Five cases of supernumerary small ring chromosomes 1: Heterogeneity and genotype–phenotype correlation

Five cases of supernumerary small ring chromosomes 1: Heterogeneity and genotype–phenotype correlation

+ MODEL European Journal of Medical Genetics 50 (2007) 94e102 http://www.elsevier.com/locate/ejmg Original article Five cases of supernumerary sma...

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European Journal of Medical Genetics 50 (2007) 94e102 http://www.elsevier.com/locate/ejmg

Original article

Five cases of supernumerary small ring chromosomes 1: Heterogeneity and genotypeephenotype correlation Laura Bernardini a, Anna Capalbo a, Maria Gabriella D’Avanzo b, Isabella Torrente a, Paola Grammatico c, Domenico Dell’Edera d, Denise Pontes Cavalcanti e, Antonio Novelli a,*, Bruno Dallapiccola a a

Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo e Istituto CSS-Mendel, Roma, Italy b Divisione di Genetica Medica, Ospedale ‘‘S.G. Moscati’’, Avellino, Italy c Dipartimento di Medicina Sperimentale e Patologia, Genetica Medica, Universita` ‘‘La Sapienza’’, Ospedale S. Camillo-Forlanini, Roma, Italy d Dipartimento Materno Infantile, U.O. Genetica Medica ASL 4, Matera, Italy e Programa de Genetica Perinatal, CAISM and Departamento de Genetica Medica, FCM-UNICAMP, Campinas, SP, Brazil Received 21 June 2006; accepted 15 November 2006 Available online 23 November 2006

Abstract Genetic counselling of patients with small supernumerary ring chromosomes (sSRCs) can be difficult, especially in prenatal testing, due to the complexity in establishing a karyotypeephenotype correlation. In fact, it has been estimated that about 10% of extra ring(1) chromosomes are associated with an unremarkable phenotype. We report on five new cases of extra ring chromosomes(1) manifesting different clinical outcome. One case was familial, segregating from a mother with mosaic karyotype, while the others were de novo. Ring chromosomes were characterised by FISH. In three subjects the involvement of the same euchromatic 1p region was demonstrated. Present observations corroborate previous results and provide some insight into the identification of the harmless ring(1) structures. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Supernumerary ring chromosome (SRC); Chromosome 1; Mosaicism; Genetic counselling

* Corresponding author. Istituto CSS-Mendel, Viale Regina Margherita, 261, 00198 Roma, Italy. Tel.: þ39 06 44160502; fax: þ39 06 44160548. E-mail address: [email protected] (A. Novelli). 1769-7212/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2006.11.001

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1. Introduction Constitutional small supernumerary marker chromosomes (sSMCs), appearing as extra centric fragments of heterogeneous origin, occur in about 0.043 per 1000 infants [12]. Small supernumerary ring chromosomes (sSRCs) are rare, accounting for 10% of all extra marker chromosomes [2]. About 40% of them are inherited [16]. Mosaicism is quite common, resulting either from postzygotic mutation or from mitotic instability [4]. Clinical outcome of sSMCs is highly variable depending on their origin, size, euchromatin content, eventual co-occurrence of uniparental disomy, and prevalence of aneuploidy in mosaic cases [9,11]. In the last 10 years FISH and molecular studies have greatly improved the characterisation and classification of sSMCs and established some karyotype/phenotype correlation [12], although in some cases clinical effects are due to mechanisms not always predictable through current diagnostic protocols. We report on five new cases of extra ring(1) chromosomes. Our study confirms the need of characterising these supernumerary markers in order to improve the estimated risk of an unfavourable outcome and to distinguish the undamaging sSMCs from all others. 2. Methods 2.1. Standard cytogenetics Chromosome analysis was carried out on circulating lymphocytes and in cultured amniocytes. Metaphases were G- and C-banded using standard procedures. One hundred cells were examined in all cases and in their parents. 2.2. FISH analysis Ring chromosomes were characterised by 24-color multi-target fluorescence in situ hybridisation (M-FISH), using the SpectraVysion kit (Vysis Inc., Downers Grove, IL), following the manufacturer’s protocol. The M-FISH results were confirmed by 1/5/19 and D1Z5 centromeric probes (Vysis Inc.). The size of ring chromosomes was established using specific BAC clones, selected on the basis of their map position (http://genome.ucsc.edu). Clones were obtained either from Sanger Institute (Cambridge, UK) or from ‘‘Resources for Molecular Cytogenetics’’ (Bari, Italy). DNA was extracted using Quantum Prep kit (BioRad, Hercules, CA), and either SpectrumGreen-dUTP or TexasRed-dUTP labelled using Nick Translation kit (Invitrogen, Carlsvad, CA), following the manufacturer’s recommendations. FISH was performed by co-denaturing probes and chromosomes at 74  C for 3 min, and keeping the slides into the hybridisation chamber (Hybrite; Vysis Inc.) at 37  C overnight. After a series of washes with 2  SSC and 4  SSC/0.05%Tween twenty, metaphases were counterstained with DAPI, viewed using an epifluorescence Nikon Eclipse E1000 microscope (Nikon Instruments, Florence, Italy), and imaged with the Genikon analyzer system (Nikon Instruments). Forty cells for each case were analysed. 2.3. Uniparental disomy (UPD) In order to exclude uniparental disomy of chromosome 1, 20 microsatellite markers, covering chromosome 1 with a resolution of approximately 10 cM (Linkage Mapping Set Version 2,

