Clinical and molecular cytogenetic studies in ring chromosome 5: Report of a child with congenital abnormalities

European Journal of Medical Genetics 55 (2012) 112e116

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Short report

Clinical and molecular cytogenetic studies in ring chromosome 5: Report of a child with congenital abnormalities Audrey Basinko a, b, c, Maria Luisa Giovannucci Uzielli d, Gloria Scarselli d, Manuela Priolo e, Giuseppina Timpani f, Marc De Braekeleer a, b, c, * a

Laboratoire d’Histologie, Cytologie et Cytogénétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, 22, Avenue Camille Desmoulins, CS 93837, F-29238 Brest cedex 3, France INSERM U-613, Brest, France c Service de Cytogénétique, Cytologie et Biologie de la Reproduction, Hôpital Morvan, CHRU Brest, Brest, France d Genetics and Molecular Medicine, University of Florence and Genetic Science, Florence, Italy e Medical Genetics Laboratory, Pediatrics Unit, General Hospital, Reggio Calabria, Italy f Neonatalogy Unit, General Hospital, Reggio Calabria, Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 July 2011 Accepted 18 November 2011 Available online 2 December 2011

We report here a child with a ring chromosome 5 (r(5)) associated with facial dysmorphology and multiple congenital abnormalities. Fluorescent in situ hybridization (FISH) using bacterial artificial chromosome (BAC) clones was performed to determine the breakpoints involved in the r(5). The 5p deletion extended from 5p13.2-3 to 5pter and measured 34.61 Mb (range: 33.7e35.52 Mb) while the 5q deletion extended from 5q35.3 to 5qter and measured 2.44 Mb (range: 2.31e2.57 Mb). The patient presented signs such as microcephaly, hypertelorism, micrognathia and epicanthal folds, partially recalling those of a deletion of the short arm of chromosome 5 and the “cri-du-chat” syndrome. The most striking phenotypic features were the congenital heart abnormalities which have been frequently reported in deletions of the distal part of the long arm of chromosome 5 and in rings leading to a 5q35-5qter deletion. However, the NKX2-5 gene, which has been related to congenital heart defects, was not deleted in our patient, nor presumably to some other patients with 5q35.3-5qter deletion. We propose that VEGFR3, deleted in our patient, could be a candidate gene for the congenital heart abnormalities observed. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Chromosome 5 Ring chromosome “Cri du chat” phenotype Congenital abnormalities

1. Introduction

2. Clinical report

Ring chromosome 5 is a cytogenetic abnormality that has rarely been reported [1e8]. Although phenotypic variability has been observed in these patients, they usually present a “cri du chat” syndrome due to the loss of band 5p15 [2,3]. A careful search in the literature failed to identify ring chromosome 5 cases that were characterized by molecular cytogenetics. We report here on a patient with “cri du chat” features who had a ring chromosome 5 that was analyzed by fluorescent in situ hybridization (FISH) using several probes.

The proband is a male baby born at 34 weeks gestation of a third pregnancy. The father and mother, who are not consanguineous, were 25 years old at the birth of the child. The anamnesis revealed no important events from a genetic and teratogenic point of view. The proband’s older sister and brother were in good health. The baby was born from an emergency Cesarean birth because of fetal suffering, with an Apgar index of 3, 5, and 8 at 1, 5, and 10 min, respectively. Birth weight was 1340 g and length 37 cm, both values well below the third percentile for the gestational age. Cranial circumference was 29 cm, much lower than the second percentile. It should be emphasized that fetal monitoring during pregnancy had revealed a severe intra-uterine growth retardation (IUGR) from the twenty-seventh week of gestation. Clinical genetic evaluation at birth showed the baby to have dysmorphic facial aspects: microcephaly, narrow biparietal diameter, hypertelorism, epicanthus, obliquity low down and outside

* Corresponding author. Laboratoire de Cytogénétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, 22, Avenue Camille Desmoulins, CS 93837, F-29238 Brest cedex 3, France. Tel.: þ33 (0)2 98 01 64 76; fax: þ33 (0)2 98 01 81 89. E-mail address: [email protected] (M. De Braekeleer). 1769-7212/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2011.11.005

