Knowledge and perceived risks in couples undergoing genetic testing after recurrent miscarriage or for poor semen quality.

Couples with recurrent miscarriage (RM) and men with poor semen quality may undergo genetic testing as part of the diagnostic work-up. This study explored their knowledge and perception of genetic testing, evaluated psychological wellbeing and identified associated variables. A prospective questionnaire study was conducted in seven clinical genetics centres and referring gynaecological departments in couples with RM or poor semen quality. Questionnaires were completed before disclosure of genetic test results. Main outcome measures were knowledge, perceived risk, anxiety and depression. Of 439 participants, 256 were not aware genetic testing was part of the diagnostic work-up. One-third (36% RM, 33% poor semen quality) indicated they had not received information about the genetic test from their doctor. Perceived risk of receiving an abnormal genetic test result was higher than objective risk. Anxiety was highly correlated with perceived risk. Women with RM were more anxious than women in the poor semen quality group or men (P<0.01). These couples undergoing genetic testing have a suboptimal understanding of the nature of testing, overestimate the risks of receiving an abnormal result and some show high levels of anxiety. The results of this study can be used to improve patient counselling before genetic testing.

Characterization of a de novo duplication of 11p14----p13, using fluorescent in situ hybridization and southern hybridization.

A de novo 11p+ chromosome was found in a child with mild mental retardation but no other remarkable dysmorphic characteristics. Banding studies suggested a duplication of regions 11p13 and 11p14 or regions 11p14 and 11p15. Using fluorescent in situ hybridization and digital imaging microscopy, we mapped probe p32.1 (D11S16) to the proximal part of region 11p14 (11p14.1) and demonstrated duplication of this probe in our patient. Southern hybridization showed duplication of p32.1 and other probes located at 11p13 and 11p14, but the gene for alpha calcitonin (CALCA), located at 11p15, was not duplicated. The application of these techniques led to the identification of the duplication as dir dup(11)(pter----p13::p15.1----qter).

Comparative genomic hybridization analysis of Wilms tumors.

In this study we have applied the technique of comparative genomic hybridization (CGH) to a large series of sporadic Wilms tumors, including six samples of the associated nephroblastomatosis. The data obtained were compared with the findings of molecular studies carried out on the same material. The aims of the study were (1) to characterize the range of genetic variation in sporadic Wilms tumor and nephroblastomatosis, (2) to determine whether changes could be found that have not been detected by commonly used techniques, and (3) to compare the sensitivity of CGH with that of conventional molecular analysis. The chromosomes that showed gains and losses by CGH were similar to those previously found in molecular and cytogenetic studies, however loss of 4q was a new event identified in 2 out of 46 tumors. We did not detect amplified genetic material. Comparison of the data from the nephroblastomatosis and tumor samples from the same patient showed that loss of 7p may be associated with malignant transformation, and that losses in 1p, 11p, 4q and gains in 1q and 12q can be early events; whilst loss in 9p and gain of 8, 10q and 18 are possible secondary changes in tumor development. The combined CGH and molecular techniques used demonstrated involvement of two specific 1p regions in the etiology of Wilms tumor.

Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype.

Current evidence suggests that aniridia (absence of iris) is caused by loss of function of one copy of the PAX6 gene, which maps to 11p13. We present the further characterisation of two aniridia pedigrees in which the disease segregates with chromosomal rearrangements which involve 11p13 but do not disrupt the PAX6 gene. We have isolated three human YAC clones which encompass the PAX6 locus and we have used these to show that in both cases the chromosomal breakpoint is at least 85 kb distal of the 3' end of PAX6. In addition, the open reading frame of PAX6 is apparently free of mutations. We propose that the PAX6 gene on the rearranged chromosome 11 is in an inappropriate chromatin environment for normal expression and therefore that a 'position effect' is the underlying mechanism of disease in these families.

Cytogenetic and molecular analysis of cellular atypical mesoblastic nephroma.

Cytogenetic and molecular analyses were performed on three cellular (atypical) congenital mesoblastic nephromas (CMNs). Two cases had trisomy 11; in one, it was the sole karyotypic abnormality, and the other had additional numerical changes as well as an isochromosome for the long arm of chromosome 1. Markers for the 11p13 and 11p15 loci were present in three copies in these two CMNs. In the third CMN, two apparently normal copies of chromosome 11 were present together with additional numerical and structural chromosome changes. Because loss of heterozygosity was observed for both 11p13 and 11p15 markers, we assume that mitotic recombination occurred. Duplication and loss of imprinting of genes at 11p15 has also been observed frequently in Wilms' tumor. We therefore propose that CMN and Wilms' tumor might share common genetic pathways.

The generation of ordered sets of cosmid DNA clones from human chromosome region 11p.

We describe progress in a continuing project aimed at the generation of an overlapping cosmid DNA clone map of the short arm of human chromosome 11. The automated procedures used to prepare DNA samples and the computerized data collection and recording systems are described. We also demonstrate the use of the clones as reagents for the rapid isolation of genomic DNAs containing smaller probed regions. We have isolated approximately 4700 human cosmid DNA clones from mouse/human hybrid cell lines that contain predominantly human chromosomal region 11p. Of the DNA in the cell lines, 60% is derived from this chromosomal region, and the remaining 40% is derived from regions of chromosomes 3, 19, and 20. A total of 4159 clones have been fingerprinted to identify potential overlaps, and we have developed 535 sets ("contigs"). Using random modeling, it is estimated that 65% of 11p must be contained in the analyzed cosmids. The database of clones has been used to identify single or overlapping clones from noncosmid DNA probes. Examples are presented. It is proposed that cosmid reference filters be distributed to requesting laboratories.

Positional cloning of genes involved in the Beckwith-Wiedemann syndrome, hemihypertrophy, and associated childhood tumors.

The Beckwith-Wiedemann syndrome (BWS) is an overgrowth malformation syndrome that occurs with an incidence of 1:13,700 births. There is a striking incidence of childhood tumors found in BWS patients. Various lines of investigation have localized "imprinted" genes involved in BWS and associated childhood tumors to 11p15. High resolution mapping of 8 rare balanced chromosomal BWS rearrangements enabled us to identify three distinct regions on chromosome 11p15 that might harbor genes involved in the above-mentioned disorders. These results suggest genetic heterogeneity that correlates with the clinical heterogeneity seen in the patients studied. Expressed candidate gene sequences from these regions have been cloned and partly sequenced. These transcripts are either disrupted by or are at least within a few kb of these BWS chromosome breakpoints. So far, zinc-finger sequences and one Kruppel-associated box (KRAB) domain were found in independent candidate genes which are compatible with a regulating function of growth promoting genes. The abundance of expression of these genes varies from low abundant in all adult and fetal tissues tested to detectable on Northern blots of adult tissues. In addition to our 11p15 studies we have analyzed additional chromosome regions, in particular 1p. Cytogenetic, loss of heterozygosity (LOH) and comparative genomic hybridization (CGH) studies have identified 1p35 as a region of interest. A positional cloning effort to identify a balanced 1p35 translocation found in a Wilms tumor has led to the isolation of a YAC, crossing this breakpoint.

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.