Ring chromosome 8 (r(8))
We obtained lymphocytes and fibroblasts from a patient with r(8) and Birk-Barel syndrome23, features of patients are briefly described in Table 1. Molecular karyotyping of lymphocytes revealed that the ring chromosome originated from a terminal deletion at 8p23.3-p23.1 (7.888 Mb) with a large (27.120 Mb) duplication at 8p23.1-p11.22 (Fig. 1a), which inverted status was confirmed by fluorescence in situ hybridization (FISH) (Fig. 1b). It is suggested that this ring chromosome 8 arose as a result of exchange between low-copy repeats24, the mechanism, which produce the normal copy region between the regions of deletion and duplication, as it was observed in our case (Fig. 1d). DNA from fibroblasts at passage 2 (P2) showed a more complex structure of the short arm of chromosome 8 with three deletions and one duplication (del8p23.3-p23.1 (7.888 Mb), del8p23.1 (3.749 Mb), del8p11.22-p11.1 (3.738 Mb), and dup8p12-p11.23 (3.244 Mb)) (Fig. 1a). GTG analysis revealed that 67% fibroblasts had r(8) and 33% fibroblasts had monosomy 8 at P2 (Fig. 1e), and interphase FISH with the centromeric probe D8Z2 showed monosomy 8 in 258 cells (51.7%) and disomy 8 in 241 cells (48.3%) (Supplementary Fig. S1). Unexpectedly, later cells with t(7;8) arose in the fibroblast culture (Fig. 1c); these cells had a proliferative advantage with more than 80% of the fibroblast at P6, P12, P17 and P24 (Fig. 1e).
We generated iPSCs from patient’s fibroblasts at P2 using episomal vectors25. The karyotypes of the six iPSC lines studied were similar to those of the initial fibroblasts in the early passages (90% of the cells had karyotype 46,XY,r(8) at P4-6), but after P8-10, cells with the normal 46,XY karyotype prevailed in all six cell lines (Fig. 1e). Interestingly, metaphases with t(7;8) were also sporadically found in two cell lines: iPSC-r(8)-1 at P9 and iPSC-r(8)-5 at P10.
SNP-array analysis revealed signs of multiple rearrangements of chromosome 8 in the cell line iPSC-r(8)-2 at P8 and the “normal” karyotype, 46,XY, at P11 with isodisomy of chromosome 8: a normal copy number of chromosome 8 with loss of heterozygosity (LOH) along nearly all chromosome (Fig. 1f). For SNP-genotyping of the lymphocytes of the father and mother of the patient and iPSCs from his fibroblasts, a total of 3499 SNP markers on chromosome 8 were analysed. A total of 210 SNPs were informative, which indicated that both homologues of chromosome 8 in iPSC-r(8)-2 at P11 were of paternal origin. The genotype distribution provided evidence of isoUPD(8)pat.
Other iPSC lines also showed sequential stages of r(8) elimination: UPD(8) was found in the iPSC-r(8)-3 and iPSC-r(8)-4 cell lines at P9, and monosomy 8 was found in the iPSC-r(8)-1 cell line at P8. Two other cell lines, analysed at P5, demonstrated evidence of multiple rearrangements (iPSC-r(8)-5) and loss of most of the chromosome 8 genetic content (iPSC-r(8)-6) (Fig. 1f). Taken together, the GTG karyotyping and SNP array data suggest that in iPSC lines with r(8), self-correction of the karyotype to normal occurs via loss of the ring and uniparental isodisomy of an intact homologue of chromosome 8.
Only one iPSC-r(8)-2 subclone, iPSC-r(8)-2-O, retained r(8) without karyotype correction. This subclone showed degeneration of colony morphology, decreases in the expression of pluripotency markers, and subsequent differentiation at P14 (Supplementary Fig. S1). The remaining cell lines had ESC-like morphology of colonies, expression of markers of pluripotency (Supplementary Fig. S1) and differentiated into cell derivatives of all three germ layers, fully consistent with iPSC status.
Ring chromosome 13 (r(13))
We obtained lymphocytes and fibroblasts from a patient with r(13), features of patients are briefly described in Table 1. Chromosome analysis revealed karyotype 46,XY,r(13)(p13q34) in lymphocytes, confirmed by FISH (Fig. 2a), and a more complex karyotype in cultured skin fibroblasts: 46,XY,r(13)/46,XY,-13,+mar/45,XY,-13. The ratio of cells with these karyotypes was mostly stable at different passages, with a low prevalence of 46,XY,r(13) metaphases (Fig. 2c).
