Individuals with abnormal phenotype and normal G-banding karyotype: improvement and limitations in the diagnosis by the use of 24-colour FISH.

The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.

Nasopharyngeal teratoma and mosaic tetrasomy 1q detected at amniocentesis. A case report and review of the literature.

The occurrence of nasopharyngeal teratomas (NPT) is an infrequent event and prenatal detection of such tumors is even rarer. We present a case report and review of the literature (N = 78 cases), in which we describe the cytogenetic, DNA, and pathological findings of a fetus with a mature NPT which was detected prenatally by ultrasound investigation following complaints of severe polyhydramnios by the mother.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment.

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.

A near false-negative finding of mosaic trisomy 21--a cautionary tale.

We report the finding of a mosaic trisomy 21 restricted to the long-term culture of chorionic villi and to one amniotic fluid culture which, if interpreted according to the standard rules for the authentication of mosaicism, would have resulted in a false-negative result. The definitive diagnosis of mosaic Down syndrome was eventually confirmed by cordocentesis and by post-abortion fibroblast cultures.

Development of a preparation and staining method for fetal erythroblasts in maternal blood: simultaneous immunocytochemical staining and FISH analysis.

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi washings, cord blood, and maternal blood samples were used for this study. After a density gradient separation and centrifugal cytology, slides were stained either with 3,3-diaminobenzidin (DAB), a marker for heme, or with antibodies against the gamma-chain of fetal hemoglobin (HbF). FISH analysis for both X- and Y-chromosomes was performed on the same slides. Cytocentrifugation provided a controlled cell density on the slides with good cell morphology. Both the DAB and HbF staining were suitable for manual screening of large numbers of slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in preparation. We were eventually able to combine HbF staining with FISH analysis, and produced a detection efficiency of >85% for both X- and Y-chromosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysis can be performed on the same cell with good image contrast, thus facilitating both manual and automated image analysis. This will facilitate the use of this approach for prenatal diagnosis.

Fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, DAB and HbF staining, and FISH analysis.

A protocol to detect fetal nucleated red blood cells (NRBCs) was tested in 217 pregnant women and in 19 nonpregnant controls. All the pregnant women were sampled after chorionic villus sampling (CVS); 20 were also sampled pre-CVS. NRBC recognition was based upon morphology by using staining of hemoglobin with 3,3-diaminobenzidin (DAB) or by immunocytochemical staining for fetal hemoglobin (HbF). This was combined with FISH analysis for both the X- and Y-chromosomes on the same cells. Progressive refinement of the methods increased the number of cases where NRBCs were detected from 53% (DAB) to 75% and 78% for DAB and HbF staining, respectively, with on average 43 NRBCs (range, 1-220). DAB gave a slightly higher yield than HbF in the lower cell count range (<25). In 6 out of 18 controls, NRBCs were detected with DAB, vs. 1 out of 19 (5%) with HbF. FISH analysis in 41 cases resulted in correct sex prediction in 80% (DAB) and 89% (HbF), respectively. Our data demonstrated an increase of cases with NRBCs (30% to 75%), as well as a rise of the mean number of NRBCs (6 to 29 cells), after CVS. We conclude that DAB staining is a straightforward way to screen for the presence of NRBCs in maternal blood, but is not specific for NRBCs of fetal origin. HbF immunophenotyping is a reliable marker for fetal NRBCs, which detected slightly fewer NRBCs than DAB-staining, but improved sex prediction and significantly reduced false-positive results. CVS at 10-13 weeks of gestation causes a significant increase of NRBCs in maternal blood. These data indicate that further refinement of NRBC detection is needed before application of noninvasive prenatal diagnosis using maternal blood is feasible.

A second case of hexasomy 8 in myelodysplastic syndrome.

Tetrasomy 8 is a rare form of acquired aneuploidy found exclusively in the myeloid leukemias. Hexasomy 8 is even rarer: only one case has been reported, thus far. We describe here the second case of hexasomy 8 as the sole abnormality in an elderly female patient with myelodysplastic syndrome (MDS).

Pitt-Rogers-Danks syndrome and Wolf-Hirschhorn syndrome are caused by a deletion in the same region on chromosome 4p 16.3.

Recently, a deletion of chromosome 4pter was found in three patients with Pitt-Rogers-Danks syndrome. We investigated two of these patients, by means of DNA and FISH studies, together with two additional patients with Pitt-Rogers-Danks syndrome, to determine the critical region of the deletion in these patients and to compare this with the critical region in Wolf-Hirschhorn syndrome. All four patients showed terminal deletions of chromosome 4p of different sizes. One of them appeared to have an unbalanced karyotype caused by a cryptic translocation t(4;8) in the mother, resulting in a deletion of chromosome 4pter and a duplication of chromosome 8pter. The localisation of the Wolf-Hirschhorn critical region has been confined to approximately 1 Mb between D4S43 and D4S115. Our study shows that the deletions in four patients with the Pitt-Rogers-Danks syndrome overlap the Wolf-Hirschhorn critical region and extend beyond this in both directions. This study, combined with the fact that our third patient, who was previously described as a Pitt-Rogers-Danks patient, but who now more closely resembles a Wolf-Hirschhorn patient, makes it likely that Pitt-Rogers-Danks and Wolf-Hirschhorn syndromes are different clinical phenotypes resulting from a deletion in the same microscopic region on chromosome 4p16.

