Noonan syndrome (NS; MIM 163950) is a common autosomal dominant disorder characterized by short stature, distinct facial features and congenital cardiac defects.1 In approximately 60% of NS cases, a heterozygous gain-of-function mutation can be identified in one of three genes: mutations are found in PTPN11, SOS1 or KRAS in 50, 10 and <5% of cases, respectively.1 The PTPN11 gene codes for SHP-2, a non-receptor tyrosine phosphatase that regulates multiple responses including proliferation, differentiation and migration. SHP-2 relays growth signals from stimulated growth factor receptors to other signaling molecules, including Ras. Ras is activated by the guanosine nucleotide exchange factor SOS1 and functions as a molecular switch, cycling between an inactive GDP-bound and an active GTP-bound state to control fundamental cellular pathways.2
Two of the three known NS genes, PTPN11 and KRAS, are well-known proto-oncogenes and specific somatic mutations of these genes are detected in cancer cells.1, 2 This favors the notion that NS predisposes to malignancy. Indeed, infants with NS that harbor specific germline mutations in PTPN11 (e.g., T73I) or in KRAS (T58I) are predisposed to develop myeloproliferative disease (MPD) resembling juvenile myelomonocytic leukemia (JMML).1 Myeloproliferation is generally self-limiting, implicating that NS-associated mutants disrupt hematopoiesis only during early stages of development. When the proliferating clone acquires additional genetic abnormalities, the disorder may become aggressive and lethal.
It is not well established whether older individuals with NS are at increased cancer risk, although there are several reports linking NS to leukemia and other malignancies.3, 4 With an estimated incidence of NS of one per 2000 life births per year, these cases of malignancy may, however, be due to chance. For two reasons, an assumed increased cancer risk in NS may not be obvious: (1) the features of NS may be subtle and overlooked in cancer patients and (2) NS-associated mutants of PTPN11 or RAS are functionally mild in contrast to the cancer-associated mutants, thus requiring several genetic alterations cooperating during transformation.
We present two childhood cases of NS with acute lymphoblastic leukemia (ALL). Both patients harbored germline mutations of PTPN11. In one patient's leukemia cells we demonstrate uniparental disomy (UPD) at the PTPN11 gene locus giving rise to loss of the wild-type and duplication of the mutant PTPN11 allele. These data provide genetic evidence that NS-associated germline mutations contribute to malignancy and that loss of the wild-type PTPN11 is an important ‘second hit’.
The first NS patient was diagnosed with hypertrophic cardiomyopathy shortly after birth. At the age of 28 months, she presented with failure to thrive, fever, malaise and progressive hepatosplenomegaly. The blood count revealed pancytopenia with hemoglobin level 7.1 g/dl, platelet count 25 000 mm3 and a leukocyte count of 3000 mm3 with 1% blasts. A bone marrow aspirate showed monomorphic infiltration with lymphoblasts. Flow cytometry was consistent with pre-B cell ALL. Genetic analysis uncovered a hyperdiploid karyotype without structural aberrations. She was treated according to the German ALL-BFM 2000 protocol (standard risk), responded well, and remains in remission 9 months after initial diagnosis.
The second NS patient presented in early childhood with poor growth and NS-like facial features. At the age of 8 years she developed fever and organomegaly. Blood count demonstrated leukocytosis with 14 800 mm3, hemoglobin 5.3 g/dl and platelets 14 000 mm3. Bone marrow aspirates showed 99% blasts. Immunophenotyping revealed early pre B-cell ALL with aberrant CD13 and CD33 expression. Genetic analyses demonstrated complex chromosomal alterations including a TEL-AML1 fusion. She was treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) intermediate risk-protocol. Three months after the end of treatment the patient relapsed. Subsequently, allogeneic hematopoietic stem cell transplantation (HSCT) was performed from an unrelated donor. One year after HSCT a second relapse was diagnosed and palliative treatment was initiated.
Mutation analysis of DNA extracted from buccal cells from the first patient revealed the presence of a heterozygous known NS-associated mutation, c.1510A>G, in PTPN11 predicting an M504V substitution in the protein (Figure 1). In this patient's leukemic blasts, we identified the same mutation and an absence of the wild-type allele (Figure 1) consistent with loss of heterozygosity. To determine whether this finding was due to loss of one copy of the gene or due to uniparental disomy, we analyzed DNA from blasts using array-CGH. In agreement with the results obtained from karyotype analysis, array-CGH showed hyperdiploidy with additional copies of chromosomes 4, 6, 14, 17, 18 and 21. Notably, array-CGH showed no evidence of a deletion in chromosome 12 band q24.13, the gene locus of PTPN11 (Supplementary Figure 1). Therefore, allele loss in this specimen presumably results from mitotic recombination and consecutive UPD (Figure 1). We next analyzed bone marrow cells obtained during remission for mutations and for genomic rearrangements and found that, with the exception of the patient's heterozygous germline PTPN11 mutation, all other genetic abnormalities were absent (Figure 1 and Supplementary Figure 1). Loss of wild-type PTPN11 allele has been previously described in a patient with isolated JMML and a somatic PTPN11A72V mutation.5 Additionally, clonal duplication of a mutant PTPN11N308D allele has recently been reported in a case of NS with therapy-related acute myeloblostastic leukemia.4 Based on these data, we hypothesize that wild-type SHP-2 has properties of a tumor suppressor and the capacity to reduce the transforming potential of oncogenically activated SHP-2 as has previously been shown for wild-type KRAS.6 Loss of the wild-type and duplication of the mutated allele due to UPD has recently been reported to occur in other oncogenes such as KRAS6 and JAK2.7 Likewise, Fitzgibbon et al. have recently identified concurrent homozygous mutations at four distinct loci (WT1, FLT3, CEBPA, and RUNX1) in myeloid leukemia specimens, indicating that mutation precedes mitotic recombination which acts as a ‘second hit’ responsible for removal of the remaining wild-type allele.8 Moreover, UPD at the NF1 tumor suppressor gene on chromosome 17 has been described as a frequent mechanism of LOH in neurofibromatosis type 1 (NF1)-associated leukemias.9, 10 Notably, NF1 shares many clinical features with NS and both syndromes are associated with JMML and aberrant Ras signaling.1
In the second patient we identified a known heterozygous NS-associated PTPN11E139D mutation. This patient's leukemia cells retained the normal allele, indicating that ALL in NS is due to heterogeneous mechanisms. In conclusion, we present genetic evidence that NS-associated germline mutations in PTPN11 may cooperate with other genetic events, such as UPD at the PTPN11 gene locus, to contribute to ALL evolution. Our study therefore suggests that, although rare, the association of NS and ALL is not a result of chance alone. Somatic mutations of PTPN11 have been described in cases of sporadic ALL.11 In similar cases the presence of NS should be carefully excluded.
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We thank Marcel Tauscher and Cornelia Klein for their excellent technical assistance. We are grateful to Dr Mwe Mwe Chao for critical comments.
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Karow, A., Steinemann, D., Göhring, G. et al. Clonal duplication of a germline PTPN11 mutation due to acquired uniparental disomy in acute lymphoblastic leukemia blasts from a patient with Noonan syndrome. Leukemia 21, 1303–1305 (2007). https://doi.org/10.1038/sj.leu.2404651
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