Background Germline mutations in  BRCA1  and  BRCA2  predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of  BRCA1  in 25 individuals from 18 families from a Greek cohort.
Background
Germline mutations in  BRCA1  and  BRCA2  predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of  BRCA1  in 25 individuals from 18 families from a Greek cohort.BRCA1BRCA2BRCA1Methods The approach used is based on dHPLC mutation screening of the  BRCA1  gene, followed by sequencing of fragments suspected to carry a mutation including intron – exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the  BRCA2  gene, followed by MLPA analysis for screening for large genomic rearrangements.
Methods
The approach used is based on dHPLC mutation screening of the  BRCA1  gene, followed by sequencing of fragments suspected to carry a mutation including intron – exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the  BRCA2  gene, followed by MLPA analysis for screening for large genomic rearrangements.BRCA1BRCA2Results A pathogenic mutation in  BRCA1  was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the  BRCA1  gene. Finally, MLPA revealed deletion of exon 20 of the  BRCA1  gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an  Alu Sp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to  Alu -mediated recombination, responsible for the majority of rearrangements in the  BRCA1  gene.
Results
A pathogenic mutation in  BRCA1  was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the  BRCA1  gene. Finally, MLPA revealed deletion of exon 20 of the  BRCA1  gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an  Alu Sp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to  Alu -mediated recombination, responsible for the majority of rearrangements in the  BRCA1  gene.BRCA1BRCA1BRCA1AluAluBRCA1Conclusions We conclude that a combination of techniques capable of detecting both single base mutations and small insertions / deletions and large genomic rearrangements is necessary in order to accurately analyze the  BRCA1  gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the  BRCA1  locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.
Conclusions
We conclude that a combination of techniques capable of detecting both single base mutations and small insertions / deletions and large genomic rearrangements is necessary in order to accurately analyze the  BRCA1  gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the  BRCA1  locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.BRCA1BRCA1

Background Germ line mutations in the  BRCA1  and  BRCA2  genes predispose individuals to breast and ovarian cancer. The lifetime risk of breast cancer in female carriers of a  BRCA1  mutation is 60–80% while that of ovarian cancer is 20–40%. The median age of diagnosis of breast cancer is 42 years, i.e. 20 years earlier than the median of unselected women in the U.S.A. and Western Europe [ 1 ].  BRCA1  is a large gene with 22 coding exons encoding a 220 kD protein [ 2 ] that functions in maintaining genomic integrity and in transcriptional regulation [ 3 , 4 ]. A multitude of mutations scattered throughout the 5592 bp coding sequence have been described. In particular, germ line mutations in  BRCA1  have been identified in 15–20% of women with a family history of breast cancer and 60–80% of women with a family history of breast and ovarian cancer [ 5 , 6 ]. The percentage of mutations identified is strongly dependent on the population studied, with strong founder effects evident in some populations [ 7 - 9 ]. The vast majority of mutations described to date are point mutations and small insertions and deletions  . Such mutations are detected by PCR-based screening methods such as the Protein Truncation Test (PTT), Single Strand Conformational Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), heteroduplex analysis (HA) and more recently denaturing high performance liquid chromatography (dHPLC) with varying degrees of sensitivity for each method. Direct DNA sequencing is used in order to confirm and characterize mutations detected by any of these approaches [ 10 , 11 ]. While in the early studies for mutation detection only single point or small insertion/deletion mutations were screened for, recent studies have shown that genomic rearrangements are also a common type of mutation in the two genes accounting for 10–30 % of all mutations identified in some populations [ 12 - 16 ]. In this study we document and extend previous work suggesting the necessity of screening for large genomic rearrangements in a complete program for mutation detection of the  BRCA1  gene by characterizing a deletion encompassing 3.2 kb of the gene including exon 20. In addition, we add more evidence supporting pathogenicity of a previously described variant, G1738R, which seems to be specific to the Greek population [ 17 ].
Background
Germ line mutations in the  BRCA1  and  BRCA2  genes predispose individuals to breast and ovarian cancer. The lifetime risk of breast cancer in female carriers of a  BRCA1  mutation is 60–80% while that of ovarian cancer is 20–40%. The median age of diagnosis of breast cancer is 42 years, i.e. 20 years earlier than the median of unselected women in the U.S.A. and Western Europe [ 1 ].  BRCA1  is a large gene with 22 coding exons encoding a 220 kD protein [ 2 ] that functions in maintaining genomic integrity and in transcriptional regulation [ 3 , 4 ].BRCA1BRCA2BRCA1BRCA1A multitude of mutations scattered throughout the 5592 bp coding sequence have been described. In particular, germ line mutations in  BRCA1  have been identified in 15–20% of women with a family history of breast cancer and 60–80% of women with a family history of breast and ovarian cancer [ 5 , 6 ]. The percentage of mutations identified is strongly dependent on the population studied, with strong founder effects evident in some populations [ 7 - 9 ]. The vast majority of mutations described to date are point mutations and small insertions and deletions  . Such mutations are detected by PCR-based screening methods such as the Protein Truncation Test (PTT), Single Strand Conformational Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), heteroduplex analysis (HA) and more recently denaturing high performance liquid chromatography (dHPLC) with varying degrees of sensitivity for each method. Direct DNA sequencing is used in order to confirm and characterize mutations detected by any of these approaches [ 10 , 11 ]. While in the early studies for mutation detection only single point or small insertion/deletion mutations were screened for, recent studies have shown that genomic rearrangements are also a common type of mutation in the two genes accounting for 10–30 % of all mutations identified in some populations [ 12 - 16 ].BRCA1In this study we document and extend previous work suggesting the necessity of screening for large genomic rearrangements in a complete program for mutation detection of the  BRCA1  gene by characterizing a deletion encompassing 3.2 kb of the gene including exon 20. In addition, we add more evidence supporting pathogenicity of a previously described variant, G1738R, which seems to be specific to the Greek population [ 17 ].BRCA1Methods Patients BRCA1  patients and their families were referred through the Oncology Departments of Hygeia Hospital and other hospitals throughout Greece. Patients were included on the basis of affected family members, types of cancer present in the family and the age at diagnosis of breast cancer in the proband. The families were subdivided into high risk when multiple cases of breast and ovarian cancer were diagnosed, medium risk if there were only 2–3 cases of breast cancer, and low risk in isolated cases of breast cancer with diagnosis before the age of 40 years. The study population consisted therefore of 12 high risk families, 2 with medium risk, and 4 families at low risk as determined by their cancer history. Ethical approval was obtained from the hospital's advisory committee and all patients signed informed consent. Screening has been completed in 25 individuals from 18 families. Testing was initially carried out on DNA from an affected family member and upon detection of an inactivating mutation the rest of the family members were directly tested for this mutation. DNA and RNA isolation Genomic DNA and RNA were purified from peripheral blood leukocytes using standard extraction protocols. PCR amplification The complete coding sequence of  BRCA1  including splice junctions was amplified by PCR. Similarly, exons 10 and 11 of the  BRCA2  gene were amplified in 3 of the patients. Primers used have been chosen from the BIC database  . In addition primers: mBRCAF: GAG TTT GTG TGT GAA CGG ACA CTG and mBRCAR: GTG CCA