Molecular cytogenetic characterization of eight small supernumerary ...

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Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8,18, and 21 in three patients.
J Appl Genet 48(2), 2007, pp. 167–175

Original article

Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8, 18, and 21 in three patients Joanna Pietrzak1, Kristin Mrasek2, Ewa Obersztyn1, Pawe³ Stankiewicz1, Nadezda Kosyakova2,3, Anja Weise2, Sau Wai Cheung4, Wei Wen Cai4, Ferdinand von Eggeling2, Tadeusz Mazurczak, Ewa Bocian1, Thomas Liehr2 1

Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland Institute of Human Genetics and Anthropology, Friedrich-Schiller-University Jena, Germany 3 Research Centre for Medical Genetics, Moscow, Russia 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA 2

Abstract. Small supernumerary marker chromosomes (sSMCs) are a morphologically heterogeneous group of additional structurally abnormal chromosomes that cannot be identified unambiguously by conventional banding techniques alone. Molecular cytogenetic methods enable detailed characterization of sSMCs; however, in many cases interpretation of their clinical significance is problematic. The aim of our study was to characterize precisely sSMCs identified in three patients with dysmorphic features, psychomotor retardation and multiple congenital anomalies. We also attempted to correlate the patients’ genotypes with phenotypes by inclusion of data from the literature. The sSMCs were initially detected by G-banding analysis in peripheral blood lymphocytes in these patients and were subsequently characterized using multicolor fluorescence in situ hybridization (M-FISH), (sub)centromere-specific multicolor FISH (cenM-FISH, subcenM-FISH), and multicolor banding (MCB) techniques. Additionally, the sSMCs in two patients were also studied by hybridization to whole-genome bacterial artificial chromosome (BAC) arrays (array-CGH) to map the breakpoints on a single BAC clone level. In all three patients, the chromosome origin, structure, and euchromatin content of the sSMCs were determined. In patient RS, only a neocentric r(2)(q35q36) was identified. It is a second neocentric sSMC(2) in the literature and the first marker chromosome derived from the terminal part of 2q. In the other two patients, two sSMCs were found, as M-FISH detected additional sSMCs that could not be characterized in G-banding analysis. In patient MK, each of four cell lines contained der(4)(:p11.1→q12:) accompanied by a sSMC(18): r(18)(:p11.2→q11.1::p11.2→q11.1:), inv dup(18)(:p11.1→q11.1::q11.1→p11.1:), or der(18)(:p11.2→q11.1::q11.1→p11.1:). In patient NP, with clinical features of trisomy 8p, three sSMCs were characterized: r(8)(:p12→q11.1::q11.1→p21:) der(8) (:p11.22→q11.1::q11.1→p21::p21→p11.22:) and der(21)(:p11.1→q21.3:). The BAC array results confirmed the molecular cytogenetic results and refined the breakpoints to the single BAC clone resolution. However, the complex mosaic structure of the marker chromosomes derived from chromosomes 8 and 18 could only be identified by molecular cytogenetic methods. This study confirms the usefulness of multicolor FISH combined with whole-genome arrays for comprehensive analyses of marker chromosomes. Keywords: array-CGH, comparative genome hybridization, genotype-phenotype correlation, FISH technique, multicolor fluorescence in situ hybridization, small supernumerary marker chromosomes.

Introduction Small supernumerary marker chromosomes (sSMCs) are a morphologically and genetically heterogeneous group of additional chromosomes

that are equal or smaller in size than chromosome 20 in the same metaphase spread. They are found in about 0.043% of the human population and have been estimated to result in abnormal phenotype in approximately 30% of sSMC carriers (Starke et al.

Received: October 2, 2006. Accepted: January 16, 2007. Correspondence: E. Bocian, Department of Medical Genetics, Institute of Mother and Child, Kasprzaka 17A, 01–211 Warszawa, Poland; e-mail: [email protected]

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2003; Liehr et al. 2004b). The sSMCs can form different chromosomal structures, e.g. inverted duplicated chromosomes, complex rearranged chromosomes, minute, ring, or neocentric chromosomes (Liehr et al. 2006) and are difficult to characterize comprehensively in both conventional and molecular cytogenetic analyses. In the last decade, development of molecular cytogenetic techniques – such as different variants of fluorescence in situ hybridization (FISH) tech-

niques, chromosome microdissection with reverse painting, and comparative genome hybridization (CGH) – enabled identification and detailed characterization of many marker chromosomes that were difficult (if not impossible) to characterize in conventional cytogenetic analysis. Here, we report the results of a comprehensive analysis of eight sSMCs from three patients, which were defined by using various multicolor FISH techniques as well as a whole-genome array.

