Further delineation of the 22q13 deletion syndrome.

A chromosomal deletion syndrome associated with a 22q13 microdeletion has previously been reported in approximately 75 children. We report six cases from Denmark with a deletion of 22q13. One was cytogenetically visible by conventional karyotyping, one was diagnosed by high resolution karyotyping after the demonstration of low arylsulfatase A activity. Two were diagnosed by high resolution CGH analysis, one was diagnosed by multisubtelomeric FISH analysis and one was diagnosed serendipitously as lack of the control signal in a FISH analysis for 22q11 deletion. One of the cases was a mosaic with 16% of cells showing two signals. The phenotype of the children included: generalized developmental delay, compromised language development, hypotonia, normal or accelerated growth and minor facial dysmorphism. Other features were partial agenesis of the corpus callosum, bilateral ureteropelvic stricture, gastroesophageal reflux and hearing loss. One case had a different phenotype, and showed a deletion as well as a duplication. The extent of the deletion was studied by quantitative PCR analysis of a number of DNA markers in the 22q13 region. The deletions varied in size, extending from 4.0 to 9.0 Mb. The clinical phenotype seemed rather similar although some specific features might be attributable to differences in deletions.

Balanced complex chromosomal rearrangements (BCCR) with at least three chromosomes and three or more breakpoints: report of three new cases.

Balanced complex chromosomal rearrangements (BCCR) encompass a heterogeneous group of rare chromosomal aberrations. In this paper, we report three cases of BCCRs. In two the probands were referred for either genetic counseling or prenatal management. One case was ascertained after chromosome analysis performed because of psychiatric manifestations; this was an isolated finding. We also outline the molecular cytogenetic techniques, which were essential in confirming and precisely delineating the BCCRs identified in these patients. In addition the various aspects of genetic counseling for this type of chromosomal rearrangement, highlighting the details particular to each individual case are discussed. We discuss the classification for this type of chromosomal mutation.

De novo complex chromosomal rearrangements (CCR) involving chromosome 1, 5, and 6 resulting in microdeletion for 6q14 in a female carrier with psychotic disorder.

A 23-year-old obese woman with a psychotic disorder was found to have a de novo apparently balanced complex chromosomal rearrangement involving chromosomes 1, 5, and 6. Molecular cytogenetic analyses using high-resolution comparative genomic hybridization (HR-CGH) showed a microdeletion at 6q14 in a der(6). Application of HR-CGH facilitated detection of micro-rearrangement of all de novo apparently balanced complex chromosomal rearrangements (CCR) and supported the localization of the breakpoint. According to our knowledge, no constitutional interstitial microdeletion of chromosome 6q14 has been found associated with a schizoid-type phenotype.

The most common chromosome aberration detected by high-resolution comparative genomic hybridization in vulvar intraepithelial neoplasia is not seen in vulvar squamous cell carcinoma.

We analyzed genetic changes in condylomas (four cases), vulvar intraepithelial neoplasia I-III (VIN I-III, eleven cases), and primary vulvar squamous cell carcinomas (VSCC, ten cases) by high-resolution comparative genomic hybridization (HR-CGH) and flowcytometry. All samples were also human papilloma virus (HPV)-genotyped. Gain of chromosome 1, the aberration most often seen in VIN III (67%), was not seen in HPV-positive or -negative VSCCs (0%). Both VIN III and VSCC frequently showed gain of 3q (56 and 70%, respectively). The VIN III samples often demonstrated gain of 20q (56%) and 20p (44%), and the VSCC samples gain of 8q (60%), loss of 3p (50%), and 8p (40%). None of the four most frequent changes in the VSCC samples occurred exclusively in the HPV-positive or -negative samples. As expected, we did not find any cytogenetic changes in condylomas and nearly any changes in VIN I-II.

Cytogenetic and molecular analysis of a family with three brothers afflicted with germ-cell cancer.

A thorough cytogenetic investigation and an analysis of detailed questionnaires were performed in a family with three brothers afflicted with germ-cell tumors (GCTs), in an attempt to detect a congenital factor related either to a hereditary genetic background or an environmental/lifestyle influence. One brother had an intracranial tumor in the pineal region and the two others had testicular tumors. Peripheral blood was studied by traditional karyotyping, multicolor-FISH, high-resolution comparative genomic hybridization (HR-CGH), and molecular analysis of selected loci on sex chromosomes (Yq11 region, TSPY, and the androgen receptor gene); however, no abnormalities were detected. The HR-CGH analysis of microdissected histological components of the overt tumors and the adjacent carcinoma in situ demonstrated a pattern of genomic imbalances characteristic for sporadic GCTs, including gain of 12p. The questionnaire and interview revealed a history of different cancers in the extended family, and a possible in utero and/or infantile exposure of the three brothers with GCTs to compounds suspected of endocrine-disrupting properties. Although no genetic aberration was detected in this family, we suspect the presence of a recessive hereditary factor pre-disposing to cancer, which probably was manifested as GCTs in the three brothers because of an adverse effect of an environmental factor on the early germ-cell differentiation.

