Clinical cytogenetic laboratories frequently identify an apparent duplication of proximal 15q that does not involve probes within the PWS/AS critical region and is not associated with any consistent phenotype. Previous mapping data placed several pseudogenes, NF1, IgH D/V, and GABRA5 in the pericentromeric region of proximal 15q. Recent studies have shown that these pseudogene sequences have increased copy numbers in subjects with apparent duplications of proximal 15q. To determine the extent of variation in a control population, we analysed NF1 and IgH D pseudogene copy number in interphase nuclei from 20 cytogenetically normal subjects by FISH. Both loci are polymorphic in controls, ranging from 1-4 signals for NF1 and 1-3 signals for IgH D. Eight subjects with apparent duplications, examined by the same method, showed significantly increased NF1 copy number (5-10 signals). 1gH D copy number was also increased in 6/8 of these patients (4-9 signals). We identified a fourth pseudogene, BCL8A, which maps to th e pericentromeric region and is coamplified along with the NF1 sequences. Interphase FISH ordering experiments show that IgH D lies closest to the centromere, while BCL8A is the most distal locus in this pseudogene array; the total size of the amplicon is estimated at ~1 Mb. The duplicated chromosome was inherited from either sex parent, indicating no parent of origin effect, and no consistent phenotype was present. FISH analysis with one or more of these probes is therefore useful in discriminating polymorphic amplification of proximal pseudogene sequences from clinically significant duplications of 15q.
Molecular cytogenetic analysis has shown that apparent duplications of proximal 15q identified by G banding are not usually associated with a duplication of DNA probes in the Prader-Willi/Angelman critical region. The majority of these cases are not duplicated for SNRPN and have a normal phenotype or inconsistent phenotypes. (1-4) They are usually inherited from a phenotypically normal parent and have been interpreted as euchromatic variants.
Two recent reports (5,6) used molecular cytogenetic techniques to show that amplification of a pseudogene cassette, mapping close to the centromere of 15q, causes the visible appearance of a duplication in banded chromosomes. The pseudogene cassette contains truncated gene sequences from a minimum of three genes transposed from other sites in the genome. One such sequence was shown to be a partial copy of the neurofibromatosis-1 (NF1) gene from chromosome 17q11.2. (5,7-10)
To determine whether increased dosage of this NF1 pseudogene correlated with observed cytogenetic duplications of 15q11.2, Barber et al (5) used FISH to show that the signals from a PAC probe containing the NF1 pseudogene were increased in intensity in two unrelated cases of chromosome 15 proximal duplications and estimated the degree of amplification from measurements of fluorescence intensities on metaphase chromosomes.
Ritchie et al (6) found that a truncated copy of the GABRA5 ([gamma]-aminobutyric acid type A receptor [alpha] 5 subunit) gene, the full length copy of which maps within the Prader-Willi/Angelman critical region, mapped to the pericentromeric region of chromosome 15, and was present in multiple copies on proximal duplication chromosomes. An estimate of the degree of amplification was made from the number of FISH signals in interphase nuclei and from released chromatin preparations. By isolating P1 clones from the region, they found that both the GABRA5 duplication sequence and one of the NF1 non-processed pseudogenes were present in the same P1 clone in the contig. A third sequence, the immunoglobulin heavy chain diversity segment gene (IgH D), had previously been mapped to proximal 15q (11-13) and was shown to be amplified in proximal duplications, but was not part of the same contig.
Ritchie et al (6) reported variation in the number of signals from GABRA5 and flanking probes in cytogenetically normal controls. In this paper, we present a more detailed study of the variation in copy number of NF1 and IgH D sequences in 20 cytogenetically normal controls and in eight proximal duplication cases. We quantified the degree of amplification by interphase FISH and established the normal range in controls. In addition, we show that a fourth pseudogene for BCL8 (Dyomin and Chaganti, unpublished data) is part of the amplified cassette. BCL8 was originally identified at the breakpoint of a translocation (t(14;15)(q32;q11-13)) in a diffuse large cell lymphoma patient. (14) Recent work has shown that the copy on chromosome 15 lacks several exons, now termed BCL8A; the full length copy, BCL8B , maps to 13q11-q12 (Dyomin and Chaganti, unpublished data). By interphase FISH mapping we have established the order of the pseudogenes within the cassette and estimated the size of the region amplified.
MATERIALS AND METHODS
The control population consists of 20 cytogenetically normal subjects from the University of Chicago Clinical Cytogenetics Laboratory. Details of the control population's ascertainment are given in table 1.
The patient population was divided into two groups: subjects in group I are phenotypically normal and were ascertained during routine cytogenetic analysis by the presence of a proximal duplication on chromosome 15, or as the parent of a fetus with a proximal duplication, while subjects in group II were ascertained on the basis of an abnormal phenotype and were found by cytogenetic analysis to have a proximal duplication on chromosome 15.
Case A was a 31 year old, phenotypically normal female, who was found to have a proximal duplication of one chromosome 15 after identification of a similar chromosome in a pregnancy loss. No evidence of a duplication was found by FISH using probes for D15S11, SNRPN, and GABRB3. Cytogenetic analysis of her parents determined that the chromosome with the duplication was inherited from her mother.
Case B was a 21 week fetus that showed extra material on chromosome 15q during routine prenatal diagnosis by amniocentesis. No evidence of …