AGG GTG AAT GAT GAA AGC, designed during the course of this study, were used for amplification of cDNA from the patients found to carry a deletion of exon 20 of the  BRCA1  gene. Reactions of 50 μl were heated on a PTC-200 MJ Research Thermocycler (MJ Research Inc., USA) at 95°C for 5 min then cycled 35 times of denaturation at 95°C for 40 sec, annealing at the appropriate temperature for 30 sec and extension at 72°C for 30 sec, followed by a final extension step at 72°C for 6 min. Reaction mixture was 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 200 μM each dNTP, 1.5 U Taq DNA polymerase (Invitrogen, Netherlands) or 2.5 U Optimase polymerase (Transgenomic, Inc., USA) and 12.5 pmol of each primer. dHPLC analysis The WAVE DNA Fragment Analysis System (Transgenomic, Inc., USA) and associated WAVE-Maker™ software were used as previously described [ 18 ]. Sequence analysis Purification of the PCR products was performed using the Concert Rapid PCR purification or gel extraction system kits (Marligen Biosciences INC, U.S.A.). Automated cycle sequencing for both strands was performed with the ABI Prism ®  310 Genetic Analyzer using the Big-Dye Terminator Cycle Sequencing Kit. Sequences obtained were aligned, using Sequencher ®  PC software, with normal sequences from Genbank ( BRCA1 : L78833,  BRCA2:  U43746) and examined for the presence of mutations. All nucleotide numbers refer to the wild-type cDNA sequence of  BRCA1  as reported in GenBank (accession number U14680). Multiplex Ligation – dependent PCR Amplification (MLPA) MLPA was carried out using the P002_BRCA1 kit (MRC-Holland, Netherlands) as instructed by the manufacturer. Fragment analysis was carried out on ABI Prism ®  310 Genetic Analyzer (Applied Biosystems, USA) using TAMRA-500 (Applied Biosystems, USA) as size standard. A peak pattern of 34 peaks ranging in size from 127 to 454 nt is obtained [ 19 ]. Long PCR The deletion in  BRCA1  exon 20 was confirmed by long PCR using the GeneAmp XL PCR System (Applied Biosystems, U.S.A.) according to the manufacturer's instructions. PCR was carried out in 50 μl reactions consisting of 1 × buffer II (supplied with the enzyme), 12 pmole each primer, 0.2 mM each dNTP and 2.4 U  rTth  DNA polymerase, XL (Applied Biosystems, U.S.A.). After 2.5 min denaturation at 95°C PCR was carried out for 19 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 8 min followed by 15 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 4 min with a time increment of 10 sec per cycle. A final extension step was carried out at 72°C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized by EtBr staining. RT-PCR Total RNA was extracted from whole blood of patients from family D using Trizol (Life Technologies, USA) according to the manufacturer's instructions. First strand synthesis was performed by denaturing approximately 500 – 1000 ng total RNA, random hexamers (5 μM final concentration) for 4 min at 70°C, followed by snap freezing on ice and addition of dNTPs (0.5 mM final concentration), 1 U/μl recombinant RNase inhibitor (Invitrogen, Netherlands) and 200 U MMLV reverse transcriptase (Invitrogen, Netherlands). The mixture was incubated at 37°C for 1 hour followed by denaturation of the enzymes at 95°C for 5 min. 4 μl of cDNA were used for subsequent PCR amplification.
Methods
Patients BRCA1  patients and their families were referred through the Oncology Departments of Hygeia Hospital and other hospitals throughout Greece. Patients were included on the basis of affected family members, types of cancer present in the family and the age at diagnosis of breast cancer in the proband. The families were subdivided into high risk when multiple cases of breast and ovarian cancer were diagnosed, medium risk if there were only 2–3 cases of breast cancer, and low risk in isolated cases of breast cancer with diagnosis before the age of 40 years. The study population consisted therefore of 12 high risk families, 2 with medium risk, and 4 families at low risk as determined by their cancer history. Ethical approval was obtained from the hospital's advisory committee and all patients signed informed consent. Screening has been completed in 25 individuals from 18 families. Testing was initially carried out on DNA from an affected family member and upon detection of an inactivating mutation the rest of the family members were directly tested for this mutation.
Patients
BRCA1  patients and their families were referred through the Oncology Departments of Hygeia Hospital and other hospitals throughout Greece. Patients were included on the basis of affected family members, types of cancer present in the family and the age at diagnosis of breast cancer in the proband. The families were subdivided into high risk when multiple cases of breast and ovarian cancer were diagnosed, medium risk if there were only 2–3 cases of breast cancer, and low risk in isolated cases of breast cancer with diagnosis before the age of 40 years. The study population consisted therefore of 12 high risk families, 2 with medium risk, and 4 families at low risk as determined by their cancer history. Ethical approval was obtained from the hospital's advisory committee and all patients signed informed consent. Screening has been completed in 25 individuals from 18 families. Testing was initially carried out on DNA from an affected family member and upon detection of an inactivating mutation the rest of the family members were directly tested for this mutation.BRCA1DNA and RNA isolation Genomic DNA and RNA were purified from peripheral blood leukocytes using standard extraction protocols.
DNA and RNA isolation
Genomic DNA and RNA were purified from peripheral blood leukocytes using standard extraction protocols.PCR amplification The complete coding sequence of  BRCA1  including splice junctions was amplified by PCR. Similarly, exons 10 and 11 of the  BRCA2  gene were amplified in 3 of the patients. Primers used have been chosen from the BIC database  . In addition primers: mBRCAF: GAG TTT GTG TGT GAA CGG ACA CTG and mBRCAR: GTG CCA AGG GTG AAT GAT GAA AGC, designed during the course of this study, were used for amplification of cDNA from the patients found to carry a deletion of exon 20 of the  BRCA1  gene. Reactions of 50 μl were heated on a PTC-200 MJ Research Thermocycler (MJ Research Inc., USA) at 95°C for 5 min then cycled 35 times of denaturation at 95°C for 40 sec, annealing at the appropriate temperature for 30 sec and extension at 72°C for 30 sec, followed by a final extension step at 72°C for 6 min. Reaction mixture was 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 200 μM each dNTP, 1.5 U Taq DNA polymerase (Invitrogen, Netherlands) or 2.5 U Optimase polymerase (Transgenomic, Inc., USA) and 12.5 pmol of each primer.
PCR amplification
The complete coding sequence of  BRCA1  including splice junctions was amplified by PCR. Similarly, exons 10 and 11 of the  BRCA2  gene were amplified in 3 of the patients. Primers used have been chosen from the BIC database  . In addition primers: mBRCAF: GAG TTT GTG TGT GAA CGG ACA CTG and mBRCAR: GTG CCA AGG GTG AAT GAT GAA AGC, designed during the course of this study, were used for amplification of cDNA from the patients found to carry a deletion of exon 20 of the  BRCA1  gene.BRCA1BRCA2BRCA1Reactions of 50 μl were heated on a PTC-200 MJ Research Thermocycler (MJ Research Inc., USA) at 95°C for 5 min then cycled 35 times of denaturation at 95°C for 40 sec, annealing at the appropriate temperature for 30 sec and extension at 72°C for 30 sec, followed by a final extension step at 72°C for 6 min. Reaction mixture was 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 200 μM each dNTP, 1.5 U Taq DNA polymerase (Invitrogen, Netherlands) or 2.5 U Optimase polymerase (Transgenomic, Inc., USA) and 12.5 pmol of each primer.dHPLC analysis The WAVE DNA Fragment Analysis System (Transgenomic, Inc., USA) and associated WAVE-Maker™ software were used as previously described [ 18 ].
dHPLC analysis
The WAVE DNA Fragment Analysis System (Transgenomic, Inc., USA) and associated WAVE-Maker™ software were used as previously described [ 18 ].Sequence analysis Purification of the PCR products was performed using the Concert Rapid PCR purification or gel extraction system kits (Marligen Biosciences INC, U.S.A.). Automated cycle sequencing for both strands was performed with the ABI Prism ®  310 Genetic Analyzer using the Big-Dye Terminator Cycle Sequencing Kit. Sequences obtained were aligned, using Sequencher ®  PC software, with normal sequences from Genbank ( BRCA1 : L78833,  BRCA2:  U43746) and examined for the presence of mutations. All nucleotide numbers refer to the wild-type cDNA sequence of  BRCA1  as reported in GenBank (accession number U14680).