Erroneous genetic sex determination of a newborn twin girl due to chimerism caused by foetal blood transfusion. A case report.

OBJECTIVE: We present a case of erroneous sex determination in a newborn twin girl (twin A) due to chimerism. CASE REPORT: Amniocentesis and ultrasound examination had pointed towards male sex of both twins. At birth, twin A presented as a phenotypically normal female with 46,XY karyotype, and 46,XY gonadal dysgenesis was suspected. Twin B was a normal male. RESULTS: In our department, further examinations of twin A included undetectable testosterone and inhibin-B and elevated FSH. Ultrasound suspected an infantile uterus, and sequencing of the SRY gene was normal. After gonadectomy, a 46,XX karyotype was demonstrated in both normal infantile ovaries and in the fibroblasts from a skin biopsy. Analysis of X-linked markers in DNA from blood lymphocytes in both twins was identical, consistent with 46,XY karyotypes. CONCLUSION: Twin A is a 46,XX female with a chimeric 46,XY blood cell line due to intrauterine transfusion from her twin brother.

High resolution comparative genomic hybridisation in clinical cytogenetics.

High resolution comparative genomic hybridisation (HR-CGH) is a diagnostic tool in our clinical cytogenetics laboratory. The present survey reports the results of 253 clinical cases in which 47 abnormalities were detected. Among 144 dysmorphic and mentally retarded subjects with a normal conventional karyotype, 15 (10%) had small deletions or duplications, of which 11 were interstitial. In addition, a case of mosaic trisomy 9 was detected. Among 25 dysmorphic and mentally retarded subjects carrying apparently balanced de novo translocations, four had deletions at translocation breakpoints and two had deletions elsewhere in the genome. Seventeen of 19 complex rearrangements were clarified by HR-CGH. A small supernumerary marker chromosome occurring with low frequency and the breakpoint of a mosaic r(18) case could not be clarified. Three of 19 other abnormalities could not be confirmed by HR-CGH. One was a Williams syndrome deletion and two were DiGeorge syndrome deletions, which were apparently below the resolution of HR-CGH. However, we were able to confirm Angelman and Prader-Willi syndrome deletions, which are about 3-5 Mb. We conclude that HR-CGH should be used for the evaluation of (1) dysmorphic and mentally retarded subjects where normal karyotyping has failed to show abnormalities, (2) dysmorphic and mentally retarded subjects carrying apparently balanced de novo translocations, (3) apparently balanced de novo translocations detected prenatally, and (4) for clarification of complex structural rearrangements.

[Detection of submicroscopic chromosome abnormalities by comparative genomic hybridization]. / Påvisning af submikroskopiske kromosomfejl med komparativ genomisk hybridisering.

INTRODUCTION: The purpose was to detect chromosome abnormalities in dysmorphic and mentally retarded individuals with normal karyotypes by means of comparative genomic hybridisation (CGH). MATERIAL AND METHODS: One hundred and forty-four individuals with normal karyotype underwent CGH analysis with a new detection technique where fixed limits are replaced by dynamic standard reference intervals. This method provides improved resolution and thereby detects minor chromosome abnormalities. RESULTS: Fifteen minor abnormalities (10%) and one trisomy 9 mosaic were found. Eleven were interstitial deletions or duplications, which cannot be detected by screening with other cytogenetic techniques. Three were terminal deletions or duplications and one was a terminal unbalanced translocation. DISCUSSION: CGH analysis with dynamic standard reference intervals is a new objective and quantitative method, which is suitable for screening for small chromosome abnormalities that can not be detected by conventional chromosome analysis. The method is recommended for use in the investigation of dysmorphic and mentally retarded individuals, in whom abnormalities are not found by ordinary karyotyping.

Comparative genomic hybridization reveals non-random chromosomal aberrations in early preinvasive cervical lesions.