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Perkin-Elmer), were genotyped. Genomic DNA was isolated from amniotic fluid and from peripheral blood cells. Microsatellite markers were amplified by polymerase chain reaction (PCR) and the fragment size was determined using an ABI PRISM 3100 (Perkin-Elmer Applied Biosystem) autosequencer. Data were collected and analysed by GeneScan version 3.5.1 (Perkin-Elmer Applied Biosystem) and Genotyper software version 2.1.5 (Perkin-Elmer Applied Biosystem). Genetic distance and marker order were established in centimorgans according to the Genethon Human Genetic Linkage Map (1996). 3. Results The five extra ring(1) cases included in this study were referred to our laboratory because of the abnormal phenotype (Case 1), reproductive failure (Cases 3 and 4), and to characterise the aneuploidy detected in amniocytes (Cases 2 and 5). Four cases were de novo mosaic sSRCs, while one subject showed a homogeneous aneuploidy inherited from the mother with mosaic karyotype (Table 1). The rings were characterised by FISH analysis using pericentromeric probes. Final karyotypes are summarised in Table 2. Case 1. The propositus was ascertained at age of 14 months because of severe psychomotor retardation and dysmorphic features (Fig. 1a). Delivery by Caesarean section was carried out at 32 weeks, after intrauterine growth retardation. Apgar scores were 8 and 9 at 1 and 5 min. Birth weight was 910 g (<3rd centile), length 34 cm (<3rd centile), head circumference (OFC) 27 cm (5th centile). At birth the baby suffered from severe respiratory distress. On examination at 14 months, weight was 5.2 kg (<3rd centile), length 67 cm (<3rd centile), and OFC 47 cm (5th centile). The child disclosed body asymmetry, with a hypoplastic right side. The face was triangular with high forehead, frontal bossing, sparse eyebrows, long eyelashes, ectropion, anteversed nostrils, long philtrum, thin upper lip, high-arched palate, and small pointed chin. Ears were low-set with flat antihelices. He also showed pectus excavatum and unilateral cryptorchidism. Giemsa-staining and G-banding analyses disclosed a ring-shaped sSRC in 15% of the metaphases (Fig. 1c). Derivation of the extra marker from chromosome 1 was supported by M-FISH analysis (Fig. 1d) and confirmed by FISH, using a specific centromeric probe (D1Z5). Further characterisation with pericentromeric clones was carried out using RP11-115N23, mapping to

Table 1 General characteristics of present extra ring(1) chromosomes Case 1

Case 2

Case 3

Case 4

Case 5

Case 6

Sex Phenotype Proportion of aneuploid cellsa

M A 15%

F N 20%

M I 70%

F I 40%

F N 100%

F N 55%

Probes

Map position (UCSC)

FISH results

RP11-110B10 RP11-115N23 a-sat 1/5/19 RP11-79E5 RP4-646P11

1p11.2: 120,769e20,926 kb 1p11.2: 120,839e20,974 kb Centromere 1q12: 127,000e141,600 kb heterochromatin 1q21.1: 142,665e142,680 kb

e þ þ þ e

e e þ e e

e e þ e e

e þ þ e e

e þ þ e e

e e þ þ e

A ¼ abnormal; I ¼ infertility; N ¼ normal. þ Indicates a positive signal onto the marker, e indicates no hybridisation signal onto the ring. a For each case 100 cells have been analysed.