Table 1. Cytogenetic and clinical description of the three presented patients Patient

Clinical symptoms

Karyotype

FISH probes/BAC clones

Identified sSMCs

RS

Delayed somatic development, small hands and feet, very "distinct" facial dysmorphism (frontal bossing, hypertelorism, low nasal bridge, small and short nose, anteverted nares, macrostomia, triangular mouth, thin upper lip, short philtrum, micrognathia), short neck, brachydactyly, short and broad hallux, hypotonia, pectus excavatum, mild pterygium colli, severe psychomotor retardation, absent speech, delayed tooth eruption.

47,XX, +mar[14]/46,XX[36]

cenM-FISH, subcen-Mix2, MCB2, wcp2, cep2 (D2Z1, Vysis)

r(2)(:q35→q36:)

MK

Moderate psychomotor retardation, short stature with shortening of upper and lower limbs, tapering fingers, facial dysmorphism (flat face, telecanthus, broad nose, prominent forehead)

mos? 48,XX,+mar1,+mar2[101]/ 47,XX,+mar1[38]/ 47,XX,+mar2[29]/46,XX[5]

multicolor FISH, subcen-Mix4, subcen-Mix18, cep4, subtel4p, subtel4q, subtel18p, subtel18q, D18Z1 (Vysis)

mar1: der(4)(:p12→q12:) mar2: r(18)(:p11.21→q11.1::p11.21→ q11.1:) inv dup(18) (:p11.1→q11.1::q11.1→p11.1:) der(18)(:p11.21→q11.1::q11.1→ p11.1:)

Intrauterine and postnatal growth retardation, microcephaly, brachycephaly, hypotonia, psychomotor retardation, partial agenesis of corpus callosum, abnormal dermatoglyphic patterns, facial dysmorphism (up-slanting palpebral fissures, anteverted nares, relatively large ears, prominent lower lip, long philtrum)

47,XY,+mar1[37]/ 48,XY,+mar1,+mar2[52]/ 49,XY,+mar2x2[7]/46,XY[4]/

NP

RP11-756J15, RP11-458J14RP11-809 H21, RP11-905B6 cenM-FISH, multicolor FISH, bC067E3 (21q11.2), subcen-Mix8, MCB8, MCB21, 21LSI, MIDI54 (for p-arm of acrocentric chromosomes), bk105N1 (in 21q21.1), bk143E1 (in 21q.21.3) , bk249H10 (in 21q22.11), subtel 8p, subtel 8q, D8Z (Vysis), D13/21Z (Q-BIOgene) RP11-501B1→RP11-9 62B15RP11-143O7→R P11-681F10RP11-47B1 3→RP11-644M

mar1: der(21)(:p11.1→q21.3:) mar2: r(8)(:p12→q11.1::q11.1→p21.1:) der(8)(:p11.22→q11.21::q11.21 →p21.1::p21.1→p11.22:)

Molecular cytogenetic characterization of sSMCs

Materials and methods Patients

Clinical description of patients and their karyotypes is given in Table 1. Written informed consent was obtained from legal guardians of the three patients. Pictures of patients RS and MK are shown in Figure 1. In all of them, a mosaic karyotype was found. In patients MK and NP, more than one marker chromosome was identified. Karyotypes of parents of all patients were normal.

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Standard techniques for DNA extraction from peripheral blood were applied. For BAC array-CGH (Cai et al. 2002), the sSMCs from patients MK and NP were microdissected, the DNA amplified by DOP-PCR (degenerated oligonucleotide polymerase chain reaction, Telenius et al. 1992) and subsequently hybridized to a genome-wide array. This array consisted of 21,658 RP11 BAC clones (7 BAC clones per 1 Mb) with unique sequences at both ends and tightly distributed insert size that completely covers the entire human genome, thereby minimizing the repeat sequence content (Li et al. 2003). A modified computer program (Biodiscovery, EI Segundo, CA) has been adapted for the analysis of this study.