Sequence analysis
Purification of the PCR products was performed using the Concert Rapid PCR purification or gel extraction system kits (Marligen Biosciences INC, U.S.A.). Automated cycle sequencing for both strands was performed with the ABI Prism ®  310 Genetic Analyzer using the Big-Dye Terminator Cycle Sequencing Kit. Sequences obtained were aligned, using Sequencher ®  PC software, with normal sequences from Genbank ( BRCA1 : L78833,  BRCA2:  U43746) and examined for the presence of mutations. All nucleotide numbers refer to the wild-type cDNA sequence of  BRCA1  as reported in GenBank (accession number U14680).BRCA1BRCA2:BRCA1Multiplex Ligation – dependent PCR Amplification (MLPA) MLPA was carried out using the P002_BRCA1 kit (MRC-Holland, Netherlands) as instructed by the manufacturer. Fragment analysis was carried out on ABI Prism ®  310 Genetic Analyzer (Applied Biosystems, USA) using TAMRA-500 (Applied Biosystems, USA) as size standard. A peak pattern of 34 peaks ranging in size from 127 to 454 nt is obtained [ 19 ].
Multiplex Ligation – dependent PCR Amplification (MLPA)
MLPA was carried out using the P002_BRCA1 kit (MRC-Holland, Netherlands) as instructed by the manufacturer. Fragment analysis was carried out on ABI Prism ®  310 Genetic Analyzer (Applied Biosystems, USA) using TAMRA-500 (Applied Biosystems, USA) as size standard. A peak pattern of 34 peaks ranging in size from 127 to 454 nt is obtained [ 19 ].Long PCR The deletion in  BRCA1  exon 20 was confirmed by long PCR using the GeneAmp XL PCR System (Applied Biosystems, U.S.A.) according to the manufacturer's instructions. PCR was carried out in 50 μl reactions consisting of 1 × buffer II (supplied with the enzyme), 12 pmole each primer, 0.2 mM each dNTP and 2.4 U  rTth  DNA polymerase, XL (Applied Biosystems, U.S.A.). After 2.5 min denaturation at 95°C PCR was carried out for 19 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 8 min followed by 15 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 4 min with a time increment of 10 sec per cycle. A final extension step was carried out at 72°C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized by EtBr staining.
Long PCR
The deletion in  BRCA1  exon 20 was confirmed by long PCR using the GeneAmp XL PCR System (Applied Biosystems, U.S.A.) according to the manufacturer's instructions. PCR was carried out in 50 μl reactions consisting of 1 × buffer II (supplied with the enzyme), 12 pmole each primer, 0.2 mM each dNTP and 2.4 U  rTth  DNA polymerase, XL (Applied Biosystems, U.S.A.). After 2.5 min denaturation at 95°C PCR was carried out for 19 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 8 min followed by 15 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 4 min with a time increment of 10 sec per cycle. A final extension step was carried out at 72°C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized by EtBr staining.BRCA1rTthRT-PCR Total RNA was extracted from whole blood of patients from family D using Trizol (Life Technologies, USA) according to the manufacturer's instructions. First strand synthesis was performed by denaturing approximately 500 – 1000 ng total RNA, random hexamers (5 μM final concentration) for 4 min at 70°C, followed by snap freezing on ice and addition of dNTPs (0.5 mM final concentration), 1 U/μl recombinant RNase inhibitor (Invitrogen, Netherlands) and 200 U MMLV reverse transcriptase (Invitrogen, Netherlands). The mixture was incubated at 37°C for 1 hour followed by denaturation of the enzymes at 95°C for 5 min. 4 μl of cDNA were used for subsequent PCR amplification.
RT-PCR
Total RNA was extracted from whole blood of patients from family D using Trizol (Life Technologies, USA) according to the manufacturer's instructions. First strand synthesis was performed by denaturing approximately 500 – 1000 ng total RNA, random hexamers (5 μM final concentration) for 4 min at 70°C, followed by snap freezing on ice and addition of dNTPs (0.5 mM final concentration), 1 U/μl recombinant RNase inhibitor (Invitrogen, Netherlands) and 200 U MMLV reverse transcriptase (Invitrogen, Netherlands). The mixture was incubated at 37°C for 1 hour followed by denaturation of the enzymes at 95°C for 5 min. 4 μl of cDNA were used for subsequent PCR amplification.Results A total of 18 families, 12 of which were at high risk of having hereditary breast cancer, have been examined for mutations at the  BRCA1  locus. A pathogenic mutation was identified in five families, where four distinct mutations have been observed. In addition, 18 polymorphisms, including a novel silent polymorphism in exon 11 of the  BRCA1  gene, have been detected (Table  1 ). Furthermore, in 3 of the families exons 10 and 11 of  BRCA2  were analyzed. Table  1  summarizes the results of single nucleotide variants detected in this study. All of the variants have been identified by dHPLC and characterized by sequencing. In family A there were four cases of breast cancer affecting four successive generations. The proband was diagnosed with breast cancer at the age of 45. The most frequently occurring mutation, 5382insC in exon 20, was identified. Analysis of the proband's daughter in whom cancer developed at the age of 38 revealed that she also carried the mutation. The same mutation was also identified in family E where breast cancer was diagnosed in 3 members in three successive generations at 42, 37 and 41 years (data not shown). In family B there were seven cases of breast cancer and one case of colorectal cancer at age 50. Mutation analysis at the  BRCA1  locus revealed a missense mutation 5331 G > A. The mutation results in substitution of a Glycine by an Arginine at codon 1738. The mutation was not detected in the patient's unaffected sister. Unfortunately, no other family members were available for analysis. In family C there were 4 cases of breast cancer in addition to cancer of the larynx, ovaries, lung and the genitals. A single base substitution, G > A at nucleotide 5586 was identified. This mutation causes a splicing defect resulting in a protein lacking exon 23. Unfortunately, no DNA was available from other family members in order to test the correlation of this mutation with the other tumor types. In Family D (Figure  1a ) there were 5 cases of breast cancer with age at onset ranging from 29 to 50 years. In addition there was one individual who had CRC. Sequencing of the complete coding region of  BRCA1,  failed to reveal a mutation. This prompted the analysis of other genes, namely  BRCA2  and  p53  in order to try and characterize the phenotype in this family, but no mutation was identified. For this reason, we decided to use the MLPA technique [ 19 ] to screen the proband for genomic rearrangements which have been shown to be responsible for a large proportion of  BRCA1  mutations. MLPA analysis of the proband (IV:10 in figure  1a ) revealed deletion of exon 20 of the  BRCA1  gene. This was confirmed by Long PCR (Figure  1b ) and RT – PCR. Sequencing of PCR products generated by RT-PCR confirmed absence of the entire exon 20 in the mRNA of the patient. Subsequently, a number of restriction endonucleases were employed in order to narrow down the deletion breakpoints and facilitate their characterization. A  Sma I-generated irregular fragment was finally isolated from agarose gel and sequenced revealing a deletion of 3.2 kb starting 21 bases into exon 20 and extending 3.2 kb into intron 20 (Figure  1c ). The 3' breakpoint lies within an  Alu Sp repeat but there are no recognizable repeat motifs at the 5' breakpoint. The deletion was also found in 4 relatives of the proband (Figure  1a ) who were tested, two of whom had breast cancer. The other two relatives had not yet developed cancer at the ages of 36 and 55 at the time of testing and are therefore assumed to be pre – symptomatic carriers. Finally, the proband's sister, who at the time of testing had not developed cancer at the age of 33 was found not to carry the mutation.