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Table 2 Overview of all reported cases with well characterised pericentromeric r(1) chromosomesa Caseb

Cytogenetic finding

Inheritance

Sized with pericentromeric clones

Clinical features

Present Case 1

Dr(1) (Tp11.2 / q12T)

de novo

Yes

[12] (Case 1e14)

Dmin(1) (:p11.1 / q12:) [40%]

de novo

No

[7]

Dr(1) (Tp12 w p11 / q12T) [15%] Dr(1) (Tp12 w 11 / q12T) [80%] þr(1) (Tp11 / q11T)(D1Z5þ) [70%] þr(1) (Tp11 / q11T)(D1Z5þ) [40%] þmin(1) (:p11.2 w 12 / q12:) [30%] þr(1) (Tp10 / q12T) [40%] þr(1) (Tp11.2 / q10T) [100%] þish der(1)(D1Z5þ) [100%] þmar(1).ish(cep1þ;wcp1) [100%] þr(1) (Tp10 / q12T) [26%]

de novo

No

de novo

Yes

de novo

Yes

Psychomotor retardation, facial dysmorphisms, body asymmetry, pectus excavatum, cryptorchidism Premature birth, mild psychomotor retardation, dysmorphisms Psychomotor retardation, dysmorphisms Right talipes equinovarus, behaviour disorder Infertility

de novo

Yes

Infertility

de novo

No

Infertility

de novo

Yes

Normal

Maternal

Yes

Normal

Maternal de novo

No No

Normal Normal

de novo

No

Normal

[8] (Case 2) Present Case 3 Present Case 4 [12] (Case 1e22) Present Case 2 Present Case 5 [10] (Case 15) [5] (Case 1) [13] (Case 1) a b

Cases collected from SMCs Page (http://mti-n.mti.uni-jena.de/whuwww/MOL_ZYTO/ssmc.htm). Cases with clinical effects are indicated in bold type.

1p11.2, and RP11-79E5, mapping to the heterochromatic 1q12 region. Both clones disclosed positive signals within the ring chromosome, while RP11-110B10, partially overlapping RP11-115N23 at 1p11.2, and RP4-646P11, mapping to 1q21.1 did not hybridise onto the marker chromosome. These results were consistent with the trisomy of less than 200 kb of an euchromatic 1p segment, and the retention of the heterochromatic 1q region (Fig. 1e,f). Microsatellite analysis showed the inheritance of one maternal and one paternal chromosome 1, excluding uniparental disomy (Fig. 1b). Case 2. This newborn female was referred for cytogenetic studies to validate the results of amniocentesis, performed because of maternal age. Karyotyping disclosed a ring-shaped supernumerary marker in 40% of lymphocytes (Fig. 2a). Pregnancy at term was uneventful and delivery was normal. Physical examination at birth and follow-up at 12 months were unremarkable, including a normal psychomotor development. The postnatal karyotype disclosed an sSRC in 20% of lymphocytes. The origin of the marker from chromosome 1 was suggested by M-FISH, and confirmed with a specific centromeric probe. FISH analysis with clones mapping to the pericentromeric regions was negative, except for RP11-79E5, indicating that the ring chromosome contained the heterochromatic 1q12 region (Fig. 2b).

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Fig. 1. (a) Case 1 at age of 14 months. (b) Chromosome 1 microsatellite analysis excludes uniparental disomy. (c) The arrow points to the small extra ring. (d) M-FISH suggests that ring derives from chromosome 1. (e,f) FISH analysis with pericentromeric clones discloses the presence of 1p region and retention of the heterochromatic 1q12 region.

