Concurrent microdeletion and duplication of 22q11.2.

Microduplication of 22q11.2 has been reported in fewer than 40 cases, all of them including the DiGeorge critical region (DGCR). We here present the characterization of a new duplication that does not include the DGCR. The duplication was initially found by multiplex ligation-dependent probe amplification analysis of 22q11.2 in a young girl with a concurrent deletion of the DGCR in 70% of her peripheral blood lymphocytes. Her phenotype included many of the features of the velocardiofacial syndrome, with velopharyngeal insufficiency, recurrent infections, learning and concentration problems as well as difficulties in social interactions. However, there were no congenital malformations, and her facial appearance was not typical for the syndrome. Further investigations included array comparative genomic hybridization (CGH) to size map the deletion/duplication and interphase fluorescent in situ hybridization to investigate mosaicism and the structure of the rearrangement. An identical duplication of this part of 22q11.2 has not been reported before, and the duplication itself seems to be associated with very mild or no symptoms. This study contributes to the growing knowledge regarding new deletions and duplications of 22q11.2, most of them mediated by the pre-disposing high number of low-copy repeats in the region.

Polymorphisms of estrogen receptor beta gene are associated with hypospadias.

INTRODUCTION: Hypospadias is a common male congenital urethral malformation, defined as the displacement of the urethral meatus ventrally from the tip of the glans penis. The importance of androgen receptor in male external genitalia development has been well recognized. Recently, the presence of active estrogen receptors (ER) in the developing male external genitalia has also been demonstrated. There are two isoforms of the human estrogen receptor, ESR1 and ESR2, which occur, with distinct tissue and cell patterns of expression. We hypothesized that modifications in these nuclear receptors' genes could lead to hypospadias. MATERIALS AND METHODS: We screened 60 boys with hypospadias for mutations in the coding regions of ESR1 and ESR2 genes, by denaturing high-performance liquid chromatography and automated sequence analysis. We also genotyped the CA repeat polymorphism in ESR2 and the TA repeat polymorphism in ESR1. RESULTS: The CA repeat polymorphism in ESR2 is prolonged in hypospadias patients compared to controls (p < 0.05). Prolongation of this CA repeat polymorphism has previously been associated with lower levels of testosterone. Six patients presented the genetic variant 2681-4A > G (rs944050) in the heterozygous form in ESR2, which was a significantly higher frequency than in the control population (p < 0.05). One of these patients also presented a 266_267insC in exon 1 of ESR2, which is also a known single nucleotide polymorphism (SNP; rs3832949). In ESR1, no significant gene alteration was found to be associated with hypospadias. CONCLUSIONS: Our results suggest that variations in the ESR2 might influence susceptibility to hypospadias.

The role of combined allelic imbalance and mutations of p53 in tumor progression and survival following surgery for colorectal carcinoma.

The role of p53 mutations in disease progression and survival of colorectal cancer is unclear, since numerous studies have reported different conclusions. However, few reports, if any, have evaluated disease progression and survival in relationship to 'functional' and 'non-functional' p53 status defined by genetic and molecular indications. Malignant colorectal tumors, from 72 unselected patients who underwent primary and potentially curative elective tumor resections, were either classified as p53 functional (p53+/+, p53+/-) or non-functional (p53-/-) based on DNA sequence analysis of all p53 exons, including determination of allelic imbalance of p53 (LOH), according to four DNA markers; 2 within the coding gene and two markers in the immediate flanking regions of p53. Tumor frequency of microsatellite instability was also analyzed according to Dukes' A-D stages. Dukes' staging predicted survival as expected, while the conceptual p53 status, functional p53 vs non-functional p53, did not clear-cut predict disease specific survival. p53 mutations alone or allelic imbalance inside the reading frame of the gene were unpredictive of survival, while allelic imbalance downstream of p53 predicted reduced survival (p < 0.05). The present study demonstrates that base mutations in combination with allelic imbalance within the reading frame of p53 do not predict survival or progression of colorectal cancer, while allelic imbalance upstream coding parts of the gene predicted disease-specific survival in univariate analysis. Thus, structural alterations within the gene seem less important than alterations in regions with potential control elements.

Analysis of a 43.6 kb deletion in a patient with Hunter syndrome (MPSII): identification of a fusion transcript including sequences from the gene W and the IDS gene.