Results
A total of 18 families, 12 of which were at high risk of having hereditary breast cancer, have been examined for mutations at the  BRCA1  locus. A pathogenic mutation was identified in five families, where four distinct mutations have been observed. In addition, 18 polymorphisms, including a novel silent polymorphism in exon 11 of the  BRCA1  gene, have been detected (Table  1 ). Furthermore, in 3 of the families exons 10 and 11 of  BRCA2  were analyzed. Table  1  summarizes the results of single nucleotide variants detected in this study. All of the variants have been identified by dHPLC and characterized by sequencing.BRCA1BRCA1BRCA2In family A there were four cases of breast cancer affecting four successive generations. The proband was diagnosed with breast cancer at the age of 45. The most frequently occurring mutation, 5382insC in exon 20, was identified. Analysis of the proband's daughter in whom cancer developed at the age of 38 revealed that she also carried the mutation. The same mutation was also identified in family E where breast cancer was diagnosed in 3 members in three successive generations at 42, 37 and 41 years (data not shown).In family B there were seven cases of breast cancer and one case of colorectal cancer at age 50. Mutation analysis at the  BRCA1  locus revealed a missense mutation 5331 G > A. The mutation results in substitution of a Glycine by an Arginine at codon 1738. The mutation was not detected in the patient's unaffected sister. Unfortunately, no other family members were available for analysis.BRCA1In family C there were 4 cases of breast cancer in addition to cancer of the larynx, ovaries, lung and the genitals. A single base substitution, G > A at nucleotide 5586 was identified. This mutation causes a splicing defect resulting in a protein lacking exon 23. Unfortunately, no DNA was available from other family members in order to test the correlation of this mutation with the other tumor types.In Family D (Figure  1a ) there were 5 cases of breast cancer with age at onset ranging from 29 to 50 years. In addition there was one individual who had CRC. Sequencing of the complete coding region of  BRCA1,  failed to reveal a mutation. This prompted the analysis of other genes, namely  BRCA2  and  p53  in order to try and characterize the phenotype in this family, but no mutation was identified. For this reason, we decided to use the MLPA technique [ 19 ] to screen the proband for genomic rearrangements which have been shown to be responsible for a large proportion of  BRCA1  mutations. MLPA analysis of the proband (IV:10 in figure  1a ) revealed deletion of exon 20 of the  BRCA1  gene. This was confirmed by Long PCR (Figure  1b ) and RT – PCR. Sequencing of PCR products generated by RT-PCR confirmed absence of the entire exon 20 in the mRNA of the patient. Subsequently, a number of restriction endonucleases were employed in order to narrow down the deletion breakpoints and facilitate their characterization. A  Sma I-generated irregular fragment was finally isolated from agarose gel and sequenced revealing a deletion of 3.2 kb starting 21 bases into exon 20 and extending 3.2 kb into intron 20 (Figure  1c ). The 3' breakpoint lies within an  Alu Sp repeat but there are no recognizable repeat motifs at the 5' breakpoint. The deletion was also found in 4 relatives of the proband (Figure  1a ) who were tested, two of whom had breast cancer. The other two relatives had not yet developed cancer at the ages of 36 and 55 at the time of testing and are therefore assumed to be pre – symptomatic carriers. Finally, the proband's sister, who at the time of testing had not developed cancer at the age of 33 was found not to carry the mutation.BRCA1,BRCA2p53BRCA1BRCA1SmaAluDiscussion A total of 18 breast cancer families have been examined for mutations at the  BRCA1  locus. In three of the families analysis was extended to exons 10 and 11 of the  BRCA2  gene. A pathogenic mutation in  BRCA1  was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. In addition, 18 polymorphisms have been found to be present in more than one family. In family B a missense mutation, 5331 G > A, was identified. The exact effect of this single amino acid change, G1738R, on the protein function is unclear. The altered glycine is located on the surface of the coil structure of the BRCT linker region and the mutation therefore may disrupt the interface or affect protein interaction [ 20 ]. This mutation has been previously described in 4 unrelated Greek patients [ 21 ]. An alternative mutation affecting the same amino acid, G1738E, has been shown to result in loss of function of the protein  in vitro  [ 22 , 23 ]. Based on this information, the family history of the individual described here and absence of the mutation in the proband's unaffected sister we hypothesize that the mutation is pathogenic although additional data are needed. Our results indicate, as has been documented by others that family history is the major determinant of the risk of breast cancer. As can be seen in Table  1  in all families where a pathogenic mutation was identified there were at least 3 cases of breast cancer. In addition, the extremely high risk suggested by the family history in some cases prompted a more intense analysis of further genes and approaches in an attempt to characterize the underling reason for such a family history. This was the case in family D where sequencing of the complete coding region of  BRCA1,  failed to reveal a mutation. This prompted the analysis of other genes, namely  BRCA2  and  p53 , but without finding a mutation. For this reason, we decided to use the MLPA technique [ 19 ] to screen the proband for genomic rearrangements which have been shown to be responsible for a large proportion of  BRCA1  mutations [ 12 - 16 ]. This led to the identification of deletion of an entire exon of the  BRCA1  gene, namely exon 20. Deletion of exon 20 has been previously described in an Italian family [ 12 ]. However, the authors of that report have localized the breakpoints of the deletion in the two flanking introns [ 12 ]. In the deletion described here the 5' breakpoint is localized inside exon 20 (21 bp upstream of the splice donor site) while the 3' breakpoint is located 3.2 kb into intron 20 within an  Alu Sp repeat. The deletion described here, therefore, is different to the majority of rearrangements described so far for the  BRCA1  gene, since they have been shown to result from homologous recombination of  Alu  repeats [ 24 , 25 ]. In this case, the 5' breakpoint does not correlate with any recognizable repeat motifs suggesting a repeat – independent recombination mechanism at play. In 2/5 pedigrees in whom a pathogenic mutation was identified there was one case each of colorectal cancer (CRC). In particular, in family D (Figure  1a ) the disease seems to have originated from a CRC patient. Data on the correlation between  BRCA1  mutations and the risk of CRC are not conclusive. Two recent studies carried out on Ashkenazi Jewish patients suggest that there is no correlation [ 26 , 27 ]. Other reports carried out on more diverse populations suggest 2- to 3-fold elevated risks of CRC among first-degree relatives of  BRCA1  mutation carriers [ 28 , 29 ]. Unfortunately, there was no DNA available for analysis from the two CRC patients in the two families. A further interesting point observed in this study is the identification of two  BRCA1  unaffected mutation carriers at the ages of 39 and 55. The penetrance and age of onset of disease in  BRCA1  mutation carriers is variable. Various reports have suggested the existence of modifying genetic and environmental factors on the penetrance of  BRCA1  and  BRCA2  mutation carriers [ 30 , 31 ]. In this respect it is interesting to examine this large pedigree for such modifying factors.
