Introduction

Cardiofaciocutaneous (CFC) syndrome (MIM 115150) is a multiple congenital anomalies/mental retardation syndrome characterised by congenital heart defects, characteristic facial appearance, short stature, ectodermal abnormalities, and mental retardation.1 A recent review has identified 54 published classical cases of CFC syndrome. All cases were sporadic, with apparently normal chromosomes, born to non-consanguineous parents and presenting the same unique combination of findings and natural history.2

The CFC phenotype comprises a characteristic facial appearance, congenital heart defects (pulmonic stenosis, 37%; atrial septal defect, 35%; hypertrophic cardiomyopathy, 11%), mental retardation/developmental delay (81%), speech delay (46%), short stature (78%) and hypotonia (28%). The typical craniofacial manifestations include relative macrocephaly, high forehead, bitemporal constriction, supraorbital hypoplasia, short nose with depressed nasal bridge and anteverted nostrils, apparently low-set and posteriorly angulated ears, hypertelorism, ptosis, epicanthal folds, and downslanting of palpebral fissures. All patients had an ectodermal abnormality consisting of sparse, slowly growing and curly hair, sparse or absent eyelashes and eyebrows, follicular keratosis or ulerythema ophryogenes, ichthyosis, hyperkeratosis, generalized hyperpigmentation, haemangiomas, hyperelastic skin, cutis marmorata, café-au-lait patches, and/or slowly growing nails.

In spite of the controversy about the distinction between CFC and Noonan syndrome (NS) most of the time a refined clinical evaluation is sufficient for the differential diagnosis. Although phenotypic similarity between CFC and NS exists, especially regarding cardiac defects, craniofacial findings, short stature and congenital lymphedema, there are clear differences in clinical manifestations and natural history that separate the two conditions.3,4,5

Autosomal dominant inheritance is evident in many NS families, and genetic heterogeneity has also been demonstrated.6 Linkage studies have defined a region of approximately 7.5 Mb on the long arm of chromosome 12 (12q24) as a candidate region for a gene(s) causing NS6,7,8,9 (Figure 1). The recent demonstration of mutations in PTPN11, the gene encoding the non-receptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2)10 (Figure 2), in 45–50% of the studied patients of NS,9,11 has enabled an initial genotype–phenotype analysis. This has also shown that pulmonic stenosis is more prevalent in the group of NS patients who had a PTPN11 mutation than in the group without a mutation (70.6 vs 46.2%; P<0.01). The identification of a PTPN11 mutation in a family segregating the Noonan-like/multiple giant-cell lesion syndrome,11 and in LEOPARD syndrome,12 suggests a considerable phenotypic range due to mutations in this gene making it necessary to study the possible role of this gene in CFC syndrome.

Figure 1
figure 1

Representation of the 12q24 chromosome region, microsatellite markers that span the Noonan syndrome critical region and results of linkage studies in NS families. Microsatellite in bold corresponds to the PTPN11 localization.

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Figure 2
figure 2

PTPN11 organization and SHP-2 structure.

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Patients

We studied ten CFC patients (nine males and one female) (Figure 3), randomly selected from a larger group of CFC patients followed by a group of clinical geneticists in Brazil, Italy and North America, experienced with CFC syndrome and its nosology with the Noonan and Costello syndromes. In all cases the clinical diagnosis was considered incontrovertible. The CFC Index2 was applied to all patients. The patients selected for this study are originally from Brazil and Italy and in each case a consent form was signed by the parents.

Figure 3
figure 3

Three of the CFC patients studied. Patient 1, male, 28 years old; patient 2, male, 3 years old; patient 3, male, 8 years old.

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Materials and methods

DNA extraction

Genomic DNA was isolated from the patients' peripheral-blood leukocytes or from Epstein–Barr virus-transformed lymphocytes using standard procedures.

Gene sequencing

The 15 exons of the PTPN11 gene, described at http:// genome.ucsc.edu//cgi-bin/hgTracks?position=NM 002834& Submit=Submit and at http://www.ncbi.nlm.nih.gov/entrez / viewer.fcgi?dispmax =1&showndispmax = 1&val = NT 009575.8&view=graph& from=758671& to=857626& sfrom=0& strand=2, and their intron–exon boundaries were sequenced bi-directionally (Table 1, Figure 2). These regions were amplified from genomic DNA using the primers shown in Table 2, which were selected by the Macintosh program Primer Express.

