**2.4 FISH analyses with BAC clones**

To delineate the extent of the deletion on chromosome 7, FISH analyses were carried out using BAC clones mapping to the long arm of chromosome 7 (7q11.23).

We identified the BAC clones of interest through the Human Genome Browser Database of the Genome Bioinformatics Group at the University of California at Santa Cruz (http://genome.ucsc.edu/). They were then ordered by Internet on the site of the Children's Hospital Oakland Research Institute in Oakland, California (http://bacpac.chori.org/).


The WBS deletion region is flanked by highly homologous clusters of genes and pseudogenes organized into low-copy-repeat (LCR) blocks. Unequal meiotic recombination during meiosis can lead to deletion of the WBS region. The unique genetic architecture of the region explains why the size of WBS deletion is almost the same in most of the patients (Baumer et al., 1998; Dutly & Schinzel, 1996; Pober, 2010; Schubert, 2009; Valero et al., 2000). Several studies have investigated the size of the WBS deletion using multiplex PCR with several microsatellite markers (Brondum-Nielsen et al., 1997; Perez Jurado et al., 1996; Wang et al., 1999; Wu et al., 1998). Most of the patients were found to carry the 1.5 or 1.8 Mb deletion. In the present study, we performed fluorescent *in situ* hybridization (FISH) with Bacterial Artificial Chromosome (BAC) clones in an attempt to map the WBS deletion in 14

Fifteen patients were referred to the cytogenetic laboratory of the Brest University Hospital by medical geneticists, pediatricians and pediatric cardiologist for suspicion of WBS between 2002 and March 2011. The reasons for referral included cardiovascular anomalies, dysmorphism or a combination of signs suspecting WBS (Table 1). However, because of limited cell pellet

Metaphase chromosomes were prepared from peripheral blood lymphocytes of the 14 patients after having obtained their parents' informed consent. Standard R banding chromosomal analyses were performed according to the standard procedures and the karyotypes described according to the International System for Cytogenetic Nomenclature

A FISH study using the Vysis Williams Region Probe - LSI ELN SpectrumOrange/LSI D7S486, D7S522 SpectrumGreen was carried out on the metaphase preparations from all 14

The Williams Region Probe consists of a probe of approximately 180 kb in size for *ELN*, *LIMK1* and the D7S613 locus located in band 7q11.23, labeled in Spectrum Orange, and a control probe for the region containing loci D7S486 and D7S522 located in band 7q31,

To delineate the extent of the deletion on chromosome 7, FISH analyses were carried out

We identified the BAC clones of interest through the Human Genome Browser Database of the Genome Bioinformatics Group at the University of California at Santa Cruz (http://genome.ucsc.edu/). They were then ordered by Internet on the site of the Children's Hospital Oakland Research Institute in Oakland, California (http://bacpac.chori.org/).

available for one patient, the study could be performed only on 14 patients.

patients, as recommended by the manufacturer (Abbott, Rungis, France).

using BAC clones mapping to the long arm of chromosome 7 (7q11.23).

**2.3 FISH analyses with commercially available probe** 

WBS patients.

**2.1 Patients** 

(ISCN 2005).

**2. Patients and methods** 

**2.2 Conventional cytogenetics** 

labeled in Spectrum Green.

**2.4 FISH analyses with BAC clones** 

Table 1. Clinical characteristics of the 14 patients with Williams-Beuren syndrome. M: month; y: year WBS suspicion: combination of signs (facial dysmorphology and cardiovascular anomaly) making the diagnosis highly probable IUGR: intra uterine growth retardatio n

NA: not available

Patient # 14 - ?: patient still too young (4 months old)


M: month; y: year WBS suspicion: combination of signs (facial dysmorphology and cardiovascular anomaly) making the diagnosis highly probable IUGR: intra uterine growth retardatio

n

NA: not available

Patient # 14 - ?: patient still too young (4 months old)

Table 1. Clinical characteristics of the 14 patients with Williams-Beuren syndrome.

When received, bacterial cultures were prepared from a single colony picked from a selective plate in the presence of chloramphenicol. Plasmids were obtained from bacterial cultures grown in the presence of chloramphenicol (10 mg/L). After having lysed bacteria using SDS1%/NaOH 0.2 N, DNA was purified from RNA, proteins and other cellular contaminants. Probes were then labeled by nick translation in Spectrum Orange (Nick Translation Kit, Abbott, Rungis, France) or in FITC (Prime-it Fluor Fluorescence Labeling Kit, Stratagene, Amsterdam, Netherlands). All BAC clones were applied to normal lymphocyte metaphases to confirm their chromosomal location.

After hybridization, the slides were counterstained with 4-6-diamino-2-phenyl-indoledihydrochloride (DAPI). The preparations were examined using a Zeiss Axio Plan Microscope (Zeiss, Le Pecq, France). Images acquisition was performed using a CCD camera and analyzed using the ISIS program (In Situ Imaging System) (MetaSystems, Altlussheim, Germany).

Twenty-two overlapping BACs covering the WBS deletion region and beyond (about 1.9 Mb) were applied on the 14 patients (Table 2).


The base pairs position (bp) are predicted on Build 39 National Center for Biotechnology Information (http://www,ncbi,nlm,nih,gov) and assembly February 2009 by The UCSC Genome Browser Database (http://genome,ucsc,edu/index,html),

Table 2. BAC library used to delimitate the deletion size in the 14 WBS patients.