Results FISH techniques

Figure 1. Pictures of patients RS (A) and MK (B), at the age of 3 and 2.5 years, respectively

Methods

All studies were performed on peripheral blood lymphocytes. Chromosome preparations, GTG banding and FISH were done according to standard procedures (Liehr et al. 1995; Verma and Babu 1998). CenM-FISH (Nietzel et al. 2001), multicolor FISH with wcp probes (Senger et al. 1998; Liehr et al. 2004), chromosome microdissection and reverse FISH (Lüdecke et al. 1989; Starke et al. 2001) were used for determination of sSMC origin. These results were confirmed by FISH with centromere-specific probes. Identification of euchromatic material was done by subcenM-FISH (Starke et al. 2003), MCB (Chudoba et al. 1999; Liehr et al. 2002), and locus-specific FISH (Weise et al. 2002). The level of sSMC mosaicism was determined by interphase-FISH. Commercial probes specific for the centromeric regions of marker chromosomes (Abbott, Q-BIOgene) were used for the estimation of the percentage of different cell lines. One hundred nuclei were counted for each probe. Microsatellite analysis was used to exclude uniparental disomy (UPD, Starke et al. 2003).

Application of different variants of M-FISH enabled a comprehensive characterization of marker chromosomes detected by GTG banding. In all analyzed cases, chromosome origin, structure, and euchromatin content were determined (Table 1). In patient RS, the chromosomal origin of the sSMC could not be determined by cenM-FISH and FISH with centromeric probes, and only chromosome microdissection and reverse painting (midi) using material from this sSMC showed clearly in one step that it was derived from chromosome 2 (Figure 2A). The same result was also obtained after application of multicolor FISH, and it was further confirmed by a single-color FISH with a wcp probe for chromosome 2 (Figure 2B), because multicolor FISH was not conclusive. FISH experiments revealed that the sSMC(2) originated from 2q35-q36 and had a ring structure. These results were confirmed by MCB analysis (Figure 2C). Thus the patient’s karyotype was described as: 47,XX,+r(2)(:q35→q36:) [14]/46,XX[36]. In patient MK, cenM-FISH showed that the sSMCs originate from chromosomes 4 and 18 (Figure 3A). Multicolor FISH, MCB, and subcenM-FISH (Figure 3B) techniques revealed four cell lines. In each of them, a der(4) was present in combination with one of three sSMCs(18), which had different structures (Table 1). Thus the patient’s karyotype was designated as: 48,XX,+der(4),+mar(18)[35%]/47,XX,+der(4) [29%]/47,XX,+mar(18)[28%]/46,XX[8%] (for details of marker chromosomes, see Table 1).

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Figure 2. Results of FISH with wcp2, reverse painting (midi), and MCB in patient RS. (A) Midi determined the 2q35-q36 origin of the sSMC. (B) FISH with the painting probe for chromosome 2 revealed the chromosome 2 origin of the sSMC. (C) MCB showed the ring structure r(2)(q35q36) of the SMC identified by midi.

Figure 3. Results of cenM-FISH and subcenM-FISH analyses in patient MK. (A) CenM-FISH enabled characterization of sSMC(4) and SMC(18) (arrowheads) in patient MK. The cenM-labeling scheme is depicted below the chromosomes. (B) SubcenM-FISH demonstrated the presence of a variant of the sSMC(4), a der(4)(:p11.1→q12:). In contrast, subcenM-FISH revealed three different variants of sSMC(18): inv dup(18)(:p11.1→q11.1::q11.1→p11.1:), der(18)(:p11.2→q11.1::q11.1→p11.1:), and r(18)(:p11.2→q11.1::p11.2→q11.1:).