We performed CGH analysis on 34 cervical lesions, which included 8 cases of koilocytosis, 6 mild dysplasias and 20 moderate dysplasias. Chromosome aberrations were detected in 11 cases of which 9 were moderate dysplasias. A total of 55 chromosome arms were involved. The most frequent aberrations were losses of 5p and Xq, each of which was present in 5/34 cases. Gain of 3q was detected in two moderate dysplasias. This aberration is the most frequent copy number change in advanced-stage cervical carcinoma. A considerable number of the aberrations found in the preinvasive cases of this study are frequently present in invasive cervical tumors. The presence of apparently non-random chromosome aberrations in early preinvasive cervical lesions has not previously been described.

Optimization of DOP-PCR amplification of DNA for high-resolution comparative genomic hybridization analysis.

BACKGROUND: Whole-genome amplification of minute samples of DNA for the use in comparative genomic hybridization (CGH) analysis has found widespread use, but the method has not been well validated. METHODS: Four protocols for degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and fluorescence labeling were applied to test DNA from normal and K-562 cells. The DNA products were used for CGH analysis. RESULTS: The DOP-PCR-amplified DNA from each protocol produced hybridizations with different qualities. These could be seen primarily as differences in background staining and signal-to-noise ratios, but also as characteristic deviations of normal/normal hybridizations. One DOP-PCR-protocol was further investigated. We observed concordance between CGH results using unamplified and DOP-PCR-amplified DNA. An example of an analysis of an invasive carcinoma of the breast supports the practical value of this approach. CONCLUSIONS: DOP-PCR-amplified DNA is applicable for high- resolution CGH, the results being similar to those of CGH using unamplified DNA.

Cryptic familial t(11;18)(q25;q23) incidentally detected by interphase FISH.

During a prospective prenatal study of numerical abnormalities of chromosomes 13, 18, 21, X and Y using locus-specific probes, we incidentally found a case with only one signal for chromosome 18 per cell in a chorionic villus sampling (CVS) associated with an otherwise apparently normal G-banded karyotype. This led us to discover a cryptic t(11;18) segregating in a four-generation family. The CVS was performed because of mental retardation in the brother to the father of the fetus. A subtelomeric chromosome 18 probe revealed one signal on 18qter and one on 11qter of the father. Thus the father had a balanced reciprocal t(11;18) in spite of the apparently normal G-banded karyotype. Using the same probes, we found an unbalanced translocation 46,XX,-18,+der (18)t(11;18)-(q25;q23)pat in the fetus. Further investigation of the family showed the translocation in balanced and unbalanced form in four generations in mentally normal and retarded individuals, respectively. The study emphasizes the need for a follow-up with molecular cytogenetic techniques in dysmorphic and retarded children.

High resolution comparative genomic hybridization detects 7-8 megabasepair deletion in PCR amplified DNA.

We investigated if any change in spatial resolution of comparative genomic hybridization analysis could be detected when using DNA amplified by degenerate oligonucleotide primed PCR (DOP-PCR) as opposed to the use of unamplified DNA. Five DNA samples from B-cell leukemias with small 11q deletions were amplified by DOP-PCR and analysed by means of high resolution comparative genomic hybridization (HR-CGH) for the evaluation of aberration size detection limit. By means of HR-CGH, we found the detection limit of DOP-PCR CGH for deletions to be between 3 Mbp and 7-8 Mbp.

Disease associated balanced chromosome rearrangements: a resource for large scale genotype-phenotype delineation in man.

Disease associated balanced chromosomal rearrangements (DBCRs), which truncate, delete, or otherwise inactivate specific genes, have been instrumental for positional cloning of many disease genes. A network of cytogenetic laboratories, Mendelian Cytogenetics Network (MCN), has been established to facilitate the identification and mapping of DBCRs. To get an estimate of the potential of this approach, we surveyed all cytogenetic archives in Denmark and southern Sweden, with a population of approximately 6.6 million. The nine laboratories have performed 71 739 postnatal cytogenetic tests. Excluding Robertsonian translocations and chromosome 9 inversions, we identified 216 DBCRs ( approximately 0.3%), including a minimum estimate of 114 de novo reciprocal translocations (0.16%) and eight de novo inversions (0.01%). Altogether, this is six times more frequent than in the general population, suggesting a causal relationship with the traits involved in most of these cases. Of the identified cases, only 25 (12%) have been published, including 12 cases with known syndromes and 13 cases with unspecified mental retardation/congenital malformations. The remaining DBCRs were associated with a plethora of traits including mental retardation, dysmorphic features, major congenital malformations, autism, and male and female infertility. Several of the unpublished DBCRs defined candidate breakpoints for nail-patella, Prader-Willi, and Schmidt syndromes, ataxia, and ulna aplasia. The implication of the survey is apparent when compared with MCN; altogether, the 292 participating laboratories have performed >2.5 million postnatal analyses, with an estimated approximately 7500 DBCRs stored in their archives, of which more than half might be causative mutations. In addition, an estimated 450-500 novel cases should be detected each year. Our data illustrate that DBCRs and MCN are resources for large scale establishment of phenotype-genotype relationships in man.

