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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.30 no.1 São Paulo  2007 



Fluorescence in situ hybridization (FISH) screening for the 22q11.2 deletion in patients with clinical features of velocardiofacial syndrome but without cardiac anomalies



Paula Sandrin-GarciaI; Antonio Richieri-CostaII; Eloiza Helena TajaraIII; Andréa Borduchi Carvalho-SallesIII; Agnes Cristina Fett-ConteIII

IDepartamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
IIDepartamento de Genética, Hospital de Pesquisa e Reabilitação de Lesões Lábio Palatais, Universidade de São Paulo, Bauru, SP, Brazil
IIIDepartamento de Biologia Molecular, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, SP, Brazil

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The velocardiofacial syndrome (VCFS), a condition associated with 22q11.2 deletions, is characterized by a typical facies, palatal anomalies, learning disabilities, behavioral disturbances and cardiac defects. We investigated the frequency of these chromosomal deletions in 16 individuals with VCFS features who presented no cardiac anomalies, one of the main characteristics of VCFS. Fluorescent in situ hybridization (FISH) with the N25 (D22S75; 22q11.2) probe revealed deletions in ten individuals (62%). Therefore, even in the absence of cardiac anomalies testing for the 22q11.2 microdeletions in individuals showing other clinical features of this syndrome is recommended.

Key words: velocardiofacial syndrome, 22q11.2 deletion.



Chromosome 22q11.2 deletions are associated with a wide spectrum of phenotypes, including velocardiofacial syndrome (VCFS), DiGeorge syndrome (DGS), conotruncal anomalies and sporadic or familial cardiac defects (Lipson et al., 1991; Scambler 2000). These deletions encompass 1.5 to 3 Mb (Lindsay et al., 1995a,b; Adeyinka et al., 2004) and are most often the result of a de novo event, although 5-10% are inherited (Sandrin-Garcia et al., 2002).

Velocardiofacial syndrome (VCFS) is characterized by a complex of clinical anomalies: typical facies, velopharyngeal insufficiency (VPI) or cleft palate, learning disabilities, behavioral disturbances and cardiac anomalies (Shprintzen, 1990; Lipson et al., 1991). The typical facies includes a prominent nose with squared nasal root and narrow alar base, relative deficiency of the malar area, vertical maxillary excess, retruded mandible, narrow palpebral fissures and minor ear anomalies. Palatal anomalies, learning disabilities and typical facies are present in all patients. Cardiac defects are found in 84% of the patients and are the main cause of morbidity and mortality (Shprintzen et al., 1981). Other less frequent features are psychiatric disorders, short stature and hyperextensibility of the digits. The estimated incidence of the syndrome is 1:4000 live births (Devriendt et al., 1998).

Cytogenetic studies using high-resolution chromosome banding have detected 22q11.2 deletions in approximately 20% of individuals with VCFS (Driscoll et al., 1992), whereas fluorescent in situ hybridization (FISH) analysis has demonstrated 22q11.2 microdeletions in the majority of these patients (Lindsay et al., 1995 a,b). A clear correlation between the extension of the deletions and the clinical variability has not become evident (Carlson et al., 1997 a,b; Edelman et al. 1999; Digilio et al., 2003).

Some studies investigated the frequency of cardiac anomalies among carriers of 22q11.2 deletions. In a European collaborative study of 558 individuals with 22q11 deletions (Ryan et al., 1997), 70% of the 545 individuals evaluated for cardiac anomalies had a significant cardiac pathology, such as Fallot's tetralogy, ventricular septal defect, interrupted aortic arch, pulmonary atresia/ventricular septal defect or truncus arteriosus. Similar frequencies were observed by McDonald-McGinn et al. (1999) and Kitsiou-Tzeli et al. (2004). However, Bassett et al. (2005) reported cardiac anomalies in 25.8% of 78 adults carrying 22q11 deletions.

To investigate the association of 22q11.2 deletions with cardiac anomalies, we searched for 22q11.2 deletions in individuals with clinical features of VCFS who did not present with cardiac anomalies.

After the approval by the Committee of Ethics in Research of the Ribeirão Preto Medical School University of São Paulo and obtaining written informed consent from the individuals or their legal guardians, we selected a group of 16 individuals (6 females, 10 males) among those referred to the Hospital de Pesquisa e Reabilitação de Lesões Lábio Palatais/USP (HPRLLP/Centrinho, Bauru, SP) with the diagnostic hypothesis of VCFS. These individuals were selected based on the main characteristics of the VCFS, i.e., typical facies, velopharyngeal insufficiency, behavioral disorders and/or learning disabilities. All of them were sporadic cases.

