Loss-of-function mutations in PTPN11 cause metachondromatosis, but not Ollier disease or Maffucci syndrome

Abstract

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.

Figure 1. Clinical, radiographic, and histologic features of metachondromatosis.

(A) Hand radiographs of participant B-IV-7, taken when 8-years-old. Exostotic lesions (white arrows) are present in the phalanges and metacarpals, and arise from the metaphysis (arrows) or epiphysis (arrowheads). Exostoses tend to point toward the adjacent joint. Endosteal lesions (red arrows) cause metaphyseal expansion. (B, C, D) Hand photograph and radiograph, and foot photograph of participant C-III-1 taken when 9-years-old, depicting mild shortening and deformity of the digits, a large exostotic lesion arising from the second metacarpal bone in the hand, and ankle enlargement superior to the malleoli due to exostoses of the tibia and fibula. (E) Ankle radiograph of participant A-IV-8 taken when 19-years-old depicting a recurrence of a previously excised exostotic lesion of the distal tibia that spans the physis. (F, G) Lateral knee radiographs of participant B-IV-7, taken at 6 years and 16 years, respectively. Note that multiple exostotic lesions of the distal femur and proximal fibula (white arrows) seen when 6-years-old (F) have regressed in the absence of surgical intervention by 16-years of age (G). (H) Ankle radiograph of participant B-IV-7, taken when 5-years-old, demonstrating radiolucency associated with endosteal lesions (red arrows) in the tibia and fibula, and mild metaphyseal flaring. Despite combined metaphyseal and epiphyseal involvement, this individual's linear growth was not affected. (I) Hip radiograph of participant A-IV-8 taken when 22-years-old depicting an endosteal lesion of the femoral neck (arrow) that has caused degeneration of the femoral head and spurring of the acetabulum. (J) Low power photomicrograph of an hematoxylin and eosin (H&E) stained section through an exostosis that had been excised from a patient with hereditary multiple exostoses. Note this exostosis is a typical osteochondroma, having a well-developed surface cartilaginous cap (arrow) and endochondral bone immediately below (arrowhead). The scale bar represents 0.15 cm (K) Photomicrograph of an H&E stained exostotic lesion excised from participant A-IV-5 when 5-years-old. This lesion is predominantly covered by a fibrous cap (arrow) and has only a small, eccentric cartilaginous cap (double arrowhead). The majority of cartilage in this, and in 14 other exostoses from patients with MC that have been analyzed, is found within a central core (arrow) and has bone formation occurring at the periphery of this cartilage core. The scale bar represents 0.5 cm. (L) High-power image of the central cartilage core shows chondrocytes with prominent cytoplasm no organization typical of a growth plate. The scale bar represents 100 µm.

Figure 2. Linkage mapping of metachondromatosis to chromosome 12q.

(A) Pedigree of the family (Family A) used to define the metachondromatosis candidate interval. Affected individuals have filled symbols. Individuals who were not examined but who were assumed to be obligate carriers have interior dots. An arrow identifies the proband. Participants whose DNA was used for linkage analysis are double underlined. Those participants with a single or double underlines were tested for the PTPN11 mutation, after the mutation had been identified in participants A-III-10 and A-IV-5 by targeted array capture and Illumina sequencing. (B) LOD score plot of chromosome 12. Only one interval, larger than 1 cM, in the entire genome attained the maximum LOD score of 2.7. Several autosomal intervals, each smaller than 1 cM, also achieved maximum or positive LOD scores (Figure S1). These likely represent either genotyping errors or short ancestral haplotypes that are coincidental with linkage. The physical coordinates shown are derived from GRCh37/hg19.

Figure 3. PTPN11 mutations identified in MC participants.

(A) Schematic of the exonic structure of PTPN11 (above) and the corresponding protein structure of SHP2 (below). The locations of mutations that were identified in MC are indicated with black lines. Predicted protein changes are indicated for the nonsense (blue) and frameshift (red) mutations, while the cDNA designation is indicated for the splice-site mutations (green). (B) Log2 values of the number of Illumina reads obtained per 50 bp window in participant S, divided by the average number of reads obtained in other participants whose DNA was captured simultaneously using the second capture array. Shown are all 50 bp windows spanning regions of PTPN11 targeted by the array, with the corresponding exonic structure of PTPN11 shown below. The red bar indicates a region spanning exon 7, in which the average log2 value is approximately −1, suggesting a heterozygous deletion. PCR amplification and sequencing of the breakpoint, using primers on either end of the deletion, indicate that 14,629 bp of sequence have been deleted and replaced with a single CA dinucleotide.

Figure 4. Loss of the wild-type PTPN11 allele in the cartilage of two exostoses.

(A) Electropherograms of PCR amplified template DNA that had been extracted from whole blood, a section of an exostosis, or the cartilage core of the same exostosis. Exostoses were available from patients A-IV-5 and A-IV-8. The site of the 5 bp deletion in exon 4 of PTPN11 in both patients is indicated with a box. Note that that the heights of the peaks corresponding to the mutant sequence are markedly reduced in amplimers from the cartilage-core compared to amplimers from blood or from a section that contains cartilage, bone and fibrous tissue. This is consistent with loss-of-heterozygosity in the cartilage component of the exostoses. (B) Electropherograms of PCR amplified template DNA extracted from blood from participants A-III-9, A-III-10 and A-IV-5, as well as DNA extracted from the cartilage core of the exostosis from participant A-IV-5 shown in (A). Blood DNA electropherograms indicate that participants A-III-9 and A-IV-10 are heterozygous at a position (asterisk) in intron 11 of PTPN11. This is the site of a known common polymorphism (rs41279092). Exostosis cartilage DNA electropherograms have a reduced adenine peak height at this position. This suggests that the wild-type PTPN11 allele inherited from the unaffected parent (A-III-9), which carries an adenine at this position, has been lost in cells that contribute to formation of the exostosis' cartilage core.