Discussion
A total of 18 breast cancer families have been examined for mutations at the  BRCA1  locus. In three of the families analysis was extended to exons 10 and 11 of the  BRCA2  gene. A pathogenic mutation in  BRCA1  was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. In addition, 18 polymorphisms have been found to be present in more than one family.BRCA1BRCA2BRCA1In family B a missense mutation, 5331 G > A, was identified. The exact effect of this single amino acid change, G1738R, on the protein function is unclear. The altered glycine is located on the surface of the coil structure of the BRCT linker region and the mutation therefore may disrupt the interface or affect protein interaction [ 20 ]. This mutation has been previously described in 4 unrelated Greek patients [ 21 ]. An alternative mutation affecting the same amino acid, G1738E, has been shown to result in loss of function of the protein  in vitro  [ 22 , 23 ]. Based on this information, the family history of the individual described here and absence of the mutation in the proband's unaffected sister we hypothesize that the mutation is pathogenic although additional data are needed.in vitroOur results indicate, as has been documented by others that family history is the major determinant of the risk of breast cancer. As can be seen in Table  1  in all families where a pathogenic mutation was identified there were at least 3 cases of breast cancer. In addition, the extremely high risk suggested by the family history in some cases prompted a more intense analysis of further genes and approaches in an attempt to characterize the underling reason for such a family history. This was the case in family D where sequencing of the complete coding region of  BRCA1,  failed to reveal a mutation. This prompted the analysis of other genes, namely  BRCA2  and  p53 , but without finding a mutation. For this reason, we decided to use the MLPA technique [ 19 ] to screen the proband for genomic rearrangements which have been shown to be responsible for a large proportion of  BRCA1  mutations [ 12 - 16 ]. This led to the identification of deletion of an entire exon of the  BRCA1  gene, namely exon 20.BRCA1,BRCA2p53BRCA1BRCA1Deletion of exon 20 has been previously described in an Italian family [ 12 ]. However, the authors of that report have localized the breakpoints of the deletion in the two flanking introns [ 12 ]. In the deletion described here the 5' breakpoint is localized inside exon 20 (21 bp upstream of the splice donor site) while the 3' breakpoint is located 3.2 kb into intron 20 within an  Alu Sp repeat. The deletion described here, therefore, is different to the majority of rearrangements described so far for the  BRCA1  gene, since they have been shown to result from homologous recombination of  Alu  repeats [ 24 , 25 ]. In this case, the 5' breakpoint does not correlate with any recognizable repeat motifs suggesting a repeat – independent recombination mechanism at play.AluBRCA1AluIn 2/5 pedigrees in whom a pathogenic mutation was identified there was one case each of colorectal cancer (CRC). In particular, in family D (Figure  1a ) the disease seems to have originated from a CRC patient. Data on the correlation between  BRCA1  mutations and the risk of CRC are not conclusive. Two recent studies carried out on Ashkenazi Jewish patients suggest that there is no correlation [ 26 , 27 ]. Other reports carried out on more diverse populations suggest 2- to 3-fold elevated risks of CRC among first-degree relatives of  BRCA1  mutation carriers [ 28 , 29 ]. Unfortunately, there was no DNA available for analysis from the two CRC patients in the two families.BRCA1BRCA1A further interesting point observed in this study is the identification of two  BRCA1  unaffected mutation carriers at the ages of 39 and 55. The penetrance and age of onset of disease in  BRCA1  mutation carriers is variable. Various reports have suggested the existence of modifying genetic and environmental factors on the penetrance of  BRCA1  and  BRCA2  mutation carriers [ 30 , 31 ]. In this respect it is interesting to examine this large pedigree for such modifying factors.BRCA1BRCA1BRCA1BRCA2Conclusions To our knowledge this is the first report of a genomic rearrangement identified as the underlying mutation of  BRCA1  in a Greek family. Although our sample group is quite small identification of 1 genomic rearrangement in 5 mutations detected, suggests that in yet another population this type of mutations contribute to the  BRCA1  mutation spectrum. This therefore warrants use of a combination of techniques capable of identifying both single base mutations in addition to large genomic rearrangements. In this respect, we have found that use of dHPLC for single base mutations and MLPA for large genomic rearrangements is a reliable combination for use as an initial screening step followed by sequencing for characterization of the mutations identified. Furthermore, our results suggest that in those families with strong evidence of linkage to the  BRCA1  locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.
Conclusions
To our knowledge this is the first report of a genomic rearrangement identified as the underlying mutation of  BRCA1  in a Greek family. Although our sample group is quite small identification of 1 genomic rearrangement in 5 mutations detected, suggests that in yet another population this type of mutations contribute to the  BRCA1  mutation spectrum. This therefore warrants use of a combination of techniques capable of identifying both single base mutations in addition to large genomic rearrangements. In this respect, we have found that use of dHPLC for single base mutations and MLPA for large genomic rearrangements is a reliable combination for use as an initial screening step followed by sequencing for characterization of the mutations identified.BRCA1BRCA1Furthermore, our results suggest that in those families with strong evidence of linkage to the  BRCA1  locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.BRCA1List of abbreviations dHPLC  denaturing High Performance Liquid Chromatography MLPA  Multiplex Ligation – dependent PCR Amplification PCR  Polymerase Chain Reaction RT-PCR  Reverse Transcription Polymerase Chain Reaction CRC  Colorectal Cancer
List of abbreviations
dHPLC  denaturing High Performance Liquid ChromatographydHPLCMLPA  Multiplex Ligation – dependent PCR AmplificationMLPAPCR  Polymerase Chain ReactionPCRRT-PCR  Reverse Transcription Polymerase Chain ReactionRT-PCRCRC  Colorectal CancerCRCCompeting interests None declared.
Competing interests
None declared.Authors contributions IB carried out mutation detection by dHPLC and sequencing for the patients analyzed. AA carried out MLPA, the molecular characterization of the deletion, and drafted the manuscript. MM contributed in the molecular studies and preparation of the manuscript and figures, ER, SL, AP, VG, AK NP, PK and DY provided the patient material, diagnosis and management,, KK and AH carried out molecular genetic analysis of the p53 gene, GN conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Authors contributions
IB carried out mutation detection by dHPLC and sequencing for the patients analyzed. AA carried out MLPA, the molecular characterization of the deletion, and drafted the manuscript. MM contributed in the molecular studies and preparation of the manuscript and figures, ER, SL, AP, VG, AK NP, PK and DY provided the patient material, diagnosis and management,, KK and AH carried out molecular genetic analysis of the p53 gene, GN conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background The lifetime prevalence of Social Phobia (SP) in European countries other than Italy has been estimated to range from 3.5% to 16.0%. The aim of this study was to assess the frequency of SP in Sardinia (Italy) in order to verify the evidence of a lower frequency of SP in Italy observed in previous studies (from 1.0% to 3.1%).
Background
The lifetime prevalence of Social Phobia (SP) in European countries other than Italy has been estimated to range from 3.5% to 16.0%. The aim of this study was to assess the frequency of SP in Sardinia (Italy) in order to verify the evidence of a lower frequency of SP in Italy observed in previous studies (from 1.0% to 3.1%).Methods A randomised cross sample of 1040 subjects, living in Cagliari, in rural areas, and in a mining district in Sardinia were interviewed using a Simplified version of the Composite International Diagnostic Interview (CIDIS). Diagnoses were made according to the 10 th  International Classification of Diseases (ICD-10).
Methods
A randomised cross sample of 1040 subjects, living in Cagliari, in rural areas, and in a mining district in Sardinia were interviewed using a Simplified version of the Composite International Diagnostic Interview (CIDIS). Diagnoses were made according to the 10 th  International Classification of Diseases (ICD-10).Results Lifetime prevalence of SP was 2.2% (males: 1.5%, females: 2.8%) whereas 6-month prevalence resulted in 1.5% (males: 0.9%, females: 2.1%). Mean age at onset was 16.2 ± 9.3 years. A statistically significant association was found with Depressive Episode, Dysthymia and Generalized Anxiety Disorder.
Results
Lifetime prevalence of SP was 2.2% (males: 1.5%, females: 2.8%) whereas 6-month prevalence resulted in 1.5% (males: 0.9%, females: 2.1%). Mean age at onset was 16.2 ± 9.3 years. A statistically significant association was found with Depressive Episode, Dysthymia and Generalized Anxiety Disorder.Conclusions The study is consistent with findings reported in several previous studies of a lower prevalence of SP in Italy. Furthermore, the results confirm the fact that SP, due to its early onset, might constitute an ideal target for early treatment aimed at preventing both the accumulation of social disabilities and impairments caused by anxiety and avoidance behaviour, as well as the onset of more serious, associated complications in later stages of the illness.
Conclusions
The study is consistent with findings reported in several previous studies of a lower prevalence of SP in Italy. Furthermore, the results confirm the fact that SP, due to its early onset, might constitute an ideal target for early treatment aimed at preventing both the accumulation of social disabilities and impairments caused by anxiety and avoidance behaviour, as well as the onset of more serious, associated complications in later stages of the illness.

Background Several epidemiological studies have attempted to describe the prevalence, socio-demographic characteristics, comorbidity, and severity of clinical manifestations of Social Phobia (SP). Quality of life and functional status of affected individuals have also been investigated [ 1 ]. Lifetime prevalence of SP in European societies other than Italy ranges from 3.5% to 16.0% [ 2 ]. As discussed in several excellent review papers, these rate differences were partly attributed to probable genetic or cultural factors [ 3 ]. Furthermore, major methodological differences (type of diagnostic criteria used, assessment tools, age of the sample) affecting the estimates have been demonstrated [ 1 ]. This study, part of an extended epidemiological investigation "Health in Sardinia," aimed to assess the prevalence rates of SP in Sardinia (Italy) in order to confirm the evidence of low SP prevalence rates ranging from 1.0% to 3.1% observed in previous research projects in Italy [ 4 - 6 ]. The study also intended to evaluate the treatments and to verify the comorbid psychiatric disorders in the identified people with Social Phobia.
