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  • Abstract Small supernumerary marker chromosomes(SMCs) are present in about 0.05% of the human popula-tion. In approximately 30% of SMC carriers (excludingthe ~60% SMC derived from one of the acrocentric chro-mosomes), an abnormal phenotype is observed. The clin-ical outcome of an SMC is difficult to predict as they canhave different phenotypic consequences because of (1)differences in euchromatic DNA-content, (2) different de-grees of mosaicism, and/or (3) uniparental disomy (UPD)of the chromosomes homologous to the SMC. Here, wepresent 35 SMCs, which are derived from all human chro-mosomes, apart from chromosome 6, as demonstrated bythe appropriate molecular cytogenetic approaches, such ascentromere-specific multicolor fluoresence in situ hybrid-ization (cenM-FISH), multicolor banding (MCB), and sub-centromere-specific multicolor FISH (subcenM-FISH). Innine cases without an aberrant phenotype, neither partialproximal trisomies nor UPD could be detected. Abnormalclinical findings, such as psychomotoric retardation and/or craniofacial dysmorphisms, were associated with sevenof the cases in which subcentromeric single-copy probeswere proven to be present in three copies. Conversely, ineight cases with a normal phenotype, proximal euchro-matic material was detected as partial trisomy. UPD wasstudied in 12 cases and subsequently detected in two ofthe cases with SMC (partial UPD 4p and maternal UPD 22in a der(22)-syndrome patient), indicating that SMC carri-ers have an enhanced risk for UPD. At present, smallproximal trisomies of 1p, 1q, 2p, 6p, 6q, 7q, 9p, and 12qseem to lead to clinical manifestations, whereas partial

    Heike Starke Angela Nietzel Anja Weise Anita Heller Kristin Mrasek Britta Belitz Christine Kelbova Marianne Volleth Beate Albrecht Beate Mitulla Ralf Trappe Iris Bartels Sabine Adolph Andreas Dufke Sylke Singer Markus Stumm Rolf-Dieter Wegner Jrg Seidel Angela Schmidt Alma Kuechler Isolde Schreyer Uwe Claussen Ferdinand von Eggeling Thomas Liehr

    Small supernumerary marker chromosomes (SMCs): genotype-phenotype correlation and classification

    Hum Genet (2003) 114 : 5167DOI 10.1007/s00439-003-1016-3

    Received: 20 May 2003 / Accepted: 4 August 2003 / Published online: 16 September 2003

    ORIGINAL INVESTIGATION

    Electronic database information: accession numbers and URLsfor the data in this article are as follows: ENSEMBL-database, http://www.ensembl.org/ National Center for Biotechnology Information (NCBI),http://www.ncbi.nlm.nih.gov/ Genome Database (GDB), http://www.gdb.org/gdb/ OMIM (Online Mendelian Inheritance in Man) Database,http://www.ncbi.nlm.nih.gov/omim

    H. Starke A. Nietzel A. Weise A. Heller K. Mrasek A. Kuechler I. Schreyer U. Claussen F. von Eggeling T. Liehr ()Institut fr Humangenetik und Anthropologie, Kollegiengasse 10, 07743 Jena, GermanyTel.: +49-3641-935533, Fax: +49-3641-935502,e-mail: i8lith@mti.uni-jena.de

    B. BelitzPraxis fr Medizinische Genetik, Frankfurter Allee 111, 10247 Berlin, Germany

    C. KelbovaPraxis fr Medizinische Genetik, Marienstrasse 27, 03046 Cottbus, Germany

    M. VollethInstitut fr Humangenetik, Leipziger Strasse 44, 39120 Magdeburg, Germany

    B. AlbrechtInstitut fr Humangenetik, Hufelandstrasse 55, 45122 Essen, Germany

    B. MitullaZentralklinikum Suhl, gGmbH, A.-Schweitzer-Strasse 2, 98527 Suhl, Germany

    R. Trappe I. BartelsInstitut fr Humangenetik, Zentrum fr Hygiene und Humangenetik, Heinrich-Dker-Weg 12, 37073 Gttingen, Germany

    S. AdolphOlgahospital, Institut fr Klinische Genetik, Bismarkstrasse 3, 70176 Stuttgart, Germany

    A. Dufke S. SingerInstitut fr Humangenetik und Anthropologie, Wilhelmstrasse 27, 72074 Tbingen, Germany

    M. Stumm R.-D. WegnerPartnerschaft fr Medizinische Genetik, Kurfrstendamm 199, 10719 Berlin, Germany

    Springer-Verlag 2003

    J. SeidelKlinik fr Kinder- und Jugendmedizin, Kochstrasse 2, 07743 Jena, Germany

    A. SchmidtPraxis fr Humangenetik, Podbielskistrasse122, 30177 Hannover, Germany

    A. KuechlerKlinik fr Strahlentherapie, Bachstrasse 18, 07743 Jena, Germany

  • proximal trisomies of 2q, 3p, 3q, 5q, 7p, 8p, 17p, and 18pmay not be associated with significant clinical symptoms.With respect to clinical outcome, a classification of SMCsis proposed that considers molecular genetic and molecu-lar cytogenetic characteristics as demonstrated by presentlyavailable methods.