Case 3. This case was ascertained in the male partner of a couple requesting chromosome analysis after two first trimester spontaneous abortions. Standard cytogenetic analysis disclosed a ring-shaped marker chromosome in 70% of lymphocytes (Fig. 2c). The marker was identified as a r(1) by M-FISH and FISH analysis, using a centromeric probe (Fig. 2f). Further characterisation with pericentromeric clones proved that the extra chromosome contained only the chromosome 1 centromeric region. Case 4. This was the female partner of a couple requesting cytogenetic analysis after a 5year history of infertility. Giemsa-staining and G-banding analyses disclosed a ring-shaped sSRC in 40% of her lymphocytes (Fig. 2d). Origin of this marker from chromosome 1 was supported by M-FISH (Fig. 2e) and corroborated by the presence of the specific centromeric probe signal. FISH characterisation with pericentromeric probes failed to show any hybridisation signal within the ring chromosome, indicating that it contained only the centromeric region. Case 5. This case was ascertained during prenatal diagnosis requested for maternal age. The karyotype of amniocytes disclosed a non-mosaic sSRC, which was proved to be inherited from the clinically normal mother, who had the same extra marker in 55% of circulating lymphocytes (Fig. 3a). Ring characterisation was carried out using M-FISH, centromeric probe and a panel of clones which proved that the marker contained the centromere and about 200 kb of 1p euchromatin, corresponding to the RP11-115N23 clone (Fig. 3b,c). FISH results in amniocytes and in maternal lymphocytes (Case 6 in Table 2) were identical, excluding further rearrangements in fetal cells. Microsatellite analysis showed the inheritance of one maternal and one paternal chromosome 1, excluding uniparental disomy (Fig. 3d). The pregnancy had ended in a normal female newborn with an unremarkable follow-up at 11 months.

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Fig. 2. (a,b) Case 2: GTG-banding discloses a ring-shaped supernumerary marker which FISH analysis demonstrates to contain the heterochromatic 1q12 region (arrow). (c,f) Case 3: the extra ring detected in GTG-banded metaphases is demonstrated to contain chromosome 1 centromeric region (arrow). (d,e) Case 4: the extra ring is detected with standard techniques and M-FISH analysis suggests it to derivate from chromosome 1 (arrow).

4. Discussion Genetic counselling of prenatally ascertained small extra marker chromosomes is problematic, particularly for the prediction of the clinical outcome. In fact, the risk associated with an abnormal phenotype for a de novo marker detected in fetal cells is about 30%, after the origin of the marker from an acrocentric chromosome is excluded. This figure is doubled when the aneuploid chromosome is a ring structure [3,5]. One third of sSMCs can be associated with distinct disorders, including PallistereKillian syndrome associated to isochromosome 12p; inv dup(15q) syndrome in cases containing the PWS/AS region; isochromosome 18p syndrome; ‘‘cat-eye’’ syndrome associated with inv dup(22q); duplication 22q syndrome, resulting from malsegregation of t(11;22) [12]. In the presence of other markers, prediction of the clinical consequences can be less obvious. Supernumerary rings derived from chromosome 1 are among the most common aneuploid small marker chromosomes [11], and display a quite controversial genotypeephenotype correlation. So far, more than 30 cases with mar(1) have been described and the clinical outcome was unremarkable in about 10% [14]. In this study, we described five new sSRCs derived from chromosome 1, including two cases with partial trisomy of pericentromeric euchromatic material. In addition, similar sized sSMCs(1) have been collected from the website http://mti-n.mti. uni-jena.de/whuwww/MOL_ZYTO/ssmc.htm, in an attempt to identify shared clinical features (Table 2).

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Fig. 3. (a) Case 5: the ring-shaped extra marker (arrow). (b) M-FISH indicates that the extra ring is derived from chromosome 1 (arrow). (c) FISH analysis with specific clones proves that extra ring has a small euchromatic 1p region. (d) Chromosome 1 microsatellite analysis excludes uniparental disomy.