A. Basinko et al. / European Journal of Medical Genetics 55 (2012) 112e116

palpebral fissures, flattened nasal bridge, medium level micrognathia. The auricular pavilion appeared “simple” with an anomalous pinna (Fig. 1). Thumbs were hypoplastic, with proximal implantation. The second finger overlapped over the third and of fifth over the fourth, on the left hand (Fig. 1). A radiographic examination of the limbs revealed a bilateral hypoplasia of the radius, an anomalous ulna, and dysplasic metacarpal and distal phalanxes bones. Upper limbs movements were reduced at the level of the elbow and the wrist, with a wrist flexion of the right hand and flexion of the fingers of both hands. Cerebral echography showed a dilatation of the two lateral ventricles and of the third ventricle, hypoplasia of the cerebellar vermis, periventricular hyperechogenicity of the ventricular horns, cavum vergae and cysts of the pellucid septum. Cardiac investigations revealed an atrial and ventricular septal defect, patent Ductus Arteriosus, hypertrophic cardiomyopathy, hypoplastic aorta with supravalvular stenosis and dilatation of the pulmonary artery. Abdominal echography revealed hypoplasia of both kidneys. X-ray investigations confirmed the bilateral radial hypoplasia. The child died at the age of 4 months and two weeks due to cardiovascular failure. Post-mortem study was not performed. 3. Cytogenetic analyses Peripheral blood lymphocytes were cultured by standard cytogenetic techniques. Q-banded (QFQ) and G banded (GAG) chromosome analysis was performed and the karyotype described following the ISCN 2009 [9]. Multiplex Ligation-dependent Probe Amplification (MLPA) at the level of subtelomeric regions of chromosome 5 was applied to confirm the result obtained by banding cytogenetics. Fluorescent in situ hybridization (FISH) was performed on metaphase chromosomes of the patient and his parents. Commercial probes applied on the patient’s metaphases were 5pter and 5qter subtelomeric probes (TelVysion 5p locus C84c11/T3 and TelVysion 5q locus D5S2907, Abbott, Rungis, France), 5p and 5q partial chromosome paints (XCAP 5 short and XCAP 5 long, Metasystems, Altlussheim, Germany), and a multicolor chromosome 5 banding (XCyte 5, Metasystems). A whole chromosome 5 paint (XCP 5, Metasystems) was tested on metaphases of the patient’s parents. The manufacturers protocols were followed for hybridization.

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In order to determine the breakpoints involved in the rearrangement, a FISH study with bacterial artificial chromosome (BAC) clones was performed on the patient’s metaphases. BAC clones were chosen according to the multicolor chromosome 5 banding results, from the UCSC genome browser site (http://genome.ucsc.edu/). They were ordered from the Children’s Hospital Oakland Research Institute (http://bacpac.chori.org/). Clones were cultured with chloramphenicol. DNA was extracted by alkaline lysis and labeled by nick translation with SpectrumGreen-dUTP (Abbott), SpectrumOrangedUTP (Abbott) or Atto425-dUTP-NT (Aqua) (Jena BioScience, Euromedex, Mundolsheim, France). Hybridizations were performed with a classic procedure. Slides were analyzed using a Zeiss Axio Plan microscope (Zeiss, Le Pecq, France). Subsequent image acquisition was performed using a CCD camera with the “in-situ imaging system” software (MetaSystems) [10]. Unfortunately, no DNA was obtained, preventing us from performing CGH analysis. 4. Results Chromosome analysis (QFQ and GAG) on patient’s peripheral blood lymphocytes, after birth, showed the presence of a mosaicism with two cell lines: mos46,XY,r(5)[183]/45,XY,-5[30] (Fig. 2). MLPA analysis confirmed the deletion of both subtelomeric regions of chromosome 5. Both parents had a normal constitutional karyotype. FISH analysis on the r(5) revealed that both 5p and 5q subtelomeres were lost (Fig. 2b). The r(5) was composed with 5p and 5q material. Loss of 5p material was larger than loss of 5q material (Fig. 2ced). The 5p breakpoint was determined between BAC clone RP11-1148B16 in 5p13.3 (deleted on the r(5)) and RP11-414K15 in 5p13.2 (present on the r(5)), whereas the 5q breakpoint was located into 5q35.3, between RP11-21I4 and RP11-825A14, respectively present and deleted on the r(5) (Fig. 2eef). According to the International System for Human Cytogenetic Nomenclature (2009), the r(5) would be described as: ish r(5)(p13.3q35.3) or r(5) (p13.2q35.3)(C84c11/T3,RP11-1148B16,pcp5pþ,RP11-414K15þ, pcp5qþ,RP11-21I4þ,RP11-825A14,D5S2907). Knowing the BAC clones location from the Human February 2009 (GRCh37/hg19) assembly, imbalances in cells with the r(5) were a monosomy from 5p13.2 (or 5p13.3) to 5pter and a monosomy from 5q35.3 to 5qter encompassing 34.61 Mb (range: 33.7e35.52 Mb) and 2.44 Mb (range: 2.31e2.57 Mb), respectively. FISH with whole chromosome 5 paint failed to identify a r(5) in the 30 metaphases analyzed from each parent.