    Introduction

    Chromosomal abnormalities, the great majority of whichare numerical, are found in 0.3%1% of newborns (Buck-ton et al. 1985; Queisser-Luft et al. 2002). The clinicaloutcomes of whole chromosome trisomies and monosomiesare well characterized. However, clinical effects for pa-tients having imbalances of small chromosomal regionsresulting from karyotypes containing small supernumerarymarker chromosomes (SMCs) are less predictable. This isattributable to the lack of rigorous cytogenetic evaluationand good clinical outcome correlation. Small SMCs arereported in 0.01%0.05% of all prenatally screened fe-tuses. Approximately half of these small SMCs are deriv-atives of chromosome 15 (Crolla 1998; Crolla et al. 1998).Among the remaining cases, there is a great variation inchromosomal origin (Callen et al. 1992; Crolla 1998;Crolla et al. 1998). According to Crolla (1998), 70% ofSMCs derived from non-acrocentric chromosomes are notassociated with clinical symptoms. Because of the vary-ing clinical outcomes attributable to the chromosomal ori-gin of the SMC, their detailed characterization is of inter-est in order to improve post-diagnostic medical care andgenetic counseling. In cases of apparently normal pheno-types, the characterization of SMCs can provide impor-tant information about regions within the human genomethat do not lead to abnormalities in the presence of genedosage imbalances (Sumption and Barber 2001) and/orare subject to gene silencing (for a review, see Li 2002).

    The origin of small SMCs is almost impossible to es-tablish by routine cytogenetics alone, whereas fluores-cence in situ hybridization (FISH) methods are highly suitedfor this purpose. SMCs have been successfully character-ized by whole-chromosome-painting (WCP) probes, cen-tromere-specific probes, or combined chromosome micro-dissection and FISH approaches (for a review, see Nietzelet al. 2001). WCP-FISH approaches are well-suited forthe determination of the chromosomal origin of marker orderivative chromosomes providing that they are largerthan 17p, whereas if they are smaller, WCP-FISH is, ingeneral, non-informative (Haddad et al. 1998; case 7, mar1, Starke et al. 1999). As recently reported, for SMCs witha euchromatic content of approximately half of the shortarm of chromosome 17p or more, characterization by themulticolor banding (MCB) technique is possible (Liehr etal. 2002b; Weise et al. 2002). For a fast and easy charac-terization of smaller SMCs, we have recently proposed thecentromere-specific multicolor FISH (cenM-FISH) method(Nietzel et al. 2001). Despite this, analyses of small SMCsto detect the presence of euchromatin have to date beenambiguous and have required further clarification. In the

    present paper, we have addressed this problem by utiliz-ing MCB probe-sets and/or a probe-set comprising of 43bacterial or yeast artificial chromosome (BAC or YAC,respectively) clones located in the proximal regions ofeach human chromosome, viz., subcentromeric multicolor-FISH (subcenM-FISH). As reported previously by us(Chudoba et al. 1999; von Eggeling et al. 2002) and oth-ers (for a review, see Kotzot 2002), after identification ofthe origin of the SMC, its normal sister-chromosomesshould be tested for their parental origin to exclude a pos-sible uniparental disomy (UPD). UPD can be tested bymolecular genetic approaches, such as microsatellite analy-sis (Salafsky et al. 2001) or methylation-specific poly-merase chain reaction (PCR; Nietzel et al. 2003).

    In this paper, molecular cytogenetics and moleculargenetics have been used for the characterization of SMCsof variable origins in 35 cases, and a correlation of theclinical phenotype with the SMC-associated genotype hasbeen made. Finally, an SMC classification system is pro-posed, which facilitates the characterization and allows aprecise description of SMCs, including their shape, theUPD of their sister-chromosomes, and the degree of mo-saicism.

    Materials and methods

    Studied cases and cytogenetics

    Cytogenetic and molecular cytogenetic studies were performed onchromosomes derived from cultivated amniotic fluid, chorionbiopsy, or peripheral blood cells. Chromosome preparations wereobtained according to standard techniques (Verma and Babu1989).

    The cases studied, the cytogenetic results, and the clinical de-tails are listed in Table 1. Cases with an additional SMC are num-bered with Arabic numerals (135) and those with structurallyaberrant markers have Roman numberals (IIII).

    Standard FISH procedures

    FISH was performed according to standard procedures (Liehr et al.1995). In some of the cases, 24-color FISH with WCP probes(Senger et al. 1998; case 12), cenM-FISH (Nietzel et al. 2001; allcases apart from I-III), or MCB (Liehr et al. 2002b; cases 1, 2, 4,7, 10, 11, 16, 17, 22, 28, 29) were applied (for technical details, seeSenger et al. 1998; Nietzel et al. 2001; Liehr et al. 2002b). Addi-tionally, in case III, the subtelomeric probe PAC 240G10 specificfor 16q was used (kindly provided by Dr. Lyndal Kearney, Lon-don, UK).

    SubcenM-FISH method

    The proximal probes included in the subcenM-FISH set (see Table 2)were chosen under the prerequisite that they were the most proxi-mal probes presently mapped in the ENSEMBL database. BAC orYAC DNA (200 ng) was amplified by degenerate oligonucleotide-primed PCR (DOP-PCR; 50-l volume; Senger et al. 1998). Eachprobe was labeled separately by using a secondary DOP-PCR (20-lvolume) either w

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