Case 1 showed prenatal and postnatal growth retardation, severe developmental delay, and dysmorphic features, which suggested a chromosomal disorder. The supernumerary ring was found in 15% of lymphocytes, and contained the heterochromatic pericentromeric 1q region, together with a small euchromatic 1p11.2 segment. Although only few known gene maps to the duplicated 1p region, it was likely that phenotype was related to trisomy of the proximal short arm segment. In fact, pericentromeric regions generally represent the less explored genomic areas because of their high content of duplicated sequences that yield difficulty in their exact sequencing [1]. Therefore, it is not possible to exclude their involvement in human diseases so far. However, the trisomy of the same 1p region was displayed also in Cases 5 and 6 that showed unremarkable phenotypes. These findings make it difficult to understand the eventual relationship between the cytogenetic unbalance and clinical outcome in Case 1, therefore, uniparental disomy was hypothesised to explain his clinical phenotype. In fact, the possible association between supernumerary marker chromosomes and uniparental disomy is well established and recommends UPD testing in sSMCs patients with clinical anomalies, since they are at increased risk of disomy for the structurally normal homologues involved in the sSMC formation [9]. Case 1 shared several features with another child carrying a mosaic sSMC(1), in combination with maternal disomy of chromosome 1 [15]. However, chromosome 1 UPD was excluded in our patient. Alternatively, the small extent of the euchromatic trisomic segment could explain the lack of any clinical effect in Cases 5 and 6, while the abnormal phenotype in Case 1 could be likely due to the additive effects of the trisomic heterochromatic 1q and euchromatic 1p regions, rather than to an altered dosage effect of one or more genes. This

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suggestion is supported by the ‘‘interactive hypothesis’’ of Wilson, arguing that some aneuploid phenotypes result from altered homeostasis of multiple contiguous or non-contiguous loci, involving some regulatory elements [18]. Case 3 was the male partner of a couple, investigated because of recurrent miscarriages and Case 4 was a subfertile woman carrying a supernumerary ring chromosome 1. Interestingly, molecular cytogenetics in Cases 3 and 4 showed identical ring structures, containing only the centromere region. These two subjects, together with the case described by Liehr et al. [12] (Case [12] 1e22 in Table 2) showed a normal phenotype, except for infertility. These results confirm the well established association between extra marker chromosomes and infertility, accounting for 0.165% of these cases [11]. The phenotype was normal as well as in Case 2, a healthy child in which the extra ring maintained both the centromere and the heterochromatic 1q region. On the whole, our results agree with literature data in regard to similar sSMC(1) and corroborate the conclusions of Liehr et al. [12] by arguing for the absence of obvious clinical effects due to dosage imbalances for the 1p11.2 / p12 or 1q12 regions. As showed in Table 2, cases showing either a centromeric region or an iuxtacentromeric region derived from 1p or the heterochromatic band 1q12, had normal phenotypes, apart from marker structure, whereas the simultaneous occurrence of the proximal 1p and 1q regions resulted in some clinical effects, including dysmorphisms and psychomotor retardation. Clinically variable features can be observed in subjects presenting cytogenetically identical mosaic markers, either because of the diverse distribution of the mosaic cell line in tissues or the different proportion of abnormal cell populations. To¨nnies et al. [17] reported on a 4-yearold child with a r(1) chromosome in about 14% of lymphocytes and 9% of buccal cells. The authors stated that it would be important to verify the proportion of the mosaic cell line in different tissues to better understand the likely correlation between aneuploidy and clinical phenotype. However, published data are controversial on this matter [6], and, although analysis of different tissues can be important to better characterise mosaic patients, this kind of study does not seem to contribute significantly to the genetic counselling. Furthermore, the present observations didn’t prove any firm correlation between the percentage of aneuploid cells and clinical consequences. In this respect, it appears more appropriate the molecular characterisation of ring structures. Additional data are needed in order to improve risk assessment, particularly during prenatal genetic counselling. However, personal experience suggests that clones RP11-110B10, mapping to 1p, and RP4-646P11, mapping to 1q, which tested negative in our cases of sSRC(1), mark the boundaries of extra rings(1) including either 1p or 1q regions, which are associated with normal phenotypes. Acknowledgements This work was supported in part by a grant from the Italian Ministry of Health (Ricerca Corrente 2006). We are grateful to Prof. Mariano Rocchi for the generous gift of BAC clones. References [1] J.A. Bailey, A.M. Yavor, H.F. Massa, B.J. Trask, E.E. Eichler, Segmental duplications: organization and impact within the current human genome project assembly, Genome Res. 11 (2001) 1005e1017.