Background
Several epidemiological studies have attempted to describe the prevalence, socio-demographic characteristics, comorbidity, and severity of clinical manifestations of Social Phobia (SP). Quality of life and functional status of affected individuals have also been investigated [ 1 ]. Lifetime prevalence of SP in European societies other than Italy ranges from 3.5% to 16.0% [ 2 ]. As discussed in several excellent review papers, these rate differences were partly attributed to probable genetic or cultural factors [ 3 ]. Furthermore, major methodological differences (type of diagnostic criteria used, assessment tools, age of the sample) affecting the estimates have been demonstrated [ 1 ].This study, part of an extended epidemiological investigation "Health in Sardinia," aimed to assess the prevalence rates of SP in Sardinia (Italy) in order to confirm the evidence of low SP prevalence rates ranging from 1.0% to 3.1% observed in previous research projects in Italy [ 4 - 6 ]. The study also intended to evaluate the treatments and to verify the comorbid psychiatric disorders in the identified people with Social Phobia.Methods The sample, which had already been examined and described in greater detail in a previously published study [ 7 ], consisted of 1040 subjects recruited throughout the island of Sardinia, Italy. 393 subjects came from the city of Cagliari, 344 from rural areas, and 303 from an industrial mining district, thus representing fairly well all socio-economic strata present on the island. The age distribution of the sample is shown in table  1 , with age range from 18 to 89 years. 79.2% out of a total of 1313 subjects approached, agreed to take part in the study, 12.5% refused to participate, and 8.3% could not be traced; the final sample did not differ respect to the population of origin with reference to the variables applied in stratification (Table  1 ). All subjects were interviewed "face to face" by trained physicians using a Simplified version of Composite International Diagnostic Interview (CIDI) [ 8 ], hence the acronym CIDI "Simplified" (CIDIS) [ 9 , 10 ]. The version used in this study had been translated into Italian and back-translated into the original French language under blind conditions respect to the first translation, by a bilingual researcher; approval of the final version was obtained from the original authors [ 11 ]. The CIDI structured interview in its various versions currently represents the most widely used diagnostic tool in psychiatric epidemiological studies conducted on the general population [ 12 ]. The CIDIS is a highly structured tool made up of 5 sections which investigate respectively: Somatoform Disorders and General Medical Conditions, Anxiety Disorders, Depressive Disorders, Substance-Related Disorders (Alcohol-Related Disorders and Substance-Related Disorders) and Eating Disorders. The computer elaboration of data obtained through application of the CIDIS enables calculation of both "lifetime prevalence" and, for the preceding six months, a series of psychiatric disorders (those more frequently observed in the general population) according to the ICD-10 diagnostic system [ 13 ]. This interview moreover enhances identification of both the type of therapist referred to and treatment already used by each patient for his or her specific problem and definition of degree of impact of the problem on the subject's daily routine. An ad hoc computer algorithm ascertained the presence of the disorders according to ICD-10 criteria [ 13 ], both in the past six months and in the lifetime. The items of the CIDIS concerning Social Phobia and the related algorithm is reported in Figure  1 . Table 1 Percentage of subdivision according to age, sex, and marital status of the sample. N (1040) MALES 461 (44.3%) FEMALES 579 (55.7%) AGE 18–24 146 (14.0%) AGE 25–44 353 (33.9%) AGE 45–64 310 (29.8%) AGE >64 231 (22.2%) UNMARRIED 370 (35.6%) MARRIED 571 (54.9%) WIDOWED/SEPARATED/DIVORCED 99 (9.5%) Figure 1 The Composite International Diagnostic Interview Simplified (CIDIS) [9, 10] algorithm for the diagnosis of Social Phobia.
Methods
The sample, which had already been examined and described in greater detail in a previously published study [ 7 ], consisted of 1040 subjects recruited throughout the island of Sardinia, Italy. 393 subjects came from the city of Cagliari, 344 from rural areas, and 303 from an industrial mining district, thus representing fairly well all socio-economic strata present on the island. The age distribution of the sample is shown in table  1 , with age range from 18 to 89 years. 79.2% out of a total of 1313 subjects approached, agreed to take part in the study, 12.5% refused to participate, and 8.3% could not be traced; the final sample did not differ respect to the population of origin with reference to the variables applied in stratification (Table  1 ). All subjects were interviewed "face to face" by trained physicians using a Simplified version of Composite International Diagnostic Interview (CIDI) [ 8 ], hence the acronym CIDI "Simplified" (CIDIS) [ 9 , 10 ]. The version used in this study had been translated into Italian and back-translated into the original French language under blind conditions respect to the first translation, by a bilingual researcher; approval of the final version was obtained from the original authors [ 11 ]. The CIDI structured interview in its various versions currently represents the most widely used diagnostic tool in psychiatric epidemiological studies conducted on the general population [ 12 ]. The CIDIS is a highly structured tool made up of 5 sections which investigate respectively: Somatoform Disorders and General Medical Conditions, Anxiety Disorders, Depressive Disorders, Substance-Related Disorders (Alcohol-Related Disorders and Substance-Related Disorders) and Eating Disorders. The computer elaboration of data obtained through application of the CIDIS enables calculation of both "lifetime prevalence" and, for the preceding six months, a series of psychiatric disorders (those more frequently observed in the general population) according to the ICD-10 diagnostic system [ 13 ]. This interview moreover enhances identification of both the type of therapist referred to and treatment already used by each patient for his or her specific problem and definition of degree of impact of the problem on the subject's daily routine. An ad hoc computer algorithm ascertained the presence of the disorders according to ICD-10 criteria [ 13 ], both in the past six months and in the lifetime. The items of the CIDIS concerning Social Phobia and the related algorithm is reported in Figure  1 .Percentage of subdivision according to age, sex, and marital status of the sample.Results Estimates of lifetime SP prevalence of 2.2% were found (males: 1.5%, females: 2.8%), with no statistically significant difference between the sexes (χ 2  = 1.2, 1DF, P = 0.25). 6-month prevalence rates were lower (total: 1.5%, males: 0.9%; females: 1%; no significant difference between the sexes, χ 2  = 1.6, 1DF, P = 0.13). Table  2  compares results obtained in this study with those of the major research projects carried out in Europe and the USA [ 4 , 5 , 14 - 23 ]. Table  3  illustrates the lifetime prevalence according to age and sex, and Table  4  shows the 6-month prevalence according to age and sex. An increased frequency of SP among the younger age groups was observed, although distribution in males resulted as being less homogeneous. In no case statistical significance was reached. Lifetime prevalence rates of SP respect to marital status of subjects studied were as follows: 3.0% among the unmarried; 1.2% among married subjects; and 3.5% among the divorced, separated and widowed (χ 2  = 5.8, 1DF, P = 0.06). Mean age at onset was 16.2 ± 9.3 years. Table 2 Lifetime prevalence of Social Phobia in the general population of Europe and USA. Country Reference Diagnostic criteria N Male Female Total Italy Faravelli et al., 1989 [4] DSM-III-R 1110 1.0 Switzerland Wacker et al., 1992 [16] DSM-III-R 470 16.0 ICD-10 9.6 ECA (USA) Schneier et al., 1992 [15] DIS 18571 2.0 3.1 2.4 Iceland Lindal and Stefansson, 1993 [32] DSM-III 862 2.5 4.5 3.5 Switzerland Degonda and Angst, 1993 [21] DSM-III 591 3.1 5.7 4.4 NCS (USA) Kessler et al., 1994 [17] CIDI 8098 11.1 15.5 13.3 France Lepine and Lellouch, 1995 [19] 2.1 5.4 4.1 Germany Wittchen et al., 1998 [23] DSM-IV 3021 2.2 4.8 3.5 Italy Carta and Rudas, 1998 [6] CIDI 783 1.7 4.6 3.1 Spain Arillo et al., 1998 [cited