The marker chromosomes in patient NP were described earlier (Bocian et al. 2006). FISH using multicolor probes confirmed that the sSMCs originate from chromosomes 8 and 21. Unexpectedly, it also identified an additional sSMC(8). The structures of both sSMCs were determined in detail (Figure 4A, B). The identified inverted duplication ring r(8)(:p12→q11.1:: q11.1→p21:) is unstable and forms a der(8)(:p11.22→q11.1::q11.1→ p21::p21→p11.22:) chromosome in a subset of cells. The sSMC(21) originates from chromosome 21p11.1→q21.3 (Figure 4A, C). The cell line with two marker chromosomes der(21) and sSMC(8) was prevalent. Thus the patient’s karyotype was described as:

47,XY,+min(21)(:p11.1→q21.3:)[30%]/ 48,XY,+min(21)(:p11.1→q21.3:)+r(8)(::p12→ q11.1::q11.1→p21::)[25%]/ 48,XY,+min(21)(:p11.1→q21.3:)+min(8) (::p11.22→q11.1::q11.1→p21::p21→p11.22:) [45%]. Microsatellite analysis

UPD was excluded only in patient MK for chromosomes 4 and 18 and in patient NP for chromosome 21; all analyzed markers for chromosome 8 were not informative.

Molecular cytogenetic characterization of sSMCs

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Figure 4. Results of FISH analysis in patient NP. (A) MCB identified two variants of the sSMC(8): r(8)(:p12→q11.1::q11.1→p12:) and der(8)(:p12→q11.1:); and one variant of the sSMC(21), der(21)(:p11.1→q21.3:). (B) SubcenM-FISH confirmed the ring structure of a variant of sSMC(8). (C) A co-hybridization of a centromeric probe specific for chromosomes 13 and 21 (green) together with a probe specific for the Down syndrome critical region (LSI 21 – red) determined the absence of this region on the sSMC(21).

Array-CGH

Array-CGH was done in patients MK and NP after microdissection, DOP-PCR and reverse painting. For microdissection of the marker chromosome in patient RS there was not enough material available. The plot results for the marker chromosomes der(18), der(4), der(21), and der(8) from patients MK and NP are shown in Figure 5 A–D. The plot for patient MK showed a gain for the region

18p11.21-q11.2 with breakpoints between BAC clones RP11-756J15 and RP11-458J14, a ~7 Mb gain in size. The second marker chromosome found in patient MK derived from chromosome 4 and presented a gain for the region 4p12-q12 defined by the BAC clones RP11-809H21 and RP11-905B6, resulting in a ~11 Mb gain in size. In patient NP, the DNA for array-CGH was obtained from the microdissection of the ring chromosome and the derivative marker chromosome 8

Figure 5. Results of array-CGH. (A) Array-CGH results in patient MK after microdissection of the marker chromosome derived from chromosome 18; the plot shows a gain for the region 18p11.21-q11.2 with breakpoints mapping at BAC clones RP11-756J15 and RP11-458J14. (B) The second marker chromosome found in patient MK originated from chromosome 4; the array-CGH showed a clear gain for the region 4p12-q12, defined by the BAC clones RP11-809H21 and RP11-905B6. (C) Array-CGH in the other marker chromosome in patient NP derived from chromosome 21 revealed a gain for the region 21p11.1-q21.3 defined by the BAC clones RP11-47B13 and RP11-644M. (D) In patient NP, the DNA for array-CGH was obtained from microdissection of the ring and the derivative structure of the marker chromosome 8 (see Table 1); a gain was observed for the region 8p21.1-q11.21 (BAC clones RP11-501B1 and RP11-962B15) with a gap for 8p12 (green arrows, BAC clone RP11-143O7 to RP11-681F10).

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(see Table 1). Therefore, a gain was observed for the region 8p21.1-q11.21 (BAC clones RP11-501B1 to RP11-962B15, ~22.4 Mb in size) with a gap in 8p12 (BAC clones RP11-143O7 to RP11-681F10, ~3.3 Mb in size). The other marker chromosome in patient NP derived from chromosome 21 and revealed a gain for the region 21p11.1-q21.3 defined by BAC clones RP11-47B13 and RP11-644M, ~15.4 Mb in size.

