Summary of supernumerary marker chromosomes
This male with wide-spaced nipples, unilateral cryptorchidism, small penis, right clubfoot and left congenital vertical talus had a peripheral blood chromosome analysis performed shortly after birth. Examination of 35 cells demonstrated a normal 46,XY karyotype. He was again evaluated at 14 months of age with findings that also included developmental delay, mild arthrogryposis, skin hyperpigmentation distributed along the lines of Blaschko, a sacral dimple, and an undescended testis on the right. Cranial MRI was normal, but lumbar spine MRI showed cord tethering with hydrosyringomyelia, anterior wedging of the T2 vertebral body, and incomplete closure of the L5 and S1 posterior vertebral elements. A scrotal skin biopsy taken at the time of orchiopexy was submitted for cytogenetic analysis. Eight of 37 cells were found to contain a large SMC. Array CGH using an expanded coverage microarray (see Methods) detected low-level gain of 100 BAC clones from 3q22.3 through 3qter (Fig. ). FISH performed on previously G-banded slides using multiple probes from the region of gain confirmed that the marker was derived from the long arm of chromosome 3, and illustrated that the marker had the configuration of an inverted, duplicated 3q (Fig. ). Also consistent with the array CGH results that the gain did not extend more centromeric than 3q22.3, the marker had no detectable chromosome 3 alpha satellite DNA by FISH (Fig. ). Although we cannot absolutely exclude the possibility that the marker contains centromeric material from another chromosome, no other gains were detected by array CGH, and G-banded analysis showed a prominent constriction at one end of the marker at band q26, indicating the likely presence of a neocentromere in that location. The combined results of G-banded analysis, array CGH, and FISH were most consistent with the following configuration of the marker: inv dup(3)(qter→q26→neo→q26→q22.3::q22.3→qter). Parental chromosome analyses were not performed.
Figure 1 Characterization of inv dup(3q) by array CGH and FISH. A. Chromosome 3 array CGH plot. The X axis represents distal p arm to distal q arm (left to right), with the centromere designated by the vertical dotted line. The blue line is a plot of the results (more ...)
A peripheral blood karyotype was performed on a 2 year-old male with moderate global developmental delay and bilateral anterior segment dysgenesis of the eye in the form of the Axenfeld-Rieger anomaly. In addition, he had macrocephaly, a past history of bilateral inguinal hernias, and several depigmented macules. An MRI was significant for a short, thick corpus callosum, prominent perivascular spaces with thinning of the gray matter, and an enlarged posterior fossa. All 20 cells examined from peripheral blood demonstrated a 48,XY,+mar1,+mar2 karyotype (Fig ). The larger marker was approximately one-half the size of a G-group chromosome, and G-banded analysis suggested the presence of satellites. The second marker consisted of a minute fragment. Array CGH using a pericentromeric microarray (see Methods) detected a single-copy gain of 20 BAC clones (approximately 5.3 Mb) from the pericentromeric region of the long arm of chromosome 13 (data not shown). Additional analysis using a targeted microarray (see Methods) revealed not only a gain of the pericentromeric region of chromosome 13, but also single copy gain of approximately 5.5 Mb from the subtelomeric region of chromosome 13 (13q33.3 to q34) (Fig. ). The intervening region of chromosome 13 covered by this array (13q14.2 to 13q33.1) did not show copy number alterations (Fig. ). FISH using probes from both the proximal (RP11-408E5, 13q12.11 and RP11-347L8, 13q12.13) and distal (RP11-63L17, 13q34) regions of 13q gain confirmed the origin of the larger SMC (Fig. and ). Both BACs from proximal 13q hybridized to one arm of the larger SMC, whereas the 13q34 subtelomeric BAC probe hybridized to the opposite arm of the larger SMC. FISH using a probe from the nucleolar organizing region (NOR) hybridized within the middle of the larger SMC, apparently flanked by the 13q proximal long arm segment on one side and by the 13q telomeric segment on the other side (data not shown). Additional FISH using a centromere probe for chromosomes 13 and 21 (D13Z1/D21Z1) showed hybridization only to the larger SMC (data not shown). Thus, none of the probes showed hybridization to the minute SMC, and its origin remains unknown. Based on the combined results of array CGH and FISH, the larger SMC appears to have the following configuration: 13qter→q33.3::p12→q12.12:.
Figure 2 SMCs from patient 2. A. G-banded normal chromosome 13 homologues and SMCs (arrows). B. Array CGH plot showing gains of both proximal and distal 13q, with normal copy number in between. C. FISH using BACs RP11-408E5 from 13q12.11 (red signal) and RP11-63L17 (more ...)