Mosaicism of the 5q deletion as assessed by interphase FISH is a common phenomenon in MDS and restricted to myeloid cells.

Interstitial deletion of chromosome 5q is a common cytogenetic abnormality observed in MDS. We have used fluorescence in situ hybridization (FISH) to determine accurately the percentage of cytogenetically abnormal peripheral blood cells. YAC and cosmid probes localized to chromosome 5q were hybridized to interphase nuclei from purified polymorphonuclear cells (PMNs) from six MDS patients with chromosome 5 deletions. Per patient, 25-67% of the cells exhibited one signal for the 5q31-q33 specific probes IL-4, D5S207 and c-fms. This percentage was constant for the various probes utilized for each patient. Hybridization of the same probes to PMNs from healthy individuals and hybridization of probes (D5S39 and D5S498) localized outside the deleted segments to PMNs of the patients, resulted in 90-95% nuclei with two signals. In addition, FACS-purified peripheral blood cells were investigated by FISH using the IL-4 cosmid. This demonstrated that the hybridization pattern in monocytes was similar to that observed in PMNs, whereas T-lymphocytes showed no loss of signals. These results indicate that a subfraction of the myeloid progenitor cells have acquired the 5q deletion.

Prenatal and postnatal investigation of a case with Miller-Dieker syndrome due to a familial cryptic translocation t(17;20) (p13.3;q13.3) detected by fluorescence in situ hybridization.

We present here a case report of a fetus with a kidney anomaly and dilated occipital horns, detected initially by echoscopy at 29 weeks' amenorrhoea. After 31 weeks of gestation, the proband was born with clinical symptoms of Miller-Dieker syndrome. This was subsequently confirmed by fluorescence in situ hybridization (FISH), but not by conventional cytogenetic analysis. FISH using a cocktail of cosmids (c197-2, c197-4, c197-9) from the Miller-Dieker critical region showed a deletion of 17p13.3 in one homologue of chromosome 17. Additional FISH studies revealed a subtle 17p;20q translocation in the father, his sister, and the paternal grandmother. Hence, our patient is a carrier of an unbalanced 17;20 translocation resulting in a partial deletion of 17p and a partial trisomy 20q. Whenever kidney anomalies and dilated occipital horns are observed together with polyhydramnios during prenatal ultrasound examination, the possibility of Miller-Dieker syndrome should be suspected. In such cases, prenatal and/or postnatal chromosome studies should also include FISH analysis with the appropriate probes.

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

A case of isodicentric 7p as sole abnormality in a patient with acute myeloid leukemia.

The detection of isochromosomes in the leukemias and in solid tumors has been well described in the literature, the most common being the i(17q), which is found in the blast crisis of CML and terminal stages of acute myeloid leukemia. Reports of isochromosome 7 have, however, been less well represented, particularly isochromosomes of the short arm of chromosome 7, which represent approximately 1% of all reported isochromosomes in neoplasia. We present here a case report of an elderly female patient with AML-M2 who manifested an idic(7p) in the majority of her bone marrow cells. Fluorescence in situ hybridization (FISH) studies with both centromere-7--and chromosome-7--specific DNA probes verified the diagnosis of idic(7p). To the best of our knowledge, this is the first report of this type of leukemia with an acquired idic(7p) as the sole cytogenetic abnormality.

Molecular, cytogenetic, and phenotypic studies of a constitutional reciprocal translocation t(5;10)(q22;q25) responsible for familial adenomatous polyposis in a Dutch pedigree.

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer caused by germline mutations in the adenomatous polyposis coli (APC) gene located on chromosome segment 5q21-q22. We detected a germline rearrangement of the APC gene in a Dutch FAP family by screening genomic DNA samples with APC cDNA probes. Subsequent molecular and cytogenetic studies revealed a constitutional reciprocal translocation t(5;10)(q22;q25) that resulted in the disruption of the APC gene. Southern blot and polymorphic marker analysis indicated that part of the APC gene had been deleted. Analysis of the APC protein product indicated that the translocation breakpoint did not lead to the formation of a detectable truncated APC protein but apparently resulted in a null allele. Evaluation of the clinical phenotypes in the patients suggested that they exhibited features of an unusual form of FAP characterized by a slightly delayed age of onset of colorectal cancer and a reduced number of colorectal polyps. The latter were mainly sessile and were located predominantly in the proximal colon. To our knowledge, this is the first description of FAP caused by a reciprocal translocation disrupting the APC gene.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Cerebellar and brainstem hypoplasia in a child with a partial monosomy for the short arm of chromosome 5 and partial trisomy for the short arm of chromosome 10.

A child with hypoplasia of the cerebellum and brainstem in association with an unbalanced translocation, resulting in a partial deletion of the short arm of chromosome 5 and a partial trisomy of the short arm of chromosome 10, is described. A balanced translocation was present in his mother and maternal grandmother. A maternal uncle had died at the age of 4 years with the same clinical picture. After reviewing the literature, we conclude that the monosomy 5p is the most likely cause for this malformation. We suggest that the possible existence of chromosomal anomalies should always be considered in the differential diagnosis of hypoplasia of the cerebellum and/or brainstem.