Discussion Characterization of marker chromosomes by conventional cytogenetic methods is difficult and often impossible. The development of molecular cytogenetic techniques (such as different variants of FISH techniques, chromosome microdissection with reverse painting, CGH, and array-CGH) has enabled identification and detailed characterization of the vast majority of marker chromosomes. In patients MK and NP, FISH with multicolor probes allowed identification of origin and precise characterization of structure of each of the sSMCs. In addition, FISH revealed the presence of an additional mosaic marker chromosome that was not seen in chromosome banding analysis. Three different forms of sSMC(18) were observed in patient MK: der, inv dup, and ring. Two forms of sSMC deriving from chromosome 8 were detected in patient NP. The different forms of sSMC(8) and sSMC(18) most likely result from their instability – opening the ring chromosomes and forming new structures. In comparison to the CGH results, in patient NP described earlier as: der(8)(:p22→q11.2:) and der(21)(pter→q21.3:) (Bocian et al. 2006), FISH with multicolor probes delineated more precisely the breakpoints of all sSMCs. In contrast to multicolor FISH techiques, CGH cannot detect chromosome material from the p arms of acrocentrics and cannot determine the SMC structure. The array-CGH results confirmed the FISH findings in patients NP and MK, and allowed delineation of the breakpoints in SMCs to single BAC clone resolution. The breakpoints in sSMC(8) in patient NP, identified previously by FISH as 8p21 and 8q11.1, were redefined by array-CGH as 8p21 and 8q11.21, respectively. In patient MK, array-CGH mapped the breakpoints to 4p12 in SMC(4) and 18p11.21 in SMC(18) (4p11.1 and 18p11.21 by FISH). Array-CGH results should be always verified by FISH analysis, because only FISH can determine the structure and level of mosaicism.

Our results in patient RS confirm that FISH analysis with centromeric and pericentromeric probes is sometimes insufficient for determination of sSMC origin. CenM-FISH and multicolor FISH did not give unambiguous results in this case. Chromosome painting showed that sSMC derives from chromosome 2. Chromosome microdissection with reverse painting demonstrated that the analyzed SMC was a neocentric fragment from the terminal part of the long arm of chromosome 2. Our results confirm that in sSMCs devoid of the α-satellite centromeric region, chromosome microdissection with reverse painting is more useful. Neocentric sSMCs are very rare and constitute 3.4% of all marker chromosomes (Liehr et al. 2004b). Recently, few hypotheses concerning the formation of neocentric chromosomes have been postulated. Liehr et al. (2004b) concluded that there are two types of the neocentric sSMCs. The more frequently observed forms originate from a U-type exchange between homologous chromosomes during meiosis. The less frequent shape of neocentric sSMCs is a ring conformation as described, e.g., for an sSMC(1) by Spiegel et al. (2003). Although many sSMCs have been identified using FISH, a detailed description of the clinical manifestations in patients with well cytogenetically characterized sSMCs is needed to assess better the recurrence risk and, in some cases, to apply appropriate medical care. Various phenotypic consequences of sSMCs are due to different chromosomal origin, euchromatin content, size, mosaicism, parental origin, potential of genomic imprinting effects, homozygosity of autosomal recessive mutations (in the case of isodisomy), and sex of sSMC carriers (Buckton 1985; Webb 1994; Crolla 1998; Langer et al. 2001; Kotzot 2002; Liehr et al. 2004a; Liehr et al. 2004c; Crolla et al. 2005). Phenotypes in the three presented patients result from sSMCs that arose de novo, contain euchromatic regions of chromosomes and have structures not found in sSMCs reported earlier. Our sSMC(2) is the second reported case with a neocentric ring derived from chromosome 2 (Petit and Fryns 1997) and the first sSMC derived from the terminal part of 2q. To date, 17 sSMCs(2) with known clinical significance have been described. None of the SMCs(2) reported in the literature contained the chromosome region present in the SMC(2) in patient RS (Plattner et al. 1993; Petit and Fryns 1997; Ostroverkhova et al. 1999;

