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Editor—At least 168 cases with a supernumerary marker chromosome (SMC) from all chromosomes not including chromosome 15 have been documented.1 Birth prevalence is estimated at 0.14 to 0.72 per 1000.2 Subjects with a SMC have a partial trisomy (duplication) and in some cases a partial tetrasomy (triplication) of the genetic material contained in the SMC. The risk of an abnormal phenotype associated with a randomly ascertained de novo SMC derived from acrocentric autosomes (excluding chromosome 15) is estimated to be approximately 7% compared with approximately 28% for SMCs derived from non-acrocentric autosomes.1 The great variability of clinical findings in patients with SMCs originating from the same chromosome is probably the result of variation in size and genetic content, the degree of mosaicism, and uniparental disomy of the normal homologues of the chromosome from which the SMC derived.
Evidence that subjects with SMCs might have an increased risk for UPD of the structurally normal homologues of the SMCs has been reported by several authors. To the best of our knowledge the coexistence of SMCs with UPD has been described for chromosomes 6, 7, 15, 20, and X.3-7 Here, we describe a further patient with multiple congenital anomalies, developmental delay, and the unique finding of coexistence of SMC 1 mosaicism and maternal uniparental disomy 1.
The female patient was born at term after an uneventful pregnancy. At her birth, her mother was 33 years old and her father was 47 years old. Birth weight was 2500 g and length 49 cm. At the age of 6 years, she was investigated because of mental retardation. Height (1.14 m) and weight (17 kg) were within the normal range, but head circumference (44 cm) was far below the 3rd centile. Additional findings were temporal narrowing, downward slanting palpebral fissures, long eyelashes, high palate, pointed chin, low set and dysplastic ears, hip dysplasia, and tapering fingers with clinodactyly of fingers 2, 4, and 5.
Chromosome preparations from blood lymphocyte cultures were performed according to standard procedures. GTG banded chromosomes were examined.
Chromosome microdissection was performed according to a slightly modified version of the protocol described by Sengeret al.8 FISH analysis was performed according to Lichter and Ried9 using the specific chromosome library generated by microdissection.
Genomic DNA from the patient and both parents was amplified by standard PCR with commercially available highly polymorphic microsatellites (Research Genetics®, Huntsville, AL, USA), loaded onto a 6% polyacrylamide/urea gel, and visualised by silver staining.
Probably owing to the low mosaicism in leucocytes, it was not possible to determine the parental origin of the SMC unambiguously by standard methods. We therefore applied a new approach recently developed in our laboratory.10 In a first step, 20 SMCs were dissected and collected in a PCR tube containing 2 μl collection drop solution (10 mmol/l Tris/HCl, 10 mmol/l NaCl, 3 mg/ml proteinase K PCR grade), and incubated for two hours at 60°C. Subsequently, proteinase K was inactivated at 90°C for 10 minutes. Whole genome amplification was performed according to an improved PEP protocol (I-PEP)11with the following modifications: no gelatine, 500 μmol/l dNTP, 1 mmol/l MgCl, and 100 μmol/l totally degenerated 15 nucleotide long primer. Finally, multiple highly polymorphic microsatellites were analysed by time release PCR (0.1 mmol/l dNTP, 0.24 μmol/l primers, 1.25 U AmpliTaq Gold® (PE Applied Biosystems, Branchburg, NJ, USA), 1.5-2.5 mmol/l MgCl, using 5 μl aliquots of the preamplified DNA in a final volume of 50 μl in a Techne Progene® thermocyler (Techne Incorporated, Princeton, NJ, USA) for 56 cycles, 60-64°C for four minutes, 94°C for one minute), run on a 6% polyacrylamide gel, and visualised by silver staining.
Chromosome preparations from blood lymphocyte cultures of the patient showed an additional small marker chromosome (SMC) in 14 of 40 metaphases (35%) investigated (fig 1A). Chromosome analysis of the mother and the father showed normal karyotypes. In order to determine the origin of the SMC, a microFISH experiment with reverse painting to normal metaphases was performed. It could be shown that the SMC was formed by a region of chromosome 1, 1p21.1→1q12 (fig 1B). Application of the “all telomeres” probe (Vysis® Inc, Downers Grove, IL, USA) showed no telomeric signal on the extra chromosome indicating that it is a ring chromosome (data not shown).
Microsatellite analysis on genomic DNA of the patient and the parents showed that the patient had inherited two maternal alleles (heterodisomy and isodisomy respectively at different loci) but no paternal allele at different loci of chromosome 1 (table 1, fig 2A, B). Investigations of several microsatellites from various other chromosomes were in agreement with correct paternity. Based on these results, the karyotype of the patient may be described as 47,XX,UPD(1)mat,+der(1)(p21.1q12)/46,XX,UPD(1)mat.