Fig. 1. Photography of the child at age 2 months showing microcephaly, narrow biparietal diameter, hypertelorism, epicanthus, obliquity low down and outside palpebral fissures, flattened nasal bridge and medium level micrognathia.

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Fig. 2. Cytogenetic analyses of the r(5). a. QFQ analysis showing the r(5). b. FISH analysis with 5pter (green) and 5qter (red) subtelomeric probes. c. FISH analysis with 5p (green) and 5q (red) partial chromosome paints. d. FISH analysis with chromosome 5 m-banding. e. FISH analysis with BAC clones RP11-1148B16 (red) and RP11-414K15 (aqua). f. FISH analysis with BAC clones RP11-21I4 (green) and RP11-825A14 (aqua).

5. Discussion Constitutional ring chromosome is a very rare abnormality that can be found in 1 in 25,000 fetuses [11]. The most frequent mechanism of ring formation involves one break in each arm followed by fusion, resulting in loss of genetic material of the distal segments of the chromosome. Many cases of rings derived from different chromosomes and variable in size have been reported. However, it is thought that large ring chromosomes are more unstable than smaller ones [12]. The r(5) reported here is large. Therefore, the high instability of its configuration could explain the presence of the 45,XY,5 cell line. Although the 45,XY,5 cell line could solely be the result of lymphocyte culture, we cannot exclude that this clone was present in vivo and explained part of the phenotype observed in this child. Detailed phenotypic description of ring chromosome 5 has usually focused on the similarities with the “cri-du-chat” syndrome, a contiguous gene deletion located in band 5p15.2-15.3 [2e5]. The patient reported here presents signs such as microcephaly, hypertelorism, micrognathia and epicanthal folds, partially recalling those of a deletion of the short arm of chromosome 5. The 5p deletion generated by the formation of the r(5) extended from 5p13.2 to pter and covered about 35 Mb. Genotypeephenotype correlation in patients with terminal 5p deletions showed a progressive severity of psychomotor retardation and clinical manifestations related to the deletion size [13,14]. However, no specific phenotypic characteristics, except for those associated with the “cri-du-chat” syndrome, could be identified. Cerebral echography showed several malformations that have already been reported in 5p deletion, without consistency between the observed abnormalities and the size of the deletion [15e20]. The most striking phenotypic features associated with the r(5) reported here are the congenital heart abnormalities, notably the atrial and ventricular septal defect and the hypoplastic aorta. A

search in the literature identified several patients with congenital heart abnormalities associated with 5p deletion spanning the region 5p13.2 to 5pter [14,21e27]. However, in most of the patients, the 5p deletion was the result of missegregation of a reciprocal translocation [22e25,27]. Therefore, the heart abnormality origin could not be definitely attributed to the 5p deletion. The same uncertainty about the origin of congenital heart defects exists for the patients recombinant of a parental pericentric inversion of chromosome 5 in whom the 5p deletion is associated with a 5q trisomy [28e32]. In these cases, a dosage effect of a gene located on the long arm cannot be excluded. Congenital heart abnormality has been frequently reported in deletions of the distal part of the long arm of chromosome 5 and in rings leading to a 5q35-5qter deletion (Table 1). Previous reports of distal 5q deletion associated with cardiac septation defects suggested that this phenotype could be related to the NKX2-5 gene (or hCSX) [33,34]. Several patients with congenital heart defect were shown to have a deletion of the NKX2-5 gene using FISH with a specific probe or by array-CGH (Table 1) [33,35e38]. Indeed, this cardiac homeobox transcription factor is located in band 5q35.1 [39]. Furthermore, mutations in the NKX2-5 gene have been found in patients with nonsyndromic congenital heart disease, including ventricular septal defects, tetralogy of Fallot, subvalvular aortic stenosis, ventricular hypertrophy and pulmonary atresia [40,41]. The 5q breakpoint in our patient is located some 5.8 Mb telomeric of the NKX2-5 gene. FISH with BAC clone RP11-466H21, containing NKX2-5, showed no deletion. Four other cases, all with del(5)(q35.3qter), have been described in the literature (Table 1). Stratton et al. (1994) reported on a 15-month-old boy with atrial septal defect [42]. Rauch et al. (2003) reported a 3.5 Mb 5q subtelomeric deletion in a 3-year-old girl with persistence of the foramen ovale [34]. Baker et al. (2002) reported on two sibs with a der(5)t(5;16)(q35.3;q24.3), one of whom having an ventricular septal defect [43].