The 16 individuals with VCFS were submitted to a study protocol including standard semiology and family history, clinical, phonoaudiological and radiological exams. Learning disabilities, behavioral disturbances and mental retardation were assessed by psychological tests. Cardiac evaluation, including electrocardiogram and echodopplercardiogram, was carried out on all 16 individuals examined and those with cardiac alterations excluded from our study. The mean age of the group ranged from 6 to 32 years with a mean of 14 ± 5.9 years. Table 1 summarizes the clinical findings in our group.

We obtained GTG-banded chromosome preparations from lymphocyte cultures (Yunis, 1976) and at least 25 metaphases/prometaphases were analyzed per individual at the resolution level of 550 to 800 bands. FISH was performed using the 90 kb N25 (D22S75) cosmid probe that encompasses the DiGeorge/VCFS minimal critical region (Oncor, Inc., Gaithersburg, MD). Control probe pH 17 (D22S39) (Oncor, Inc., Gaithersburg, MD) was co-hybridized. For immunodetection, anti-digoxigenin fluorescent isothiocyanate-conjugated antibody was used. Chromosomes were counterstained with propidium iodide. Analyses were performed by two independent observers using a Carl Zeiss Axioskop Microscope equipped with a standard fluorescence isothiocyanate (FITC) filter. Photographs were taken with Kodak Ektachrome 400 film.

A 22q11.2 deletion was identified in three (19%) individuals (02, 06 and 13) after GTG-banding. No other chromosomal abnormalities were detected. When FISH was used, ten (62%) individuals (01, 02, 03, 05, 06, 07, 10, 11, 13 and 14) showed the deletion (Table 1).

The critical VCFS region (DGCR) encompasses about 3 Mb, which is deleted in about 90% of individuals with VCFS (Morrow et al., 1995). In addition to the 3 Mb deletion, another 8% of cases have been found to present a smaller deletion of about 1.5 Mb (Carlson et al., 1997a), and it has also been shown that in familial cases these smaller deletions were predominant (Adeyinka et al., 2004). The probe we used covered ~90 kb of the critical region and detected the majority of deletions. Nevertheless, the presence of deletions, which did not involve this segment, cannot be ruled out in our non-deletion individuals.

All our patients presented the characteristic clinical features of VCFS except for cardiac anomalies and, apart from this, there were no significant clinical difference between individuals with or without the 22q11.2 deletion, agreeing with previously reported data (Lindsay et al., 1995a, Digilio et al 2003).

Frequencies of individuals without cardiac anomalies ranging from 25 to 70% have previously been reported among carriers of 22q11.2 deletions (Ryan et al 1997; McDonald-McGinn, 1999; Kitsiou-Tzeli et al., 2004; Bassett et al., 2005). To our knowledge this is the first study that determined the frequency of deletions in individuals with VCFS who had been selected on the basis of the absence of cardiac defects, and revealed the relatively high frequency of 62% deletions.

Testing for the presence of the 22q11.2 deletion is recommended for patients with two or more relevant clinical features of VCFS in any combination (typical facies, cardiac anomalies, palatal anomalies and learning disabilities), particularly when the typical facies is associated with palatal anomalies (Scambler, 2000). The high frequency of 22q11.2 deletion found in our small series of 16 individuals with VCFS without cardiac defects indicate that testing for deletions is recommended in such individuals.



We thank the medical staff of HPRLLP (Centrinho) for selecting the patients, Dr. Paulo Roberto Franciscone for cardiac evaluation of the patients and Dr. Robert F. H. Dekker, Dr. Peter James Harris and Dr. José Fernando Garcia for revising the text. We also thank the individuals tested and their families for their collaboration.



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Send correspondence to:
Agnes Cristina Fett-Conte
Departamento de Biologia Molecular
Faculdade de Medicina de São José do Rio Preto
Hospital de Base, Av. Brigadeiro Faria Lima 5544
15090-000 São José do Rio Preto, SP, Brazil

Received: November 4, 2005; Accepted: August 9, 2006.



Editor: Angela M. Vianna-Morgante

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