Diagnosis of a complex chromosomal rearrangement using fluorescent in situ hybridisation.

We report the use of fluorescent in situ hybridisation (FISH) to clarify a complex chromosomal rearrangement (CCR) carried by a woman presenting with recurrent miscarriages. CCRs are rare cytogenetic rearrangements involving three or more chromosomes, which can be difficult to interpret using routine cytogenetic studies with GTG banding. FISH was used to establish a correct interpretation of the maternal karyotype before amniocentesis in a present pregnancy.     

Familial complex chromosomal rearrangement resulting in a recombinant chromosome

Familial complex chromosomal rearrangements (CCRs) are rare and tend to involve fewer breakpoints and fewer chromosomes than CCRs that are de novo in origin. We report on a CCR identified in a child with congenital heart disease and dysmorphic features. Initially, the child's karyotype was thought to involve a straightforward three-way translocation between chromosomes 3, 8, and 16. However, after analyzing the mother's chromosomes, the mother was found to have a more complex rearrangement that resulted in a recombinant chromosome in the child. The mother's karyotype included an inverted chromosome 2 and multiple translocations involving chromosomes 3, 5, 8, and 16. No evidence of deletion or duplication that could account for the clinical findings in the child was identified.     

Miller-Dieker syndrome resulting from rearrangement of a familial chromosome 17 inversion detected by fluorescence in situ hybridisation.

We report a case of Miller-Dieker syndrome (MDS) owing to an unbalanced rearrangement of a familial pericentric inversion of chromosome 17 (inv(17) (p13.3q25.1)). In addition to lissencephaly and the facial features of MDS, the affected child had other congenital malformations consistent with distal 17q duplication. Initial cytogenetic analysis failed to show any abnormality and fluorescence in situ hybridisation (FISH) studies confirmed the 17p deletion in the proband and identified the chromosome 17 inversion in his mother. FISH studies were performed in other relatives and enabled first trimester prenatal diagnosis by chorionic villus sampling in a subsequent pregnancy of the proband's mother. These findings underline the value of FISH in the investigation of MDS families.     

Imbalances of chromosome 17 in medulloblastomas determined by comparative genomic hybridisation and fluorescence in situ hybridisation.

AIMS: To investigate the status of chromosome 17 in a series of medulloblastomas using comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). METHODS: Frozen tissue and formalin fixed, paraffin was embedded tissue from 27 medulloblastomas were analysed by CGH and FISH, respectively. CGH ratio profiles for chromosome 17 were compared with the results of FISH, for which loss or gain of 17p or 17q was assessed in two distinct ways using a combination of differentially labelled subtelomeric and centromeric probes and analysing 200 nuclei in each tumour. RESULTS: CGH revealed imbalances consistent with isochromosome 17q in eight of 27 tumours. Either loss of 17p or gain of 17q was identified in a further nine tumours, whereas 10 tumours were apparently balanced. Using control results from preparations of paraffin wax embedded tonsils, thresholds for the detection of abnormalities by FISH were established, either by determining the dominant pattern of signals in each case, or the mean ratio of subtelomeric to centromeric signals. Results by CGH and FISH were concordant in 21 of 27 tumours. In the remainder, most discrepancies related to methodological differences. CONCLUSIONS: CGH has a role in disclosing common, genome wide chromosomal gains or losses in tumours, the clinical relevance of which can then be studied in large archival series of paraffin wax embedded tumours using FISH.     

Familial complex chromosome rearrangement giving rise to balanced and unbalanced recombination products

We report on a family ascertained through a 14-month-old girl with a terminal deletion of chromosome 8p23.1. Analysis of the karyotype of other relatives showed that the mother is the carrier of a balanced complex 4-break chromosome rearrangement, which she and her brother inherited from their father following recombination. This complex chromosome rearrangement (CCR) was confirmed by fluorescence in-situ hybridization (FISH) using libraries for chromosomes 1, 8, and 9, and telomeric probes for the long arm of chromosome 9. The karyotype of the maternal grandfather was 46,XY,t(1;8) (p31;q21.1),t(8;9)(p23.1;q34). The karyotype of his daughter is 46,XX,rec(8)t(1;8) (p31;q21.1)t(8;9)(p23.1;q34)pat. The karyotype of the proposita is 46,XX,rec(8)t(8;9) (p23.1;q34)mat, and that of her abnormal elder sister is 46,XX,t(1;8)(p31;q21.1)rec(8) t(8;9)(p23.1;q34)mat,der(9)t(8;9)(p23.1;q34) mat. Unbalanced segregation and/or recombination during maternal meiosis gave rise to the two abnormal sisters, one effectively with 8p trisomy and the other with monosomy for that same 8p segment. To our knowledge, this is the first case of a familial CCR giving rise to unbalanced recombination products. Am. J. Med. Genet. 79:30–34, 1998. © 1998 Wiley-Liss, Inc.     

