALL development.

Identification of novel genomic markers related to progression to glioblastoma through genomic profiling of 25 primary glioma cell lines.

Identification of genetic copy number changes in glial tumors is of importance in the context of improved/refined diagnostic, prognostic procedures and therapeutic decision-making. In order to detect recurrent genomic copy number changes that might play a role in glioma pathogenesis and/or progression, we characterized 25 primary glioma cell lines including 15 non glioblastoma (non GBM) (I-III WHO grade) and 10 GBM (IV WHO grade), by array comparative genomic hybridization, using a DNA microarray comprising approx. 3500 BACs covering the entire genome with a 1 Mb resolution and additional 800 BACs covering chromosome 19 at tiling path resolution. Combined evaluation by single clone and whole chromosome analysis plus 'moving average (MA) approach' enabled us to confirm most of the genetic abnormalities previously identified to be associated with glioma progression, including +1q32, +7, -10, -22q, PTEN and p16 loss, and to disclose new small genomic regions, some correlating with grade malignancy. Grade I-III gliomas exclusively showed losses at 3p26 (53%), 4q13-21 (33%) and 7p15-p21 (26%), whereas only GBMs exhibited 4p16.1 losses (40%). Other recurrent imbalances, such as losses at 4p15, 5q22-q23, 6p23-25, 12p13 and gains at 11p11-q13, were shared by different glioma grades. Three intervals with peak of loss could be further refined for chromosome 10 by our MA approach. Data analysis of full-coverage chromosome 19 highlighted two main regions of copy number gain, never described before in gliomas, at 19p13.11 and 19q13.13-13.2. The well-known 19q13.3 loss of heterozygosity area in gliomas was not frequently affected in our cell lines. Genomic hotspot detection facilitated the identification of small intervals resulting in positional candidate genes such as PRDM2 (1p36.21), LRP1B (2q22.3), ADARB2 (10p15.3), BCCIP (10q26.2) and ING1 (13q34) for losses and ECT2 (3q26.3), MDK, DDB2, IG20 (11p11.2) for gains. These data increase our current knowledge about cryptic genetic changes in gliomas and may facilitate the further identification of novel genetic elements, which may provide us with molecular tools for the improved diagnostics and therapeutic decision-making in these tumors.

Molecular mechanisms underlying the MiT translocation subgroup of renal cell carcinomas.

Renal cell carcinomas (RCCs) represent a heterogeneous group of neoplasms, which differ in histological, pathologic and clinical characteristics. The tumors originate from different locations within the nephron and are accompanied by different recurrent (cyto)genetic anomalies. Recently, a novel subgroup of RCCs has been defined, i.e., the MiT translocation subgroup of RCCs. These tumors originate from the proximal tubule of the nephron, exhibit pleomorphic histological features including clear cell morphologies and papillary structures, and are found predominantly in children and young adults. In addition, these tumors are characterized by the occurrence of recurrent chromosomal translocations, which result in disruption and fusion of either the TFE3 or TFEB genes, both members of the MiT family of basic helix-loop-helix/leucine-zipper transcription factor genes. Hence the name MiT translocation subgroup of RCCs. In this review several features of this RCC subgroup will be discussed, including the molecular mechanisms that may underlie their development.

The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of synpolydactyly.

Molecular analysis of the reciprocal chromosomal translocation t(12;22)(p11.2;q13.3) cosegregating with a complex type of synpolydactyly showed involvement of an alternatively spliced exon of the fibulin-1 gene (FBLN1 located in 22q13.3) and the C12orf2 (HoJ-1) gene on the short arm of chromosome 12. Investigation of the possible functional involvement of the fibulin-1 protein (FBLN1) in the observed phenotype showed that FBLN1 is expressed in the extracellular matrix (ECM) in association with the digits in the developing limb. Furthermore, fibroblasts derived from patients with the complex type of synpolydactyly displayed alterations in the level of FBLN1-D splice variant incorporated into the ECM and secreted into the conditioned culture medium. By contrast, the expression of the FBLN1-C splice variant was not perturbed in the patient fibroblasts. Based on these findings, we propose that the t(12;22) results in haploinsufficiency of the FBLN1-D variant, which could lead to the observed limb malformations.