in 33] DIS 237 8.9 Netherlands Bijl et al., 1998 [20] DSM-III-R 7076 5.9 9.7 7.8 France Lépine and Pélissolo, 1999 [22] DSM-IV 7.3 Italy Faravelli et al., 2000 [5] FPI/CIDI 2355 1.9 4.0 3.1 Italy Carta et al., 2002 [7] CIDI 1040 1.5 2.8 2.2 Table 3 Lifetime prevalence N (%) of Social Phobia according to age and sex. Age Male OR Female OR Total OR <25 1 (1.3) 0.9 4 (5.0) 2.3 5 (3.4) 1.7 25–44 4 (2.5) 2.5 4 (2.1) 0.7 8 (2.2) 1.1 45–64 1 (0.7) 0.4 4 (2.4) 0.8 5 (1.6) 0.6 >65 1 (1.1) 0.7 4 (2.8) 1.1 5 (2.2) 0.9 Male according to age χ 2  with Yathes correction = 1.6, 3 df, p = 0.89; female according to age χ 2  with Yathes correction = 2.2, 3 df, p < 0.71; total sample according to age χ 2  with Yathes correction = 1.8, 3 df, p = 0.82 Table 4 Six month prevalence N (%) of Social Phobia according to age and sex. Age Male OR Female OR Total OR <25 1 (1.5) 1.8 3 (3.7) 2.0 4 (2.7) 2.1 25–44 1 (0.6) 0.6 3 (0.7) 0.5 4 (1.1) 0.6 45–64 1 (0.7) 0.7 2 (1.1) 0.5 3 (0.9) 0.5 >65 1 (1.1) 1.4 4 (2.8) 1.4 5 (2.1) 1.6 Male according to age χ 2  = 0.6, 3 df, p = 0.98; female according to age χ 2  with Yathes correction = 3.4, 3 df, p = 0.44; total sample according to age χ 2  with Yathes correction = 3.4, 3 df, p = 0.44 During the week prior to the study, 8 (50.0%) out of the 16 subjects who had been diagnosed with SP over the past six months had been taking low doses of anxiolytics (less than the equivalent of 2 mg of Lorazepam). 3 (18.7%) were on antidepressants, one of whom (6.2%) at non-therapeutic doses, and 1 subject (6.2%) was undergoing cognitive behavioural psychotherapy. The remaining 6 subjects (37.5%) were not on any treatment. Six out of the 10 treated subjects (60%) were being supervised by their general practitioners (GP), 1 (10%) by a neurologist and 2 (20%) by psychiatrists. All subjects presented some degree of comorbidity with Depressive Episodes (DE), Panic Attack Disorder (PAD), and agoraphobia (AP). Only 1 subject (10%) was undergoing cognitive behavioural therapy with a psychologist/psychotherapist. Table  5  illustrates the rate of comorbidity with major psychiatric disorders observed in the general population, as well as the degree (OR) of associated disorders observed with regard to frequency reported for the latter in populations not affected by SP. A statistically significant difference was revealed for association with DE, Dysthymia (DD), and Generalized Anxiety Disorder (GAD). In spite of their increased frequency among patients affected by SP, disorders such as PAD and Specific Phobia do not represent a statistically significant association. The mean age at onset of comorbid DE was 6.5 ± 6.6 years subsequent to onset of SP, whereas GAD was manifested at a mean of 4.3 ± 7.8 years later. Table 5 Lifetime comorbidity of Social Phobia. N (%) OR χ 2 Depressive Episode (DE) 9 (39.1) 4.3 11.1* Dysthymia (DD) 5 (21.7) 7.1 14.1* Generalized Anxiety Disorder (GAD) 10 (43.4) 6.5 20.9* Panic Attack Disorder (PAD) 2 (8.7) 3.3 1.1 Specific Phobia 1 (4.3) 8.6 1.6 *p < 0.001
Results
Estimates of lifetime SP prevalence of 2.2% were found (males: 1.5%, females: 2.8%), with no statistically significant difference between the sexes (χ 2  = 1.2, 1DF, P = 0.25). 6-month prevalence rates were lower (total: 1.5%, males: 0.9%; females: 1%; no significant difference between the sexes, χ 2  = 1.6, 1DF, P = 0.13). Table  2  compares results obtained in this study with those of the major research projects carried out in Europe and the USA [ 4 , 5 , 14 - 23 ]. Table  3  illustrates the lifetime prevalence according to age and sex, and Table  4  shows the 6-month prevalence according to age and sex. An increased frequency of SP among the younger age groups was observed, although distribution in males resulted as being less homogeneous. In no case statistical significance was reached. Lifetime prevalence rates of SP respect to marital status of subjects studied were as follows: 3.0% among the unmarried; 1.2% among married subjects; and 3.5% among the divorced, separated and widowed (χ 2  = 5.8, 1DF, P = 0.06). Mean age at onset was 16.2 ± 9.3 years.Lifetime prevalence of Social Phobia in the general population of Europe and USA.Lifetime prevalence N (%) of Social Phobia according to age and sex.Male according to age χ 2  with Yathes correction = 1.6, 3 df, p = 0.89; female according to age χ 2  with Yathes correction = 2.2, 3 df, p < 0.71; total sample according to age χ 2  with Yathes correction = 1.8, 3 df, p = 0.82Six month prevalence N (%) of Social Phobia according to age and sex.Male according to age χ 2  = 0.6, 3 df, p = 0.98; female according to age χ 2  with Yathes correction = 3.4, 3 df, p = 0.44; total sample according to age χ 2  with Yathes correction = 3.4, 3 df, p = 0.44During the week prior to the study, 8 (50.0%) out of the 16 subjects who had been diagnosed with SP over the past six months had been taking low doses of anxiolytics (less than the equivalent of 2 mg of Lorazepam). 3 (18.7%) were on antidepressants, one of whom (6.2%) at non-therapeutic doses, and 1 subject (6.2%) was undergoing cognitive behavioural psychotherapy. The remaining 6 subjects (37.5%) were not on any treatment.Six out of the 10 treated subjects (60%) were being supervised by their general practitioners (GP), 1 (10%) by a neurologist and 2 (20%) by psychiatrists. All subjects presented some degree of comorbidity with Depressive Episodes (DE), Panic Attack Disorder (PAD), and agoraphobia (AP). Only 1 subject (10%) was undergoing cognitive behavioural therapy with a psychologist/psychotherapist.Table  5  illustrates the rate of comorbidity with major psychiatric disorders observed in the general population, as well as the degree (OR) of associated disorders observed with regard to frequency reported for the latter in populations not affected by SP. A statistically significant difference was revealed for association with DE, Dysthymia (DD), and Generalized Anxiety Disorder (GAD). In spite of their increased frequency among patients affected by SP, disorders such as PAD and Specific Phobia do not represent a statistically significant association. The mean age at onset of comorbid DE was 6.5 ± 6.6 years subsequent to onset of SP, whereas GAD was manifested at a mean of 4.3 ± 7.8 years later.Lifetime comorbidity of Social Phobia.*p < 0.001Discussion Several epidemiological studies carried out in Europe (Switzerland [ 16 , 21 ], France [ 19 , 22 ], Germany [ 23 ]) and in the USA [ 15 , 17 ], recently reviewed by Furmark [ 2 ], suggest that SP is one of the more frequently observed anxiety disorders in the general population in Western countries. However, frequency rates reported in the various studies differ from country to country and according to time and evaluation methods used. Indeed, the two American studies [ 15 , 17 ] carried out at an interval of approximately 15 years, illustrate distinctly contrasting results, and it is hard to establish what factors really determine variance in findings. The present study is consistent with the tendency towards rather low lifetime prevalence rates of SP observed in other Italian research projects. If we take into account only those European researches that adopted ICD-10 diagnosis, our results seem to indicate a lower frequency than a study carried out in Formentera, Spain (lifetime prevalence of 2.8% against 8.9% in females [cited in [ 33 ]]) and Basel, Switzerland (lifetime prevalence in the total sample 2.2 against 9.6% [ 16 ]). However, lower frequencies emerged also in Italian surveys conducted using different methods [ 5 , 7 ]. The Italian studies were carried out over a considerable period of time and in two distinct areas: the Florence area [ 4 , 5 ] and Sardinia [[ 6 ], the present study]. It is therefore quite likely that the lower frequencies observed may be the result of an effectively reduced vulnerability of Italians to SP. The results of this study can indeed be assumed as being influenced by several cultural variables – the genetic diversity of the two populations examined, as well as the considerable genetic variance of the Sardinian population respect to other European populations [ 24 , 25 ]. Findings for six-month prevalence show lower frequencies than those evidenced in the recent studies: 1.5% against 4.0% in Iceland [ 26 ] and against 4.0% in Munich [ 14 ]. Our rates are lower than E.C.A. findings: 2.7% in Duke [ 27 ] and 2.2% in Baltimore, New Haven, and Saint Louis [ 28 ]. The 6-month prevalence rates obtained in Sardinia are similar only to data reported for Edmonton (1.2%) from a survey carried out more than 15 years ago [ 29 ] and published in 1994 [ 30 ]. However, the prevalence data emerging from this study further justify the considerable interest shown in this disorder from both a medical and a social point of view. This research confirms the fact that onset of the illness occurs primarily during childhood and adolescence [ 31 ], thus underlining the suitability of the condition as a candidate for early treatment aimed at preventing both the slow accumulation of social disabilities and impairment caused by anxiety and by avoidance behaviour, as well as the onset of more serious complications (e.g., DE or GAD), which may be manifested many years after onset of SP. Indeed, subjects affected by social phobia presented a high risk of comorbidity with both the latter disorders and DD [ 1 ] . It should be underlined that 60% of subjects undergoing treatment (not all affected subjects) chose their general practitioner (GP). This view is corroborated by the fact that those patients treated by a psychiatrist invariably presented comorbidity with DE, PAD, and AP, thereby representing the more severely affected from a psychopathological point of view. Overall however, the low rate of patients with SP treated with first line-treatments is alarmingly low. In the future, serious attempts should be made to improve the GPs' abilities to recognise SP in order to prevent the use of inappropriate treatments, such as insufficient doses of benzodiazepines, which may be linked to the physician's incorrect diagnosing of the disorder. Due to the fact that subjects affected by SP most frequently refer to their GP, the importance of preventing SP as opposed to other types of disorders, as well as the markedly incapacitating nature of SP reinforce this necessity for a better training of GPs. It is however mandatory to briefly acknowledge some potential limitations of this study. First, the number of cases and the sample size is too small to allow firm conclusions to be drawn concerning the true rate and the degree to which they might actually differ from previous studies with higher estimates. Secondly, differences in the assessment strategy might have resulted in a diminished comparability.
Discussion
Several epidemiological studies carried out in Europe (Switzerland [ 16 , 21 ], France [ 19 , 22 ], Germany [ 23 ]) and in the USA [ 15 , 17 ], recently reviewed by Furmark [ 2 ], suggest that SP is one of the more frequently observed anxiety disorders in the general population in Western countries. However, frequency rates reported in the various studies differ from country to country and according to time and evaluation methods used. Indeed, the two American studies [ 15 , 17 ] carried out at an interval of approximately 15 years, illustrate distinctly contrasting results, and it is hard to establish what factors really determine variance in findings.The present study is consistent with the tendency towards rather low lifetime prevalence rates of SP observed in other Italian research projects. If we take into account only those European researches that adopted ICD-10 diagnosis, our results seem to indicate a lower frequency than a study carried out in Formentera, Spain (lifetime prevalence of 2.8% against 8.9% in females [cited in [ 33 ]]) and Basel, Switzerland (lifetime prevalence in the total sample 2.2 against 9.6% [ 16 ]). However, lower frequencies emerged also in Italian surveys conducted using different methods [ 5 , 7 ]. The Italian studies were carried out over a considerable period of time and in two distinct areas: the Florence area [ 4 , 5 ] and Sardinia [[ 6 ], the present study]. It is therefore quite likely that the lower frequencies observed may be the result of an effectively reduced vulnerability of Italians to SP. The results of this study can indeed be assumed as being influenced by several cultural variables – the genetic diversity of the two populations examined, as well as the considerable genetic variance of the Sardinian population respect to other European populations [ 24 , 25 ].Findings for six-month prevalence show lower frequencies than those evidenced in the recent studies: 1.5% against 4.0% in Iceland [ 26 ] and against 4.0% in Munich [ 14 ]. Our rates are lower than E.C.A. findings: 2.7% in Duke [ 27 ] and 2.2% in Baltimore, New Haven, and Saint Louis [ 28 ]. The 6-month prevalence rates obtained in Sardinia are similar only to data reported for Edmonton (1.2%) from a survey carried out more than 15 years ago [ 29 ] and published in 1994 [ 30 ]. However, the prevalence data emerging from this study further justify the considerable interest shown in this disorder from both a medical and a social point of view. This research confirms the fact that onset of the illness occurs primarily during childhood and adolescence [ 31 ], thus underlining the suitability of the condition as a candidate for early treatment aimed at preventing both the slow accumulation of social disabilities and impairment caused by anxiety and by avoidance behaviour, as well as the onset of more serious complications (e.g., DE or GAD), which may be manifested many years after onset of SP. Indeed, subjects affected by social phobia presented a high risk of comorbidity with both the latter disorders and DD [ 1 ] .It should be underlined that 60% of subjects undergoing treatment (not all affected subjects) chose their general practitioner (GP). This view is corroborated by the fact that those patients treated by a psychiatrist invariably presented comorbidity with DE, PAD, and AP, thereby representing the more severely affected from a psychopathological point of view. Overall however, the low rate of patients with SP treated with first line-treatments is alarmingly low.In the future, serious attempts should be made to improve the GPs' abilities to recognise SP in order to prevent the use of inappropriate treatments, such as insufficient doses of benzodiazepines, which may be linked to the physician's incorrect diagnosing of the disorder. Due to the fact that subjects affected by SP most frequently refer to their GP, the importance of preventing SP as opposed to other types of disorders, as well as the markedly incapacitating nature of SP reinforce this necessity for a better training of GPs.It is however mandatory to briefly acknowledge some potential limitations of this study. First, the number of cases and the sample size is too small to allow firm conclusions to be drawn concerning the true rate and the degree to which they might actually differ from previous studies with higher estimates. Secondly, differences in the assessment strategy might have resulted in a diminished comparability.Conclusions The study is consistent with findings reported in several previous studies of a lower prevalence of Social Phobia in Italy and confirms the fact that onset of the illness occurs primarily during childhood and adolescence. Furthermore, the results confirm the fact that SP, due to its early onset, might constitute an ideal target for early treatment aimed at preventing both the accumulation of social disabilities and impairments caused by anxiety and avoidance behaviour, as well as the onset of more serious, associated complications in later stages of the illness, or many years after onset of SP.
Conclusions
The study is consistent with findings reported in several previous studies of a lower prevalence of Social Phobia in Italy and confirms the fact that onset of the illness occurs primarily during childhood and adolescence. Furthermore, the results confirm the fact that SP, due to its early onset, might constitute an ideal target for early treatment aimed at preventing both the accumulation of social disabilities and impairments caused by anxiety and avoidance behaviour, as well as the onset of more serious, associated complications in later stages of the illness, or many years after onset of SP.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions MGC participated in the design of the study, performed the statistical analysis and drafted the manuscript. UHW participated in the statistical analysis and drafted the manuscript. MCH, MC, BC, LDO, MAR conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Authors' contributions
MGC participated in the design of the study, performed the statistical analysis and drafted the manuscript. UHW participated in the statistical analysis and drafted the manuscript. MCH, MC, BC, LDO, MAR conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: