Phenotypic Analysis of Individuals With Costello Syndrome due to HRAS p.G13C

Pregnancy and Delivery

Polyhydramnios was noted in 7/11 pregnancies, and 2 individuals had fetal pleural effusion. The pregnancy of individual CS#295 was complicated by thickened nuchal fold, scalp and body edema, and right-sided fetal pleural effusion noted at 19 weeks gestation. The pleural effusion persisted on subsequent ultrasound studies, fetal measurement remained large throughout the pregnancy with estimated fetal weight >97th centile, and polyhydramnios was diagnosed at 34 weeks gestation. A second individual, CS#IT-2, was born at 29 weeks gestation by Cesarean section for fetal distress and had left pleural effusion and ascites after delivery. In this cohort preterm delivery occurred between 29 and 36 weeks gestation in six individuals, and term delivery in six (Table I).

Feeding and Growth

Significant feeding difficulties occurred in all individuals, and seven used feeding tubes. Four of these seven used only nasogastric tubes, and three required percutaneous gastrostomy. Whereas length at birth was within or above the normal range, the most recently obtained height measurement was at or below average in all. Short stature with height below −2 SD was seen in 5/12 individuals, none used growth hormone prior to this measurement. In comparison, 19/19 individuals with p.G12S who never used growth hormone had a length below the 3rd centile [Gripp et al., 2006], resulting in a statistically significant difference. Growth hormone deficiency had been diagnosed in two individuals, but none had received GH replacement therapy at the time of the last measurement. Weight was below average for age in all individuals, commensurate with the decreased height. Head circumference was significantly larger than height for age in all individuals (Table I), allowing for the diagnosis of relative macrocephaly.

Development, Behavior, and Neurologic Issues

Developmental delay was apparent in all individuals after age 1 year, with walking unassisted from age 14 months to 6 years in those who achieved this skill. Words were first used between age 14 months and 2 years in those for whom this information was available (Table I). Formal brief IQ assessment [Axelrad et al., 2009] performed in three individuals showed a range from 67 to 83. Additional IQ data provided by the school psychologist in CS#204 documented a verbal IQ of 77; performance IQ of 77 and full scale IQ of 68. Thus, two individuals fall into the mild intellectual disability range, and two others tested within the low normal IQ range. Development and cognitive ability were assessed in individuals CS#314, CS#PT-1, and CS#DE-1 using different measures as indicated (Table I), and while not directly comparable, their results were in a similar range. Overall cognitive development was described as delayed in this cohort, and school placement ranged accordingly from mainstream classes to a full time special education setting. Individual CS#314 uses a communication device due to limited speech production. Neurologic abnormalities included irritability in infancy in 5/12 individuals. In the most severely affected, CS#204, it was described as “almost constant screaming, irritability, and arching of her back; she ate and slept poorly during this time”. Seizures required medication in one individual, and in another an isolated seizure occurred when she had pneumonia at age 18 years. Hypotonia was noted in 9/12 individuals, and appeared to improve with age.

Brain MRI studies were available for review in six, detailed information was provided in one additional patient and limited information in three. Posterior fossa crowding was present in all with sufficient data (Table I; Fig. 2). Individual CS#255 showed an increase in his OFC crossing centiles upward during his first year of life. Imaging studies revealed a progressive Chiari 1 abnormality, and posterior fossa decompression was performed at age 9 months, followed by VP shunt placement for increasing hydrocephalus, also at age 9 months. While there was no statistically significant difference regarding crowding of the posterior fossa or Chiari 1 malformation, the p.G13C cohort required significantly fewer neurosurgical procedures (Table I).

Cardiac Findings

HCM was reported in eight individuals and was mild in seven. Patient CS#253 had several congenital heart defects including ASD, VSD, PDA, and an unusual long segment coarctation, requiring cardiac surgery at age 6 days. One individual required surgical repair of valvar and supravalvar pulmonic stenosis. Supraventricular tachycardia, including ectopic atrial tachycardia, or multifocal atrial tachycardia (MAT) was not reported. Individual CS#022 had mild concentric left ventricular hypertrophy at age 9 years, which remodeled into mild interventricular septal thickening without left ventricular outflow tract obstruction at age 13 years. At 9 years, aortic dilatation at the sinus of Valsalva was noted without bicuspid aortic valve or hypertension. He developed a tachycardia at age 10 years which was diagnosed as atrial fibrillation. At his current age of 17 years, these issues are stable while treated with losartan and aspirin. While congenital heart defects and HCM occurred with equal frequency in the p.G13C and p.G12S cohorts, the absence of MAT in the p.G13C cohort is notable. Even including the atrial fibrillation as arrhythmia, there is a statistically significant difference.

Ectodermal Findings

Two individuals (CS#270 and CS#IT-1) were formally diagnosed with loose anagen hair (LAH), a condition defined as easily pluckable, sparse, thin, and slow growing hair with abnormal hair bulb lacking inner and outer root sheaths on microscopy. The hair was described as sparse and slow growing in others and on inspection of photographs (Figs. 1 and 3) the hair appears uncombed and less curly than in most individuals with Costello syndrome with HRAS p.G12S (Fig. 3C,D). A strikingly unusual finding of extremely long eyelashes was noted on photograph review and, while not quantified, is demonstrated in Figure 3. Individuals CS#253, CS-#270, CS#314, and CS#DE-1 required regular trimming of their eye lashes. The unusual length of the lashes was noted in early infancy in CS#314, and trimming started around age 3 years because the lashes touched the eye glasses and their length and curliness caused irritation. Monthly cutting of the lashes continues to his current age of 13 years. Lashes grew notably faster at the lateral aspect than medially in individuals CS#314 and CS#DE-1. In individual CS#DE-1, disproportionately long eye lashes were first noted at age 14 months, trimming started around age 2¹/₂ years and continues, approximately every 3 months, at age 6 years. Additional ectodermal findings include a facial café-au-lait macule in one and hemangiomata in two individuals. Distal digital creases [Ørstavik et al., 2007] typical for individuals with rasopathies [Rasopathy Network, 2010] were present in CS#295 (Fig. 4).

Tumors

No individual developed the benign skin tumors known as papillomata, or other tumors typical for Costello syndrome such as embryonal rhabdomyosarcoma, neuroblastoma, or transitional cell carcinoma of the bladder. In individuals with Costello syndrome due to p.G12S, papillomata arise throughout childhood and are noted as early as age 3 years [Gripp et al., 2006]. While the stated age (Table I) in our cohort refers to the age at last height measurement, only two patients are currently younger than 3 years and we confirmed that neither recently developed papillomata.

Musculoskeletal Findings

Typical Costello syndrome findings noted in this cohort include tight Achilles tendons, pectus excavatum or carinatum, scoliosis, hip dysplasia, and joint laxity. Individual CS#253 has a wide neck (Fig. 1). Low bone density was reported in one older individual. None showed ulnar deviation of the wrists and fingers, resulting in a statistically significant difference (Table I).

Facial Findings

Review of the facial photographs (Figs. 1 and 3) suggests less coarseness of the facial features, compared to individuals with Costello syndrome due to p.G12S. While the forehead is tall and the face shape relatively square, the nasal bridge may be less depressed. The ear lobes are less prominent and anteverted. Individuals with p.G13C have a relatively large mouth, but their lips are not as prominent and thick as seen in many individuals with p.G12S. The philtrum is long, wide, and prominent. Ptosis (CS#253), epicanthus (CS#295; CS#314), and prominent ear lobes (CS#IT-2) can occur, but the overall impression reported by experienced clinical geneticists is different from Costello syndrome due to p.G12S and may suggest a diagnosis of Noonan syndrome.

Loose Anagen Hair and Long Eye Lashes

The hemangiomata, hyperpigmented lesions, and distal digital creases seen in one or more of the individuals with p.G13C are typical for rasopathies [Kratz et al., 2007]. Two individuals were formally diagnosed with LAH, a condition not previously reported in patients with a documented HRAS mutation. However, LAH is a characteristic finding for Noonan syndrome with LAH [Mazzanti et al., 2003] caused by mutations in SHOC2 [Cordeddu et al., 2009]. In individuals with Noonan syndrome and LAH, the easy hair pluckability often resolves in late childhood, and similarly in individual CS# IT-1 the hair loss improved with age. While other individuals in our cohort were not formally diagnosed with LAH, their hair is less curly than typical for most Costello syndrome individuals, and it appears very sparse in early childhood and uncombable thereafter (Figs. 1 and 3). Thus, it resembles the “matted,” thin, sparse hair characteristic for LAH [Cantatore-Francis and Orlow, 2009]. In this context it is important to remember that the tight curly hair shown in Figure 3 for individuals with p.G12S is not universally present as we know of a few individuals with this mutation who have straight hair [see photograph in Gripp and Lin, 2009]. Further, the patients with the very rare p.T58I and p.A146V HRAS mutations do not show the tight curly hair [Gripp et al., 2008].