High resolution comparative genomic hybridisation analysis reveals imbalances in dyschromosomal patients with normal or apparently balanced conventional karyotypes.

A sensitive technique is needed for screening whole genome imbalances in dyschromosomal patients when G-banding shows normal karyotypes or apparently balanced translocations. In this study we performed highly sensitive comparative genomic hybridisation analysis on a number of such cases and revealed chromosomal imbalances in all.

Rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization: a one-year clinical experience with high-risk and urgent fetal and postnatal samples.

OBJECTIVES: To evaluate the clinical utility of rapid prenatal and postnatal detection of common chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) analysis with DNA probes. DESIGN: Four hundred and seventy-seven high-risk and/or urgent amniotic fluid, chorionic villus and fetal and postnatal blood samples were prospectively examined by FISH with probes specific for chromosomes 13, 18, 21, X, and Y and results were reported within 48 hours. All FISH results were followed by conventional chromosome analysis, if possible. SETTING: Cytogenetic service laboratory at the tertiary referral center, Rigshospitalet in Copenhagen. MAIN OUTCOME MEASURES: The fraction of clinically significant chromosome aneuploidies that was detected by FISH analysis, and the fraction of terminations that was based on FISH and ultrasound results rather than on conventional cytogenetic results. RESULTS: The FISH assay detected 76% of the clinically significant chromosome abnormalities as determined by subsequent cytogenetic analysis. Seventy-two percent of the terminations of the chromosomally abnormal pregnancies were based on FISH and ultrasound results rather than on conventional cytogenetic results. CONCLUSION: FISH analysis is a clinically useful adjunctive tool to conventional pre- and postnatal cytogenetic analysis. The assay rapidly detects the majority of clinically significant chromosome abnormalities, thus facilitating difficult pre- and postnatal clinical decisions.

Deletions below 10 megabasepairs are detected in comparative genomic hybridization by standard reference intervals.

Comparative genomic hybridization (CGH) is a widely used technique for studying chromosomal imbalances. The sensitivity of the technique is, however, relatively low. Deletions down to a size of 10-12 Mbp have been detected by the use of fixed diagnostic thresholds. In this study, we applied standard reference intervals as detection criteria on a number of deletions in the range of 3 Mbp to 14-18 Mbp. All deletions were detected. Thus, detection by standard reference intervals confers a considerably higher sensitivity to CGH analysis compared to fixed diagnostic thresholds. Genes Chromosomes Cancer 25:410-413, 1999.

Comparison of cell cultures, chromosome quality and karyotypes obtained after chorionic villus sampling and early amniocentesis with filter technique.

548 cell cultures and karyotypes obtained by early amniocentesis with filtration technique (EAF) at a mean gestational age of 12 1/2 weeks were compared with 555 obtained by transabdominal chorionic villus sampling (CVS) at a mean gestational age of 11 weeks. The number of abnormal karyotypes, culture failure rate and harvest time were evaluated. The results were then compared with three similar studies from the literature evaluating early amniotic fluid cultures obtained with conventional techniques compared with chorionic villus cultures. Further, the quality of the chromosome preparations from chorionic villi and early amniotic fluid respectively was compared. More abnormal karyotypes were found among the CVS cultures, which was expected due to earlier sampling and presence of confined placental mosaicism. No ambiguous results were present after EAF. The lowest culture failure rate of 0.2 per cent was found after EAF compared with 0.9 per cent among CVS. EAF also showed a significantly lower culture failure rate when compared with the literature, where early amniocentesis (EA) had been carried out by standard techniques. The time from sampling to harvest was longer in the EAF group (mean 9.5 days) compared with CVS (mean 6.1 days), in accordance with the literature. Nevertheless, the culture time of EAF was significantly shorter than the mean of EA from the comparative studies, whereas CVS culture times showed no differences. Rates of pseudomosaicism, maternal cell contamination, single cell aberrations, number of chromosome bands, mitoses counted and number of primary cultures needed for each karyotype were also compared. We concluded that EAF carried out around 12 1/2 weeks of gestation is a successful method with a lower culture failure rate compared with CVS cultures from 11-week gestations and with a significantly lower culture failure rate when compared with EA from similar studies. EAF provides chromosome preparations of high quality, and the risk of ambiguous karyotypes is very low.