A complex chromosome rearrangement with at least five breakpoints studied by fluorescence in situ hybridization

A newborn infant with multiple congenital anomalies was diagnosed with an unbalanced translocation of chromosomes 1 and 5. Studies of parental chromosomes revealed a complex rearrangement in the patient's mother involving the exchange of terminal long arms between chromosomes 1 and 5 and the insertion of an interstitial segment from the same chromosome 5q into chromosome 2q by high-resolution G-banding. Further study of the mother's chromosomes by fluorescent in situ hybridization (FISH) detected an additional insertion between the rearranged chromosomes 2 and 5, which was not revealed by G-banding. This led to the identification of a complex translocation-insertion between 3 chromosomes with at least 5 breaks [t(1;5;2)(1pter→1q42.3::5q23.2→5qter;5pter→5q21.2::2q33→2q35::1q42.3→1qter;2pter→2q33::5q21.2→5q23.2::2q35→2qter)] and illustrates the value of FISH as an adjunct to standard cytogenetics, particularly in cases of complex rearrangements. Am. J. Med. Genet. 68:417–420, 1997. © 1997 Wiley-Liss, Inc.     

Sensitive in situ hybridisation technique using biotin-streptavidin-polyalkaline phosphatase complex.

A sensitive in situ hybridisation technique, using a biotin-streptavidin-polyalkaline phosphatase complex detection system, was successfully applied to smears of fresh cultured cells, frozen sections, and formalin fixed paraffin processed tissue: the procedure was successful for DNA-DNA hybridizations using a variety of DNA probes. The detection method is rapid, reliable, and economical producing a purplish-blue precipitate at the site of hybridisation and clearly visible by low power light microscopy.     

Investigation of chromosome 21 aneuploidies in breast fibroadenomas by fluorescence in situ hybridisation

Abstract Fibroadenoma (FA) is a benign breast tumour that occurs in about 25% of women. Cytogenetic studies suggest that numerical chromosomal aberrations may contribute to tumorigenesis, but chromosomal instability is still poorly characterised in breast cancer. The aim of this study was to investigate numerical alterations of chromosome 21 in 15 breast FAs. All samples were analysed by classical cytogenetics and by fluorescence in situ hybridisation (FISH) for chromosome 21 DNA sequences. Classical cytogenetics analysis showed that all cells were diploidies with modal number varying between 43 and 47 chromosomes, and clonal chromosome alterations in 46.7% of tumours. Clonal numerical alterations involved, preferentially, chromosomes 8, 10, 12, 16 and 21. FISH analysis showed a statistically significant difference for chromosome 21 monosomy between seven samples and control group. This monosomy varied from 24.5% to 43.5% of analysed cells. The presence of chromosomal alterations in FAs may be a consequence of the proliferation process and is probably not related to the aetiology of this type of lesion. The study of benign proliferations and comparison with chromosome alterations in their malignant counterparts should result in an understanding of the genes acting in cell proliferation alone and those that cause these cells to both undergo malignant transformation and become invasive.     

Maternal complex chromosome rearrangement ascertained through a del (13)(q12.1q14.1) detected in her mildly affected daughter.

A maternal complex chromosome rearrangement (CCR) involving chromosomes 2, 13, and 20 was ascertained in a normal female through the diagnosis of a deletion of 13q in her daughter. The child has mild clinical features and developmental delay consistent with proximal deletions of 13q that do not extend into band q32 and a del(13)(q12q14.1) that does not involve the retinoblastoma locus by FISH. Maternal studies by GTG banding and FISH showed a complex karyotype with bands 13q12.3-->13q12.1::20p13 translocated to 2p13 and bands 2pter-->2p13::13q12.3-->13q14.1 translocated into band 20p13. This would be the first report of an interstitial deletion of 13q inherited from a parental complex chromosome rearrangement.     

In situ hybridisation in perspective.

In the introduction to this review two questions were posed: is the technology associated with ISH ready for general use, and will the method become an important investigative tool? With the exception of the demonstration of some single and low copy sequences, non-radioactive ISH is now sufficiently developed and simplified to make it a routine technique. It is also clear that ISH will continue to have an important research role. In diagnostic pathology the technique is already providing valuable information and the present decade should see the development of many more diagnostic applications.     

Trisomy 10qter confirmed by in situ hybridisation.

We report a boy with multiple congenital anomalies compatible with trisomy for the distal region of the long arm of chromosome 10 and a male karyotype with one 18p+. In situ hybridisation with a cDNA for ornithine aminotransferase (OAT), whose locus maps to 10q26, confirmed the clinical suspicion of distal trisomy 10q. Subterminal localisation of the labelling signals on chromosome 10 and on the der(18) indicated the localisation of the OAT locus in the proximal part of 10q26. Two clusters of labelling signals were also found on the pericentromeric and proximal portion of the X chromosome short arm, thus confirming the presence in this region of two non-adjacent OAT pseudogenes. The phenotypic similarities of this patient to previously reported cases provide further support for the delineation of trisomy 10qter as a specific, clinically recognisable syndrome.     

A complex,five breakpoint intrachromosomal rearrangement ascertained through two recombinant offspring

Intrachromosomal rearrangements usually result from three or fewer breaks. We report a complex intrachromosomal rearrangement resulting from five breaks in one chromosome 10 of a phenotypically normal father of two developmentally delayed children. GTG-banding analysis of the father's rearranged chromosome 10 suggested an initial pericentric inversion followed by an insertion from the short arm into the terminal band of the long arm [der(10)(pter→p13::q21.2→p12.2::q22.1→q26.3::q22.1→q21.2::p12.2→p13::q26.3→qter)]. To our knowledge, this rearrangement is the most complex ever reported in a single chromosome. Both children inherited a recombinant chromosome 10 with loss of the insertion and the segment distal to it [rec(10)der(pter→p13::q21.2→p12.2::q22.1→q26.3:)]. Mechanisms for both rearrangements are proposed. © 1996 Wiley-Liss, Inc.     

Disclosure of five breakpoints in a complex chromosome rearrangement by microdissection and FISH.

Microdissection and fluorescence in situ hybridisation (FISH) were used to elucidate the nature of a complex chromosome translocation, after GTG banding failed in the complete characterisation of the structural rearrangement between chromosomes 6 and 12. These chromosomes were painted with chromosome specific paints and one of the chromosome regions involved in the translocation was isolated by microdissection. Ten copies of the microdissected region were collected with microneedles from GTG banded metaphases, transferred to a collecting drop, and amplified by means of DOP-PCR. The PCR product was labelled with biotin-14-dATP and used as a FISH probe for hybridisation to normal metaphase chromosomes and metaphase chromosomes of the patients (microFISH). FISH with this chromosome region specific painting probe and with chromosome band specific probes enabled the characterisation of a complex chromosome rearrangement with five breakpoints in two chromosomes. This resulted in the following karyotype: 46,XY,t(6;12)(6pter--> 6q12::12q24.1-->12qter;12qter-->12q13.3:: 6q16.2-->6q26::12q13.3-->12q24.1::6q12--> 6q16.2::6q26-->6qter).     

Familial mental retardation in a family with an inherited chromosome rearrangement

A family of three generations has been described with an insertional type of chromosome rearrangement involving chromosomes 11 and 18[46,XX or XY, ins(11;18)(p15;q11q21)] detected by G-banding using a trypsin digestion method. Four members of this family with clinical features of 18q− have inherited the der(18) from their father and are thus deficient for (18)(q11q21). Three other family members have inherited the der(11) and thus have a duplication of the same segment [(18)(q11q21)]. Genetic marker studies on this family, show no significant segregation of any of the markers studied with either the der(11) or der(18). Eight family members had the PepA⁸PepA¹ genotype and four of these were carrying the der(18), indicating that the PepA locus which had been previously assigned to chromosome 18, does not lie in the segment q11→q21.     

Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes.

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convenient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.     

Kallmann syndrome associated with complex chromosome rearrangement

We report on a male with Kallmann syndrome (KS) and an apparently balanced complex chromosome rearrangement (CCR): 46,XY,t(3;9)(9;12)(q13.2;q21.2p13;q15). This is the first known report of a CCR in the KS and the second reported case of a definitive autosomal chromosome abnormality with KS. Possible relationships between the cytogenetic abnormality and KS are discussed. © 1993 Wiley-Liss, Inc.