Table 1 Intron–exon boundaries for the PTPN11 gene, exons length and their position in the mRNA (NM_002834). Intronic sequences are represented in lower case, and exonic, in upper case
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Table 2 Primers and PCR conditions used to amplify the coding region of the PTPN11 gene
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PRC reactions were carried out under standard conditions, containing: 50 ng of genomic DNA, 1 mM of each forward and reverse primers and 10 μl of Promega TAX Master Mix, as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C (1 min), annealing at 53–60°C (1 min) and extension at 72°C (1 min), followed by final extension at 72°C for 5 min, using the Gene Amp PCR System 9600 - Perkin Elmer.

PRC products were purified with a PCR product pre-sequencing kit, composed of exonuclease 1 and shrimp alkaline phosphatase, by USB Corporation, according to their instructions, and later using the Centri-Sep Spin Columns by Applied Biosystems. Sequencing reactions were carried out using the ABIPRISM dye terminator cycle sequencing kit, and the products were analysed on an ABIPRISM 310 Genetic Analyzer DNA sequencer. Sequences were analysed manually.

Study of the cDNA

In order to detect the presence of deletions within the coding region, which could be overlooked by analysing the sequences, we constructed five pairs of primers to amplify overlapping cDNA fragments of about 550 bp (Table 3). These fragments were amplified by PCR, carried out under standard conditions, containing: 50 ng of cDNA, 1 mM of each forward and reverse primers and 10 μl of Promega TAX Master Mix, as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C (1 min), annealing at 55–64°C (1 min) and extension at 72°C (1.5 min), followed by extension period of 10 min, using the Gene Amp PCR System 9600 - Perkin Elmer. Every PCR reaction included 10 CFC patients and one control subject. PCR products were electrophoresed on a 2% agarose gel to verify the actual size of the fragments obtained from each patient.

Table 3 Primes and PCR conditions to amplify overlapping fragments of PTPN11 cDNA
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Results

The CFC Index of all ten patents included in this study ranged from −1 SD to +1 SD. Three facial pictures are shown in Figure 3.

The coding-region of PTPN11 and its intron–exon boundary regions were sequenced in 10 CFC patients and no base changes were identified.

The sizes of the amplified cDNA fragments were as expected and single bands were detected from all patients in all five fragments.

Discussion

The actual relationship between CFC and Noonan syndromes has been the object of a heated controversy, and many opinions have been expressed as to whether they are different genetic entities, allelic conditions, a contiguous gene syndrome, or even different phenotypes of the same condition.2,3,4,5,13,14,15 The finding of one of the causes of Noonan syndrome is a very important step towards the clarification of this matter.

The protein encoded by PTPN11 is a member of the protein tyrosine phosphatase (PTP) family. PTPs regulate a variety of cellular processes, including cell growth, differentiation, mitotic cycle and oncogenic transformation and, in mouse, is involved in cardiac semilunar valvogenesis.16 The finding of 45–50% PTPN11 mutation prevalence in a sample of Noonan syndrome patients with almost 100% penetrance9,11 demonstrates that it is one of the genes causing Noonan syndrome. Mutations found in NS patients were all missense changes affecting amino acid residues that are conserved among the vertebrate SHP-2 orthologs.

PTPN11 mutations have also been found in conditions such as the Multiple-Lentigines/LEOPARD syndrome12 and Noonan-like/multiple giant-cell lesion syndrome.11 These data are of great importance in extending the phenotype of Noonan syndrome. It is also important to better classify some patients whose diagnosis is controversial. The finding of the same PTPN11 mutation in the two patients from a large family with NS who were previously diagnosed as having CFC syndrome17,18 demonstrates that those patients also have Noonan syndrome as already evident by their published phenotype. Therefore, to date, there are no familial cases of CFC syndrome. We have demonstrated that CFC syndrome patients do not have missense changes on the coding region of the PTPN11, nor small or large intragenic deletions, which we regard as molecular evidence that CFC and Noonan syndromes are distinct genetic entities.

We encourage PTPN11 mutation screening in other CFC patients to confirm our results.