A. de Klein

Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

Genetic Factors in Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect associated with high mortality and morbidity. Although the exact etiology of most cases of CDH remains unknown, there is a growing body of evidence that genetic factors play an important role in the development of CDH. In this review, we examine key findings that are likely to form the basis for future research in this field. Specific topics include a short overview of normal and abnormal diaphragm development, a discussion of syndromic forms of CDH, a detailed review of chromosomal regions recurrently altered in CDH, a description of the retinoid hypothesis of CDH, and evidence of the roles of specific genes in the development of CDH.

Congenital Diaphragmatic Hernia and Chromosome 15q26: Determination of a Candidate Region by Use of Fluorescent In Situ Hybridization and Array-Based Comparative Genomic Hybridization

Congenital diaphragmatic hernia (CDH) has an incidence of 1 in 3,000 births and a high mortality rate (33%-58%). Multifactorial inheritance, teratogenic agents, and genetic abnormalities have all been suggested as possible etiologic factors. To define candidate regions for CDH, we analyzed cytogenetic data collected on 200 CDH cases, of which 7% and 5% showed numerical and structural abnormalities, respectively. This study focused on the most frequent structural anomaly found: a deletion on chromosome 15q. We analyzed material from three of our patients and from four previously published patients with CDH and a 15q deletion. By using array-based comparative genomic hybridization and fluorescent in situ hybridization to determine the boundaries of the deletions and by including data from two individuals with terminal 15q deletions but without CDH, we were able to exclude a substantial portion of the telomeric region from the genetic etiology of this disorder. Moreover, one patient with CDH harbored a small interstitial deletion. Together, these findings allowed us to define a minimal deletion region of approximately 5 Mb at chromosome 15q26.1-26.2. The region contains four known genes, of which two--NR2F2 and CHD2--are particularly intriguing gene candidates for CDH.

C-abl and bcr are rearranged in a Ph1-negative CML patient.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c‐abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c‐abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1‐negative patients may in fact belong to the clinical entity of Ph1‐positive CML.

Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma.

Background:Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin.Methods:GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH.Results:Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS.Conclusion:The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.

Molecular characterization of the testis specific c-abl mRNA in mouse.

The c‐abl gene encodes a protein tyrosine kinase and is transcribed from at least two promoters giving rise to transcripts of two size classes of approximately 5 and 6 kb in length. These mRNAs only differ in their most 5′ exon and encode proteins of similar size but with different N‐termini. In the mouse testis an additional abundant c‐abl mRNA of 4 kb is detected. This mRNA was shown to be expressed in the haploid male germ cells of the adult mouse. Here we describe the cloning and molecular characterization of a cDNA representing the testis specific c‐abl transcript. We show that the 4 kb c‐abl mRNA arises from alternative polyadenylation of an RNA transcribed from the same promoter as the 5 kb mRNA. The site of polyadenylation is unusual in this shorter transcript as it is not preceded by the highly conserved hexanucleotide AAUAAA. The use of this polyadenylation site removes 1.2 kb of 3′ sequences present in the somatic c‐abl mRNAs, but does not affect the main open reading frame of the transcript. Using in situ hybridization on whole testis sections it is shown that the 4 kb c‐abl mRNA is most abundant in the elongating spermatids.

Genetic and environmental factors in the etiology of esophageal atresia and/or tracheoesophageal fistula: An overview of the current concepts

Esophageal atresia and/or tracheoesophageal fistula (EA/TEF) are severe congenital anomalies. Although recent years have brought significant improvement in clinical treatment, our understanding of the etiology of these defects is lagging. Many genes and genetic pathways have been implicated in the development of EA/TEF, but only a few genes have been shown to be involved in humans, in animals, or in both. Extrapolating data from animal models to humans is not always straightforward. Environmental factors may also carry a risk, but the mechanisms are yet to be elucidated. This review gives an overview of the current state of knowledge about both genetic and environmental risk factors in the etiology of EA/TEF.

Prenatal detection and outcome of congenital diaphragmatic hernia (CDH) associated with deletion of chromosome 15q26: two patients and review of the literature.

Congenital diaphragmatic hernia (CDH) is a severe birth defect characterized by a defect in the diaphragm with pulmonary hypoplasia and postnatal pulmonary hypertension. Approximately 50% of CDH cases are associated with other non‐pulmonary congenital anomalies (so called non‐isolated CDH) and in 5–10% of cases there is a chromosomal etiology. The majority of CDH cases are detected prenatally. In some cases prenatal chromosome analysis reveals a causative chromosomal anomaly, most often aneuploidy. Deletion of 15q26 is the most frequently described structural chromosomal aberration in patients with non‐isolated CDH. In this paper we report on two patients with a deletion of 15q26 and phenotypes similar to other patients with CDH caused by 15q26 deletions. This phenotype consists of intra‐uterine growth retardation, left‐sided CDH, cardiac anomalies and characteristic facial features, similar to those seen in Fryns syndrome. We propose that when this combination of birth defects is identified, either pre‐ or postnatally, further investigations to confirm or exclude a deletion of 15q26 are indicated, since the diagnosis of this deletion will have major consequences for the prognosis and, therefore, can affect decision making.

Congenital diaphragmatic hernia associated with duplication of 11q23-qter

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect with a high mortality. Although little is known about its etiology, there is increasing evidence for a strong genetic contribution. Both numerical and structural chromosomal abnormalities have been described in patients with CDH. Partial trisomy 11q and partial trisomy 22 associated with the common t(11;22) has been reported in several cases of CDH. It has been assumed that the diaphragmatic defect seen in these individuals was primarily due to duplication of material from chromosome 22q11. However, in this report we describe a family with a t(11;12) in which one of two brothers with partial trisomy 11q has a left sided posterolateral CDH. This is the second case of CDH in partial trisomy 11q due to an unbalanced translocation other than t(11;22). Using array‐based comparative genomic hybridization and fluorescent in situ hybridization, we mapped the breakpoints in both brothers and their mother who is a balanced translocation carrier. Our results suggest that duplication of one or more genes on a ∼19 Mb region of 11q23.3‐qter predisposes to the development of CDH. These effects may be the primary cause of CDH in individuals t(11;22) or may be additive to effects from the duplication of chromosome 22 material. We also conclude that the partial trisomy 11q syndrome has a variable phenotype and that CDH should be added to the spectrum of anomalies that can be present in this syndrome.

Translocation of oncogene c-sis from chromosome 22 to chromosome 11 in a Ewing sarcoma-derived cell line.

Somatic cell hybrids, obtained after fusion of translocation (11;22)-positive Ewing sarcoma cells and Chinese hamster fibroblasts, were assayed for the presence of immunoglobulin C lambda, Philadelphia chromosome breakpoint cluster region, and c-sis oncogene sequences. It was found that c-sis was translocated from chromosome 22 to chromosome 11 in the Ewing sarcoma cells used, indicating that the breakpoint must be proximal to this locus. Moreover, we found that the chromosome 22-linked C lambda and breakpoint cluster region sequences are not translocated. This result confirms an earlier cytogenetic observation that the Ewing sarcoma-associated breakpoint in chromosome 22 is distal to those observed in translocation (8;22)-positive Burkitt lymphoma and in Philadelphia chromosome-positive chronic myeloid leukemia.