Materials and methods

FAMILY

The diagnosis of CS was made using the international Cowden’s Consortium CS diagnosis criteria. The diagnosis of BZS was made according to accepted clinical criteria. Blood samples were obtained after informed consent from subjects with CS.

DNA EXTRACTION

Genomic DNA was extracted from whole blood using a QIAamp Blood Maxi Kit (Qiagen). DNA concentration was measured by OD₂₆₀and purity was checked by the ratio of OD₂₆₀/OD₂₈₀.

PCR AMPLIFICATION AND MUTATION DETECTION

Genomic DNA was amplified with primers flanking the exons of PTEN/MMAC1 as described.7 24 For mutation detection, PCR products were sequenced using an Applied Biosystem 310 automated sequencing system.

ALLELE SPECIFIC HYBRIDISATION

Oligonucleotides (20 base pairs) were synthesised with either the wild type or the mutant nucleotide located in the centre. DNA from subjects with a mutation and normal controls were amplified and blotted onto the nylon membranes. Oligonucleotides were then labelled with³²PγATP and used as probe in the hybridisation reactions. Two identical membranes containing mutant and normal PCR products were hybridised with wild type and mutant oligonucleotides respectively in a buffer (5 × Denhardts, 5 × SSPE, 0.5% SDS) at 48°C for two hours. Blots were then washed in 2 × SSPE, 0.1% SDS at 48°C for 30 minutes. The blots were then air dried and autoradiology performed at –70°C with Kodak XAR film and intensifying screens.

Results

As shown in the pedigree (fig 1), the MHSF066 family consists of affected family members in three generations, two subjects with CS and two with BZS. MHSF066-01 and her mother, MHSF066-02, both phenotypically fulfil the criteria for CS, by the presence of classical skin lesions and benign and malignant neoplasms. The germline mutations in PTEN and associated phenotypes are listed in table 1. MHSF066-01, who has been reported previously,31 is a 31 year old white female with a unique presentation of CS. She was diagnosed with a follicular thyroid carcinoma at the age of 10, which later metastasised to the left lung and was treated successfully with surgical resection. At the age of 12, she developed an osteosarcoma of the left proximal tibia and a large lipoma (10 × 15 cm) in the mediastinum. Intraductal breast carcinoma was diagnosed at the age of 21 and multiple sclerosis at the age of 26. In addition to having a complex medical history, she gave birth to two male infants subsequently identified as having BZS. MHSF066-03 and MHSF066-04, currently 9 and 6 years old respectively, were both born with macrocephaly (>95th centile) with normal ventricular size. During their early childhood years, they showed signs of developmental delay, hypotonia, and lentigines of the penis, all characteristic of BZS.

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Figure 1

(A) DNA sequence analysis of the PTEN gene in four members from three generations in this family. A C→T transition was evident in all four affected subjects which resulted in a premature stop at codon 335. (B) Analysis of PCR products flanking exon 8 of the PTEN gene by allele specific hybridisation with mutant and wild type oligonucleotides showed the presence of the two alleles in the affected subjects but not in the unaffected population.

The affected and unaffected members of family MHSF066 were analysed for germline PTEN mutations. Direct sequencing analysis of the PCR product flanking exon 8 showed a transition of C to T at nucleotide 1003, resulting in a change from arginine to a stop codon (R335X). This nonsense mutation was identified in all four affected subjects with CS and BZS in this family.

The mutation was subsequently confirmed by allele specific oligonucleotide (ASO) analysis (fig 1), which showed both the wild type and the mutant allele from each of the affected subject’s genomic DNA. By contrast, ASO of 30 alleles from unaffected, unrelated subjects failed to identify the mutant allele.

Discussion

PTEN has been identified as the susceptibility gene for two hamartoma syndromes, CS and BZS.20 Although distinct phenotypically, these two disorders display partial clinical overlap.21 We have identified a germline mutation, R335X, in the PTEN coding sequence, in a family with two members with the classical phenotypic findings of CS and two members with the classical phenotypic findings of BZS. To our knowledge, this is the first report that contains subjects with the CS and the BZS phenotype in a single family resulting from the same PTEN mutation. Since patients with CS usually develop characteristic skin findings in the second decade, it will be crucial to follow the two affected male children with BZS in this family to evaluate whether the features of CS develop. The severe tumour phenotype of proband MHSF 066-01 also deserves comment. In addition to developing malignant tumours at a young age, including osteosarcoma, follicular thyroid carcinoma with metastasis to the lung, and breast carcinoma, she also shows an unknown association of CS, that is, multiple sclerosis.

To date, nine families with BZS have been screened for PTEN mutations, of which five were found to exhibit mutations in this gene.20 28 30 32 These are summarised in table 2. Of interest, two of the mutations identified in BZS families, R233X and Y68H, were also independently reported in unrelated families with CS.20 28 30 R335X represents another example of a particular germline PTEN mutation being present in both syndromes.20 30 Although the mutation reported herein, R335X, has not been previously reported in BZS, it has been observed in three unrelated families with CS. Moreover, we have recently identified R335X in an unrelated female proband with BZS (J Toket al, unpublished data), suggesting that this truncating PTEN mutation is common in both hamartoma syndromes. Additionally, we have tested a frameshift mutation, 915del13, resulting in a premature stop at codon 312 in exon 8, and identified almost a complete loss of the PTEN phosphatase activity (unpublished data). Similar functional changes are predicted to occur with the truncating PTEN mutation R335X.

These data, together with our own, clearly indicate that an identical mutation, found in a single germline, in the PTEN gene can confer susceptibility to two distinct phenotypes, CS and BZS. These observations suggest that increased susceptibility to breast and thyroid cancer may be found in members of BZS families associated with germline PTEN mutations. Moreover, it also suggests that women with CS may give birth to offspring with the findings of BZS as well as CS. While the notion that these two diseases may be within the spectrum of the same primary disorder awaits further study, we cannot exclude the presence of epigenetic factors, such as sex hormones or other modifying genes playing a role in the pathogenesis of the phenotypic findings of both syndromes.