Probably owing to the patient's relatively low level mosaicism of leucocytes with the SMC, the microsatellite analysis with pericentromeric markers did not result in an unambiguous determination of the parental origin of the SMC. Although some markers showed a weak paternal band, this was not obvious enough for unequivocal parental determination. Therefore, a new approach combining microdissection of the SMC with subsequent PEP-PCR and conventional microsatellite PCR was performed. All investigated markers mapping to the duplicated region clearly showed one paternal band and no maternal band and were thus in agreement with paternal origin of the SMC (fig 2B). In addition, by investigating the genomic DNA with these pericentromeric markers, three markers mapping within the duplicated region on the short arm of chromosome 1 within a 10 cM distance from the centromere and also two markers at the distal end of the short arm of chromosome 1 showed maternal isodisomy (table 1).
The great variability of clinical symptoms in patients with a SMC of the same chromosomal origin is probably the result of variation of the genetic content, the degree of mosaicism (especially, as in our case, in ring chromosomes), and uniparental disomy of the normal homologues from which the SMC is derived.1 12
To allow any karyotype-phenotype correlation, comparison of a sufficient number of accurately investigated cases is necessary. The best way to characterise a SMC is a multistep approach: (1) classical cytogenetic procedures, which provide information about the degree of mosaicism by investigating many cells, if possible, from different tissues; (2) microFISH, which gives detailed information about the chromosomal content of the SMC; (3) microsatellite analysis of the normal homologues, which gives information about uniparental disomy and a clue towards the mechanism of formation; and (4) the absence of telomeres indicated that the SMC is a ring.
In most cases with an SMC originating from chromosome 1, neither FISH analysis with locus specific probes nor molecular investigations with microsatellites was performed.12 The patients described show great variability of their phenotypes and so far no distinct syndrome has been associated with the group of cases with a supernumerary marker chromosome originating from chromosome 1.
The phenotype of four patients with maternal UPD(1) reported so far13-16 is normal and therefore suggests that maternally imprinted loci on chromosome 1, if existing at all, probably do not influence the phenotype. Thus, the congenital anomalies in our patient are probably not the result of the maternal UPD of chromosome 1, but are more likely related to the mosaic duplication of chromosome 1 (p21.1→q12).
Several authors assumed that subjects with SMCs might have an increased risk for UPD of the structurally normal homologues from which the SMCs derived. However, the incidence of UPD associated with a SMC is not known, and so far only one case each of chromosomes 6, 7, 20, and X,3 5-7 as well as nine cases of chromosome 15, seven with Prader-Willi syndrome, and two with Angelman syndrome, have been reported.4 17-22
Coexistence of UPD with a SMC is mainly explained by the following two mechanisms.21 First, duplication of the normal homologue in a zygote which has inherited a SMC in place of the normal corresponding chromosome “rescues” an aneuploidy. In this case, UPD arises by mitotic non-disjunction and therefore complete isodisomy should always be observed. Second, the zygote may have originated as a trisomy with the single parental chromosome being lost through a breakage event or, alternatively, a disomic gamete was fertilised by a gamete with a SMC formed during meiosis.
To the best of our knowledge, 10 cases of supernumerary marker chromosome 1 (SMC 1) mosaicism have been reported so far. Most of these ring shaped markers were identified by fluorescence in situ hybridisation (FISH) with centromere specific probes.
We report another case with SMC 1 mosaicism identified by combining chromosome microdissection with reverse painting to normal metaphases (microFISH).
Microsatellite analysis showed heterodisomic maternal uniparental disomy (UPD) of the normal chromosomes 1. In addition, a new approach combining chromosome microdissection, primer-extension-preamplification polymerase chain reaction (PEP-PCR) and standard PCR of highly polymorphic microsatellites showed paternal alleles only within the region of the SMC. This indicates that the SMC is of paternal origin.
Heterodisomy, as reported in our case, can only be explained by the second mechanism. The distribution of isodisomic and heterodisomic segments in our case, pericentromeric and more distal, respectively, indicates an error in maternal meiosis II resulting in the maternal UPD(1). Whether the SMC was formed during paternal meiosis or in an early somatic cell division cannot be differentiated. If it was formed during paternal meiosis, the coexistence of a SMC with UPD may be considered as a coincidence. Even if it was formed postzygotically, our case might at best not contradict the hypothesis that subjects being uniparentally disomic might have an increased risk of bearing a SMC of the same homologue (rather than the reverse). In other words, the presence of two maternal (or paternal) chromosomes in the zygote might constitute a risk for the formation of a paternal (or maternal) SMC through a breakage event.
Parental origin of the normal chromosomes and of the SMC is sometimes difficult to determine. In many instances, only quantitative results can be obtained (mitotically formed mosaicism, supernumerary marker chromosomes). The new technical approach applied in the case reported here allows the unequivocal tracing back of informative alleles from a dissected abnormal chromosome to one of the parental homologues.
In conclusion, we present a case with coexistence of a SMC 1 with maternal UPD(1) and hence mosaicism for complete maternal uniparental disomy and partial trisomy for a small pericentromeric segment of chromosome 1 combined with uniparental disomy for the rest of the chromosome. By a new technical approach, which allows the parental origin of the marker chromosome to be determined, even in cases with low level mosaicism, we were able to unequivocally show the paternal origin of the marker.
The authors are grateful to the family for their cooperation. The study was supported by the Swiss National Foundation, grants 32-45604.95, 32-56053.98, and 71P51778.