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Table 1 Congenital heart abnormality associated with distal 5q deletion. Reference

Chromosomal abnormality

Technique(s) used

Size (Mb)

NKX2-5 deletion

ASD

VSD

PDA

PFO

TOF

[51] [52] [53] [54] [27] [33] [33] [55] [35] [42] [34] [43] [43] [36] [37,38] [4] [5] Present case

del(5)(q33q34) del(5)(q33.3q35.1) del(5)(q35.1qter) del(5)(q35.2qter) del(5)(q33q35) del(5)(q34q35.4) del(5)(q34q35.4) del(5)(q35.qter) del(5)(q35.1q35.3) del(5)(q35.3qter) del(5)(q35.3qter) del(5)(q35.3qter) del(5)(q35.3qter) del(5)(q35.1q35.2) del(5)(q35.1q35.1) r(5)(p15q35) r(5)(p14q35) r(5)(p13.3q35.3)

CC þ FISHa CC þ FISHa CC CC CC CC CC CC CC CC BAC CC CC BAC Array-CGH CC CC BAC

ND ND ND ND ND ND ND ND ND ND 3.5 ND ND 2.2 2.8 ND ND 2.44

ND ND ND ND ND þ þ ND þ ND ND ND ND þ þ ND ND 

e e e e e þ þ þ þ þ e e e þ e e e þ

e e e e þ þ e þ e e e e þ e e þ þ þ

e e þ e þ e e e þ e e e e e e e e þ

e e e e e e e e e e þ e e e e e e e

e e e e e e e e e e e e e e þ e e e

CC: conventional cytogenetics; FISH: fluorescent in situ hybridization; BAC: Bacterial Artificial Chromosome; CGH: comparative genomic hybridization; ND: not determined; ASD: atrial septal defect; VSD: ventricular septal defect; PDA: patent ductus arteriosus; PFO: persistent foramen ovale; TOF: tetralogy of Fallot. a In these cases, FISH was used to confirm the interstitial nature of the deletion.

The r(5) observed in our patient resulted in a 2.44 Mb (range: 2.31e2.57 Mb) deletion in the subtelomeric region of the long arm and let to the loss of several genes (http://genome.ucsc.edu/). Among them, VEGFR3 (also known as FLT4, fms-related tyrosine kinase 4) could be a candidate gene for the congenital heart defects observed in our patient. The VEGFR3 protein is thought to be involved in lymphangiogenesis and maintenance of the lymphatic endothelium [44,45]. However, the VEGFR3 gene in mice is involved in blood vessel development in early embryos and its inactivation results in large vessels becoming abnormally organized with defective lumens, leading to cardiovascular failure at embryonic day 9.5. Thus, VEGFR3 has an essential role in the development of the embryonic cardiovascular system [46]. VEGFR3 encodes a tyrosine kinase receptor for vascular endothelial growth factors C and D (VEGFC and VEGFD), two members of the VEGF (vascular endothelial growth factor) family, among which one, VEGFA, binding to VEGFR1 and VEGFR2, is involved in embryonic endocardial cushion formation and potentially contributes to congenital heart defects [47,48]. Although the role of VEGFR3 in heart morphogenesis is still hypothetical, VEGFR3 and VEGFC were shown to be expressed in heart of 12.5 and 14 day-old murine embryos [49,50]. Therefore, VEGFR3 could be a candidate gene for the congenital heart abnormalities observed in our patient and in others with 5q35.3-5qter deletion. Acknowledgements The authors thank Nathalie Douet-Guilbert, Nadia Guéganic, Marie-Josée Le Bris, Frédéric Morel, Matthieu Talagas, Lisa Di Medio, Stefania Melchiorri and Debora Nannini for excellent technical skills and helpful discussions. References [1] R.E. Carrel, R.S. Sparkes, S.W. Wright, Chromosome survey of moderately to profoundly retarded patients, Am. J. Ment. Def. 77 (1973) 616e622. [2] Y. Nakagome, K. Iinume, K. Taniguchi, Points of exchange in a human no. 5 ring chromosome, Cytogenet. Cell Genet. 12 (1973) 35e39. [3] E. Suerinck, B. Noel, M.O. Rethore, Ring chromosome 5 in two malformed boys with Cri du Chat syndrome, Clin. Genet. 14 (1978) 125e129. [4] K. Kula, S. Patil, J. Hanson, A. Nowak, H. Zellweger, Ring chromosome 5 with dental anomalies, Pediatr. Dent 3 (1981) 329e333.

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