Molecular cytogenetic characterization of sSMCs

Villa et al. 2001; Giardino et al. 2002; Lasan et al. 2003; Starke et al. 2003; Guancialli-Franci et al. 2004; Mrasek et al. 2005). In addition, a patient with trisomy 2q35-q37 (due to insertion of 2q material into 17q25) had a few similar clinical symptoms, such as psychomotor retardation, speech delay, short neck and fingers (found in the presented case), suggesting that they are consequences of the additional copy of this region of chromosome 2 (Fritz et al. 1999). Differences between clinical pictures in that case and our patient probably resulted from the fact that the sSMC(2) in patient RS contains a smaller fragment of chromosome 2q (2q35-q36). The clinical outcome in patients with marker chromosomes is more complicated if the sSMC carriers have complex mosaic karyotypes with more than one marker chromosome. This is the case in patients MK and NP with four and three sSMCs, respectively. In patient MK, multicolor FISH techniques showed trisomy of 4q12 and trior tetrasomy of 18p11.2, whereas in patient NP, trisomy or tetrasomy of chromosome fragments on 8p, and trisomy of 21p11.1→q21.3 were determined. The association of sSMC(4) and sSMC(18) found in patient MK has not been reported previously. Only two phenotypically abnormal patients with sSMCs similar to those of patient MK, showing an isolated min(4)(:p10→q12:) and min(4) (:p10→q13:), have been described (Fang et al. 1995; Bonnet et al. 2006). Motor developmental delay and narrow fingers were common in these three patients, suggesting a correlation between the trisomy of the pericentromeric fragment of chromosome 4q and the clinical symptoms. Based on the comparison of clinical features of sSMC(18) carriers described earlier, we propose that tri- or tetrasomy of pericentromeric region of chromosome 18 results in tapering of fingers and motor developmental delay – features found also in patient MK (Viersbach et al. 1998; Baumer et al. 2002; Starke et al. 2003; Guanciali-Franci et al. 2004; Timur et al. 2004; Bartsch et al. 2005). Abnormal phenotype in patient NP results from the presence of two sSMCs(8) deriving from different regions of chromosome 8, as well as from min(21). Some of known sSMCs(8) with breakpoints in 8p11 and 8q11 have not been associated with any abnormalities in their carriers (Nietzel et al. 2001; Daniel and Malafiej 2003; Starke et al. 2003; Bartsch et al. 2005; Liehr et al. 2006). In other patients with sSMCs(8), various clinical pictures have been observed (Blennow et al. 1993; Plattner et al. 1993; Butler et al. 1995;

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Hastings et al. 1999; Batanian et al. 2000; Anderlid et al. 2001; Loeffler et al. 2003). Based on the comparison of clinical phenotypes of patients with trisomy of 8p23→q10 due to sSMCs(8) (Demori et al. 2004) or with tri- and tetrasomy of region 8p21→q11 (Liehr et al. 2006), we propose that corpus callosum, lower lip, and developmental delay result from the presence of these sSMCs. The psychomotor retardation, facial anomalies, and brachycephaly present in patient NP, are clinical symptoms that can be found in patients with partial trisomy of 8p (Engelen et al. 1995). The other sSMC in patient NP derives from chromosome 21. Among clinical symptoms of some patients with marker chromosomes derived from the q arm of chromosome 21 (Daniel 1979; Park et al. 1987; Sun et al. 1995; Vierschbach et al. 1998), only microcephaly, and hypotonia is present in our patient NP. In summary, our data confirm the usefulness of multicolor FISH techniques and chromosome microdissection with reverse painting for the comprehensive analysis of sSMCs: determination of chromosomal origin of sSMCs (including neocentric marker chromosomes), characterization of their structures, identification of sSMCs not ascertained by any conventional or cytogenetic technique, and demonstrate that array-CGH is a reliable method for fast and easy determination of the breakpoints of sSMCs. Further studies are necessary for better phenotype-genotype correlations and assessment of genetic risk for various marker chromosomes. Acknowledgments. Supported in parts by Dr. Robert Pfleger-Stiftung, the Deutsche Forschungsgemeinschaft (436 RUS 17/135/03; 436 RUS 17/109/04, 436 RUS 17/22/06, WE 3617/2-1), the Deutscher Akademischer Austausch Dienst, the Schering Foundation, the Boehringer-Ingelheim-Fonds, and the Evangelische Studienwerk e.V. Villigst. REFERENCES Anderlid BM, Sigrid Sahlén S, Schoumans J, Holmberg E, Chsgren I, Mortier G, et al. 2001. Detailed characterization of 12 supernumerary ring chromosomes using micro-FISH and search for uniparental disomy. Am J Med Genet 99: 223–233. Batanian JR, Huang Y, Gottesman GS, Grange DK, Blasingame AV, 2000. Preferential involvement of the short arm in chromosome 8-derived supernumerary markers and ring as identified by chromosome arm painting. Am J Med Genet 90: 276–282. Bartsch O, Loitzsch A, Kozlowski P, Mazauric ML, Hickmann G, 2005. Forty-two supernumerary marker chromosomes (SMCs) in 43273 prenatal samples: chromosomal distribution, clinical find-

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