Molecular karyotyping of lymphocytes revealed two deletions at 13q34 (223 kb and 1.16 Mb) on chromosome 13 (Fig. 2b), but in fibroblast culture at P2, array-based comparative genomic hybridization (aCGH) recorded monosomy 13 in addition to a terminal deletion at 13q34 (2.099 Mb) (Fig. 2b).
Four iPSC lines with r(13), derived from patient’s fibroblasts26 using lentiviral vectors, showed a variety of karyotypes. Two cell lines, iPSC-r(13)-1 and iPSC-r(13)-2, consisted predominantly of cells with r(13), cells with monosomy 13 or with a marker chromosome (13 derivative) were minor classes. The other two cell lines, iPSC-r(13)-3 and iPSC-r(13)-4, showed pronounced variability in the frequencies of cells with different karyotypes, and on average, the major sub-population was composed of cells with a marker chromosome or even with monosomy 13 (Fig. 2c).
Molecular karyotyping of the cell lines iPSC-r(13)-1, iPSC-r(13)-2, iPSC-r(13)-3, and iPSC-r(13)-4 was performed at P25, P11, P12, and P9, respectively (Fig. 2d). The cell line iPSC-r(13)-1 showed amplification of 13q31.3 (26 kb) affecting a single gene, GPC5, that has not been described in fibroblasts; a terminal 13q34 deletion (2.53 Mb); and trisomy of chromosome 17. In the iPSC-r(13)-2 cell line, only a 13q34 terminal deletion (1.987 Mb) was identified. One of the most puzzling profiles of chromosome 13 was obtained in the cell line iPSC-r(13)-3, which exhibited five large deletions covering most of the long arm and a copy-neutral region of 5.333 Mb surrounded by the deleted regions. Surprisingly, this copy-neutral region was located within the LOH region of chromosome 13 (80.116 Mb) (Fig. 2d). The cell line iPSC-r(13)-4 also revealed multiple deletions on chromosome 13 as well as three LOH regions of 16.692 Mb, 32.472 Mb, and 20.627 Mb, respectively. Thus, two cytogenetically stable cell lines, iPSC-r(13)-1 and iPSC-r(13)-2, retained the terminal deletion similar to that in the initial ring chromosome, but two “unstable” cell lines revealed significant losses of the chromosome 13 genetic content (Supplementary Fig. S2). This outcome may have been caused by the coexistence of cell sub-populations with various lengths of deletions, especially as cytogenetic analysis revealed a wide range of marker chromosome variants of different sizes in these iPSC lines (Fig. 2e). The presence of the copy-neutral region surrounded by the deleted regions in iPSC-r(13)-3 may be a consequence of chromothripsis. However, the LOH in this area may indicate the probability of repair of the normal copy number due to recombination with the intact chromosome 13 homologue. We also found sporadic numerical amplifications of r(13) (Fig. 2f).
Despite the fact that the cell lines iPSC-r(13)-3 and iPSC-r(13)-4 were functionally similar to monosomy 13, they retained the normal ESC-like morphology of colonies and all the features of iPSCs, including the expression of pluripotency markers and the ability to differentiate into cell derivatives of all three germ layers in the embryoid bodies (Supplementary Fig. S2). At late passages (near P25), three of four cell lines with r(13) acquired trisomy of chromosome 12 or 17, each of which is a recurrent form of chromosomal aneuploidy in human pluripotent stem cell cultures27,28,29.
Thus, we found that in iPSC lines with r(13), both loss and fragmentation of the ring chromosome can occur; in addition, we found that pluripotency is compatible with a wide range of derivative karyotypes. Noticeable differences were found between the cell lines in the stability of r(13), which raises a question: why do isogenic iPSC lines with r(13) differ so much in their levels of mitotic instability?
Ring chromosome 18 (r(18))
Lymphocytes and fibroblasts were obtained from a patient with r(18), features of patients are described in Table 1. Microarray analysis revealed two deletions on chromosome 18: a small (1.238 Mb) deletion of the terminal part of the long arm at 18q23 and a large (13.52 Mb) deletion that affected almost the entire short arm at 18p11.32p11.21 (Fig. 3a). The formed ring chromosome was stable: the lymphocyte karyotype was 46,XY,r(18)(p11.1q23)/46,XY,der(18)r(18;18)(p11.1q23;q23p11.1). In fibroblasts, 100% of metaphases had karyotype 46,XY,r(18) at P4 and P11 (Fig. 3b). The deletions determined by aCGH were similar in lymphocytes and fibroblasts (Fig. 3a).