Analysis of 60 peripheral blood metaphase cells from both parents did not show either of the SMCs.
A peripheral blood karyotype from this term newborn female with low birthweight, hydrocephalus, possible partial agenesis of the corpus callosum, preauricular pits, and total anomalous pulmonary venous return showed a small mono-satellited SMC in all 20 metaphase cells examined (Fig. ). Array CGH using the pericentromeric microarray detected a single-copy gain of approximately 3.8 Mb of the proximal long arm of chromosome 22 from q11.1 to q11.21 (data not shown), including the VCFS/DiGeorge syndrome critical region (DGS1). Additional analysis using the expanded coverage microarray revealed not only the gain of proximal 22q, but also single-copy gain of approximately 5.2 Mb from 22q13.31 to 22q13.33 (Fig. ). The intervening region of chromosome 22, from q11.21 to q13.31, showed no copy number alterations. FISH using BAC clones from both the proximal (RP11-1037C4, 22q11.1 and CTD-2593O4, 22q11.21) and distal (RP11-676E13, 22q13.33) regions of 22q gain confirmed the array findings (Fig. and ). Both probes hybridized to the marker in all 60 cells examined. Thus, the most likely configuration of this SMC is: 22pter→q11.21::q13.31→qter. Parental chromosome analyses were not performed.
Figure 3 Chromosome 22 SMC from patient 3. A. G-banded normal chromosome 22 homologues and SMC (arrow). B. Array CGH plot showing gains of both proximal and distal 22q, but not the intervening region between 22q11.21 and q13.31. C. FISH using BACs RP11-1037C4 (more ...)
A prenatal ultrasound performed on this female fetus revealed bilateral cleft lip anomaly, ventriculomegaly, and possible agenesis of the corpus callosum. A 48,XX,+2mar/49,XX+3mar karyotype was found at amniocentesis performed at another institution. Interphase FISH to screen for aneuploidies of chromosomes 13, 18, 21, X, and Y showed three X chromosome centromere signals in 40 out of 50 nuclei scored, suggesting that one of the marker chromosomes was derived from the X chromosome. At birth, bilateral cleft lip anomaly, a flattened nasal profile (nasomaxillary hypoplasia), and upslanting palpebral fissures were noted. MRI of the brain showed fusion of the frontal lobes and thalami, as well as partial agenesis of the corpus callosum, consistent with a semilobar holoprosencephaly. No renal anomalies were detected by ultrasound. An echocardiogram showed normal intracardiac structure with a patent ductus arteriosus. A peripheral blood karyotype confirmed the amniocentesis finding of multiple marker chromosomes, but showed four SMCs in the majority of 20 cells examined. A few cells possibly contained five SMCs, although the minute size of some of the markers made them difficult to distinguish from debris. The SMCs ranged in size from minute to one that was approximately one-third the size of a G-group chromosome, and had the appearance of a ring in some cells. Array CGH using the targeted microarray demonstrated gains of the pericentromeric regions of 1p, 4q, 7p, and 11q (Fig. ). Numbering of the SMCs from 1 to 4, with SMC 1 representing the largest, and SMC 4 representing the smallest, SMC 1 hybridized with both a chromosome 11 centromere probe (D11Z1) and BAC RP11-601I15 from 4q12 (Fig. ), but did not hybridize with a chromosome 4 alpha satellite probe (not shown). SMC 2 hybridized with BAC RP11-1324A7 from 7p11.1 (Fig. ), while SMC3 hybridized with BAC RP11-527D19 from 1p12 (Fig. ). SMC 4 hybridized with an X chromosome centromere probe (DXZ1) as expected based on the results from amniocentesis. X chromosome alpha satellite sequence is not represented on the targeted microarray that was used for array CGH, and the X chromosome did not show any obvious gains, suggesting that this marker may contain only X centromere material, or a very limited amount of euchromatin. Thus, combined array CGH and FISH results revealed the following configurations of the SMCs in this patient:
Figure 4 Characterization of multiple marker chromosomes in patient 4 by array CGH and FISH. A. Chromosome plots showing pericentromeric gains of 1p, 4q, 7p, and 11q detected by array CGH. B. – D. G-banded and corresponding destained FISH images of the (more ...)
SKY was also performed by an independent laboratory. Results from five cells, each containing two to three SMCs, showed tentative assignments from chromosomes 1, 5, 7, 11, and 16 (data not shown). FISH did not confirm the presence of chromosome 5 or 16 material in the SMCs. Thus, although SKY classified SMCs derived from chromosomes 1, 7, and 11 material, it did not detect chromosome 4 material in the der(11) SMC, and SMCs in two cells were incorrectly identified as derived from chromosomes 5 and 16. Parental karyotypes were normal.