Mucopolysaccharidosis type II (Hunter syndrome) is an X-linked lysosomal storage disorder. A novel mutation is described in an MPS II patient in whom the disorder is caused by a 43.6 kb deletion. Southern blot analysis, PCR analysis and subsequent sequencing of the deletion junction revealed that the deletion spans exons 1-7 of the iduronate-2-sulfatase (IDS) gene, the IDS-2 locus and exons 3-5 of the recently identified gene W. Short direct repeats of 12 bp were identified at both deletion breakpoints, suggesting that the deletion is the result of an illegitimate recombination event. A sequence motif (TGAGGA) which is identical to a consensus sequence frequently associated with deletions in man was identified at both breakpoints. This further supports the notion that this motif is a hot spot for recombination. Gene expression studies by RT-PCR analysis of total RNA derived from fibroblasts of the patient revealed the presence of a novel fusion transcript. DNA sequence analysis of the cDNA demonstrated that it consists of exons derived from both the gene W and the IDS gene. A similar but longer fusion transcript containing exons 2-4 of the gene W and exons 4-9 of the IDS gene could also be detected in RNA of normal cell lines originating from different tissues. This result further demonstrates the complex gene expression profile of the IDS region, which may contribute to the observed genomic instability of this region.

Two distinct deletions in the IDS gene and the gene W: a novel type of mutation associated with the Hunter syndrome.

A novel mutation has been identified in a patient with the Hunter syndrome (mucopolysaccharidosis type II), in whom the disorder is associated with two distinct deletions separated by 30 kb. The deletions were characterized by Southern blot and PCR analyses, and the nucleotide sequences at both junctions were determined. The first deletion, corresponding to a loss of 3152 bp of DNA, included exons 5 and 6 of the iduronate-2-sulfatase (IDS) gene. The second deletion was 3603 bp long and included exons 3 and 4 of gene W, which is located in the DXS466 locus telomeric of the IDS gene. Both deletions are the result of nonhomologous (illegitimate) recombination events between short direct repeats at the deletion breakpoints. An interesting finding was the presence of the heptamer sequence 5'-TACTCTA-3' present at both deletion junctions, suggesting that this motif might be a hot spot for recombination. We propose that the double deletion is the result of homology-associated nonhomologous recombinations caused by the presence of large duplicated regions in Xq27.3-q28.

Double-strand breaks may initiate the inversion mutation causing the Hunter syndrome.

We have previously shown that patients with the Hunter syndrome frequently have suffered from a recombination event between the IDS gene and its putative pseudogene, IDS-2, resulting in an inversion of the intervening DNA. The inversion, which might be the consequence of an intrachromosomal mispairing, is caused by homologous recombination between sequences located in intron 7 of the IDS gene and sequences located distal of exon 3 in IDS-2. In order to gain insight into the mechanisms causing the inversion, we have isolated both inversion junctions in six unrelated patients. DNA sequence analysis of the junctions showed that all recombinations have taken place within a 1 kb region where the sequence identity is >98%. An interesting finding was the identification of regions with alternating IDS gene and IDS-2 sequences present at one inversion junction, suggesting that the recombination event has been initiated by a double-strand break in intron 7 of the IDS gene. The results from this study suggest that homologous recombination in man could be explained by mechanisms similar to those described for Saccharomyces cerevisiae. The results also have practical implications for diagnosis of patients with the Hunter syndrome.

Molecular and phenotypic variation in patients with severe Hunter syndrome.

Severe Hunter syndrome is a fatal X-linked lysosomal storage disorder caused by iduronate-2-sulphatase (IDS) deficiency. Patients with complete deletion of the IDS locus often have atypical phenotypes including ptosis, obstructive sleep apnoea, and the occurrence of seizures. We have used genomic DNA sequencing to identify several new genes in the IDS region. DNA deletion patients with atypical symptoms have been analysed to determine whether these atypical symptoms could be due to involvement of these other loci. The occurrence of seizures in two individuals correlated with a deletion extending proximal of IDS, up to and including part of the FMR2 locus. Other (non-seizure) symptoms were associated with distal deletions. In addition, a group of patients with no variant symptoms, and a characteristic rearrangement involving a recombination between the IDS gene and an adjacent IDS pseudogene (IDS psi), showed normal expression of loci distal to IDS. Together, these results identify FMR2 as a candidate gene for seizures, when mutated along with IDS.