Strikingly long eye lashes were noted in several individuals with p.G13C, and required regular trimming in at least four individuals. Typically long eye lashes are not considered a cosmetic or medical problem. In other individuals, for example CS#295, there is a notable discrepancy between the short and sparse scalp hair and the long and full eye lashes. Long eye lashes have previously been noted in other conditions, such as Cornelia de Lange syndrome and allergic vernal keratoconjunctivitis [Pucci et al., 2005]. In the latter, eye lash measurements documented increased length but none required trimming. We propose the term dolichocilia, combining the meaning of “unusually long” and “eye lashes,” to differentiate this state requiring eye lash trimming from the milder long appearing eye lashes. The nomenclature guidelines [Hall et al., 2009] suggest the term ciliary trichomegaly as a synonym for long eye lashes; however, “tricho” refers to all hair and not only lashes, and “megaly” generally implies enlargement, not specifically increased length. The distinctive condition noted in the individuals with HRAS p.G13C is more specifically termed dolichocilia. Dolichocilia is not seen in Costello syndrome patients with p.G12S, or patients with LAH without a syndromic diagnosis, or patients with Noonan syndrome with LAH [L. Mazzanti, personal communication]. At present, dolichocilia appears to be a unique finding associated with HRAS p.G13C. Given the variability of Costello syndrome, and the overall similarity of findings between the individuals with p.G13C and other germline HRAS mutations, the clinical diagnostic term “Costello syndrome” appears appropriate. Considering the distinct and statistically significant differences, the value of an “axis diagnosis” as proposed by Robin and Biesecker [2001] specifying the clinical diagnosis on axis 1, molecular change on axis 2, and environmental factors on axis 3, is apparent. This approach equally values the clinical diagnosis and the specific molecular cause, rather than stressing one over the other. For the individuals reported here, the diagnosis would be specified as axis 1: Costello syndrome; axis 2: HRAS p.G13C; and axis 3: N/A. We encourage all clinicians to obtain molecular test results and share them with affected families.

Functional Effects of HRAS p.G13C

Functional effects of HRAS changes can be assessed through their transforming activity, however, such data are not available for the missense change resulting in p.G13C [Fasano et al., 1984]. The COSMIC database (COSMIC, 2010) lists HRAS sequencing information derived from 19,157 unique malignant tumor samples, and of 57 samples with a missense mutation affecting p.G13, only 5 carry a p.G13C change. In comparison, 408 samples had a change affecting the p.G12 position, with 52 resulting in a p.G12S change. Taken together, the relative rarity of HRAS p.G13C in malignant tumors suggest a much lower transforming capacity, compared to the more common HRAS mutations.

The HRAS missense mutations with transforming activity typically result in decreased intrinsic hydrolysis, maintaining the active GTP-bound state [for review, see Sol-Church and Gripp, 2009]. Functional studies performed on Costello syndrome patient fibroblasts confirmed the increased amount of GTP-bound HRAS, compared to normal control fibroblasts [Rosenberger et al., 2009]. Interestingly, downstream effects measured through MEK1/2 and ERK phosphorylation were normal under basal conditions and only slightly prolonged after EGF stimulation. In contrast, the alternate PI3K/AKT signaling pathway showed increased baseline AKT phosphorylation and an enhanced late effect after EGF stimulation [Rosenberger et al., 2009]. No statistically significant differences were found for p.G13C compared to p.G12S, however, neither were differences found between p.G12S and p.G12V, the most strongly activating and transforming HRAS mutation. While these functional studies do not offer an explanation for the distinctive ectodermal effects seen with p.G13C, they may support a lesser transforming activity accounting for the absence of papillomata and tumors. It is possible that these distinctive findings reflect cell type specific effects of this particular missense mutation, for example, through altered interaction with SHOC2. Myristolation of the abnormal SHOC2 protein seen with the SHOC2 mutation resulting in Noonan syndrome with LAH results in abnormal protein targeting to the plasma membrane [Cordeddu et al., 2009]. As two of the individuals reported here were formally diagnosed with LAH, and in others had the sparse, matted hair typical of older individuals with a past history of LAH, this phenotypic similarity suggests a similar cell type specific effect of the altered protein product. Further functional studies may benefit from using patient derived cells, rather than transfecting over-expressing clones, and focusing on perturbations of other signaling cascades controlled by the GTPase.