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[2] E. Blennow, T.H. Bui, U. Kristoffersson, M. Vujic, G. Anneren, E. Holmberg, M. Nordenskjold, Swedish survey on extra structurally abnormal chromosomes in 39105 consecutive prenatal diagnoses: prevalence and characterization by fluorescence in situ hybridization, Prenat. Diagn. 14 (1994) 1019e1028. [3] E. Blennow, E. Tillberg, Small extra ring chromosome derived from chromosome 10p: clinical report and characterization by FISH, J. Med. Genet. 33 (1996) 399e402. [4] H. Chen, C.M. Tuc-Muller, D.A.S. Batista, W. Wertellecki, Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization, Am. J. Med. Genet. 56 (1995) 219e223. [5] J.A. Crolla, F.L. Long, H. Rivera, N.R. Dennis, FISH and molecular study of autosomal supernumerary marker chromosomes excluding those derived from chromosomes 15 and 22: I. Results of 26 new cases, Am. J. Med. Genet. 75 (1998) 355e366. [6] A. Daniel, P. Malafiej, A series of supernumerary small ring marker autosomes identified by FISH with chromosome probe arrays and literature review excluding chromosome 15, Am. J. Med. Genet. 117A (2003) 212e222. [7] A.J. Dawson, D. Konkin, D. Riordan, A.E. Chudley, Mosaic trisomy of a small r(1) with an abnormal phenotype, Am. J. Med. Genet. 103 (2001) 32e35. [8] D. Giardino, D. Bettio, G. Gottardi, N. Rizzi, M. Pierluigi, C. Perfumo, A. Cali, F. Dagna Bricarelli, L. Larizza, FISH characterization of two supernumerary r(1) associated with distinct clinical phenotypes, Am. J. Med. Genet. 84 (1999) 377e380. [9] D. Kotzot, Supernumerary marker chromosomes (SMC) and uniparental disomy (UPD): coincidence or consequence? J. Med. Genet. 39 (2002) 775e778. [10] M.M. Li, P.N. Howard-Peebles, L.D. Killos, L. Fallon, E. Listgarten, W.S. Stanley, Characterization and clinical implications of marker chromosomes identified at prenatal diagnosis, Prenat. Diagn. 20 (2000) 138e143. [11] T. Liehr, U. Claussen, H. Starke, Small supernumerary marker chromosomes (sSMC) in humans, Cytogenet. Genome Res. 107 (2004) 55e67. [12] T. Liehr, K. Mrasek, A. Weise, A. Dufke, L. Rodriguez, N. Martinez Guardia, A. Sanchis, J.R. Vermeesch, C. Ramel, A. Polityko, O.A. Haas, J. Anderson, U. Claussen, F. von Eggeling, H. Starke, Small supernumerary marker chromosomesdprogress towards a genotypeephenotype correlation, Cytogenet. Genome Res. 112 (2006) 23e34. [13] K. Michalski, M. Rauer, N. Williamson, A. Perszyk, J.J. Hoo, Identification, counselling, and outcome of two cases of prenatally diagnosed supernumerary small ring chromosomes, Am. J. Med. Genet. 46 (1993) 88e94. [14] L. Rodriguez, H. Starke, M. Guardia, H. To¨nnies, H. Neitzel, P. Kozlowski, A.H. Mazauric, F.L. Grondona, E. Mansilla, M.J.S. Mun˜oz, T. Liehr, M.L. Martinez-Frias, Three new cases with a supernumerary ring chromosome 1, Clin. Dysmorphol. 14 (2005) 169e175. [15] B. Ro¨thlisberger, T. Zerova, D. Kotzot, T.I. Buzhievskaya, D. Balmer, A. Schinzel, Supernumerary marker chromosome (1) of paternal origin and maternal uniparental disomy 1 in a developmentally delayed child, J. Med. Genet. 38 (2001) 885e888. [16] E.S. Sachs, J.O. Van Hemel, J.C. Den Hollander, M.G.J. Jahoda, Marker chromosome in a series of 10,000 prenatal diagnoses. Cytogenetic and follow-up studies, Prenat. Diagn. 7 (1987) 81e89. [17] H. To¨nnies, L.M. Neumann, B. Gr}uneberg, H. Neitzel, Characterization of a supernumerary ring chromosome 1 mosaicism in two cell systems by molecular cytogenetic techniques and review of the literature, Am. J. Med. Genet. 121A (2003) 163e167. [18] G.N. Wilson, Karyotype/phenotype controversy: genetic and molecular implications of alternative hypotheses, Am. J. Med. Genet. 36 (1990) 500e505.