Genotype and phenotype spectrum of NRAS germline variants

Abstract

RASopathies comprise a group of disorders clinically characterized by short stature, heart defects, facial dysmorphism, and varying degrees of intellectual disability and cancer predisposition. They are caused by germline variants in genes encoding key components or modulators of the highly conserved RAS-MAPK signalling pathway that lead to dysregulation of cell signal transmission. Germline changes in the genes encoding members of the RAS subfamily of GTPases are rare and associated with variable phenotypes of the RASopathy spectrum, ranging from Costello syndrome (HRAS variants) to Noonan and Cardiofaciocutaneous syndromes (KRAS variants). A small number of RASopathy cases with disease-causing germline NRAS alterations have been reported. Affected individuals exhibited features fitting Noonan syndrome, and the observed germline variants differed from the typical oncogenic NRAS changes occurring as somatic events in tumours. Here we describe 19 new cases with RASopathy due to disease-causing variants in NRAS. Importantly, four of them harbored missense changes affecting Gly12, which was previously described to occur exclusively in cancer. The phenotype in our cohort was variable but well within the RASopathy spectrum. Further, one of the patients (c.35G>A; p.(Gly12Asp)) had a myeloproliferative disorder, and one subject (c.34G>C; p.(Gly12Arg)) exhibited an uncharacterized brain tumour. With this report, we expand the genotype and phenotype spectrum of RASopathy-associated germline NRAS variants and provide evidence that NRAS variants do not spare the cancer-associated mutation hotspots.

Figure 1

NRAS domain structure and spectrum of germline variants. Upper panel: The sequences of amino acids 1–70 of three RAS proteins are aligned. RASopathy-associated and germline variants in NRAS are represented above the sequence alignment. Length of bars and numerals at their basis indicate the numbers of unrelated observations for each disease-causing variant. Bold font indicates disease-causing variants described previously, which were also identified in our cohort. Novel changes identified in this study are written in italics. Variants previously reported to cause NS but not found in our cohort are written in normal font. *Notably, the reported case with the c.38G>A (p.(Gly13Asp)) variant had a primary diagnosis of JMML and mild Noonan-like features were only noted retrospectively. Bars and numerals below the alignment represent the numbers of somatic variants associated with cancer at the respective position (according to COSMIC, Sept 2016). Lower panel: Domain structure of entire NRAS protein. Numbers represent amino acids, black boxes: coding exons, dark grey boxes: functional domains, HVR, hypervariable region; P-L, P-Loop; SW I, switch I region; SW II, switch II region.

Figure 2

Functional characterization of the NS-causing NRAS^Thr58Ile variant: (a) ERK1/2 and AKT phosphorylation assays. HEK293T cells were transfected with the indicated FLAG-tagged NRAS construct. Following starvation (18 h) and EGF stimulation (30 ng/ml, 5 min), ERK (anti p-ERK1/2) and AKT (anti p-AKT) phosphorylation levels were evaluated. Normalization for transfection efficiency and total protein amount was determined by the use of anti-β-tubulin and anti-FLAG antibodies, respectively. Representative blots of three performed experiments are shown. (b) Determination of basal GTP-bound NRAS level in 293 T cells transiently expressing wild-type or mutant FLAG-tagged NRAS. Normalization was determined by using an anti-FLAG antibody on aliquots of the corresponding cell lysates. Assays were performed in serum-free condition. A representative blot of three performed experiments is shown.

Figure 3

Clinical photographs documenting the craniofacial phenotype of patients with heterozygous NRAS variants. The patients’ ID numbers (according to Table 1) are given in the upper left corner of each photograph. Written consent was obtained from the patient or his/her legal guardian for publication of the images. P1: p.(Gly12Ser), 4 years old; P3–1, P3–2, P3-3: p.(Ile24Asn), familial observation with affected family members shown at age 10, 13, and 36 years, respectively; P5-1: p.(Gly60Glu), 17 years old; P7: p.(Glu37dup), 6 month old; P10-1: p.(Gly60Glu), 18 years old; P11: p.(Thr50Ile), 11 years old; P13: p.(Gly12Asp), 1 year old.