We generated iPSCs from patient’s fibroblasts at P3 using episomal vectors30. Three iPSC lines with r(18) were characterized by relatively high karyotype stability. At an early passage (P4), there was a small proportion of metaphases with monosomy 18 (6%, 30%, and 11% in cell lines iPSC-r(18)-1, iPSC-r(18)-2, and iPSC-r(18)-3, respectively), but after P7, almost 100% of the cells had karyotype 46,XY,r(18) (Fig. 3b). However, the mitotically stable ring chromosome was preserved until up to the 20th passage only in cell line iPSC-r(18)-1, for which aCGH at P12 also revealed the absence of any new rearrangements (Fig. 3c). In iPSC-r(18)-2 at P13, aCGH revealed the loss of most of the chromosome 18 genetic material, although metaphase analysis at P12 found a marker chromosome—derivative 18—in only 10% of the metaphases. As expected, at P20, 54% of the cells in this cell line had the karyotypes 46,XY,-18,+mar or 47,XY,-18,+mar,+mar (Fig. 3d), indicating the predominance of the process of r(18) fragmentation at later passages (Fig. 3b,c). In iPSC-r(18)-3, GTG analysis revealed 100% of the cells with the ring at P12; 92% with the ring and 8% with monosomy 18 at P20 (Fig. 3b). Nevertheless, microarray analysis at P13 detected the presence of multiple deletions interspersed with copy-neutral areas and even duplication (Fig. 3c). The different GTG and microarray data may be explained by methodical differences: metaphase analysis registers mainly actively proliferating cells, whereas microarray analysis displays the quantitative ratio of cell sub-populations in the total DNA pool. Most likely, in iPSC-r(18)-3, a considerable proportion of cells underwent multiple rearrangements. Variants of ring chromosomes and markers, registered in these cell lines, are in Fig. 3e and Supplementary Fig. S3. Despite the difference in stability of the ring chromosome, all these cell lines had normal iPSC characteristics, including morphology of colonies and expression of pluripotency markers (Supplementary Fig. S3).
Ring chromosome 22 (r(22))
We obtained lymphocytes and fibroblasts from a Phelan–McDermid syndrome patient with r(22)31, features of patients are briefly described in Table 1. Metaphase analysis of the G-banded chromosomes from peripheral blood lymphocytes revealed 46,XX,r(22)/45,XX,-22 mosaicism with 8% monosomic cells. The skin fibroblast culture showed high-grade dynamic mosaicism of cells with and without r(22): from P15 to P42, the percentage of 46,XX,r(22) metaphases decreased from 73 to 56%, and the percentage of metaphases with monosomy 22 increased from 18 to 42%, respectively (Fig. 4a). Morphology of r(22) is shown in Fig. 4b and c, and in Supplementary Fig. S4. In lymphocytes, aCGH identified a small (180 kb) microduplication at 22q13.32 and a subtelomeric microdeletion 2.024 Mb in size at 22q13.32-q13.33, and a similar microdeletion was observed in skin fibroblasts from the patient (Fig. 4d). At the microduplication region of 22q13.32, aCGH demonstrated a shift in the chromosomal profile towards duplication; however, this shift did not reach the significance level due to the high variance of the fluorescence intensity among the chromosomes.
Only two iPSC lines32 were obtained from the patient’s fibroblasts using lentiviral vectors, and in both, metaphase analysis showed a stable prevalence of cells with r(22): karyotype 46,XX,r(22) was found in 68–97% of the metaphases in iPSC-r(22)-1 from P5 to P60 and 58–90% of the metaphases in iPSC-r(22)-2 from P6 to P38 (Fig. 4a). The remaining cells mainly had monosomy 22. Microarray analysis of the cell lines iPSC-r(22)-1 (P8) and iPSC-r(22)-2 (P5) showed only microdeletion of the 22q13.32-q13.33 region, which is in accordance with that detected in lymphocytes and fibroblasts (Fig. 4e). We found a rather stable structure of r(22) during iPSC culture without any fragmentation at the sub-microscopic level. Thus, iPSCs with r(22) have a relatively stable karyotype, cells with r(22) persist as a modal sub-population for dozens of passages, and the ring structure remains virtually invariable. This variant of r(22) is even more stable in iPSCs than in cultured fibroblasts.