Prenatal and infantile diagnosis of craniosynostosis in individuals with RASopathies

Fetuses with RASopathies can have a wide variety of anomalies including increased nuchal translucency, hydrops fetalis, and structural anomalies (typically cardiac and renal). There are few reports that describe prenatal‐onset craniosynostosis in association with a RASopathy diagnosis. We present clinical and molecular characteristics of five individuals with RASopathy and craniosynostosis. Two were diagnosed with craniosynostosis prenatally, 1 was diagnosed as a neonate, and 2 had evidence of craniosynostosis noted as neonates without formal diagnosis until later. Two of these individuals have Noonan syndrome (PTPN11 and KRAS variants) and three individuals have Cardiofaciocutaneous syndrome (KRAS variants). Three individuals had single suture synostosis and two had multiple suture involvement. The most common sutures involved were sagittal (n = 3), followed by coronal (n = 3), and lambdoid (n = 2) sutures. This case series confirms craniosynostosis as one of the prenatal findings in individuals with RASopathies and emphasizes the importance of considering a RASopathy diagnosis in fetuses with multiple anomalies in combination with craniosynostosis.

Craniosynostosis occurs in 1:2500 live births and involves the premature fusion of one or more cranial sutures (Boulet et al., 2008).This leads to abnormal head shape and can also result in other complications such as increased intracranial pressure, hearing and vision problems, and developmental delay (Boulet et al., 2008;Cunningham et al., 2007).
The aim of this study is to expand on current knowledge of craniosynostosis in individuals with a RASopathy.Specifically, this study assesses and describes the phenotypic and genetic heterogeneity of this group of conditions and highlights craniosynostosis as a recurrent prenatal finding in multiple individuals with a RASopathy diagnosis.

| Editorial policies and ethical considerations
This research study was approved by the Cincinnati Children's Hospital Medical Center (CCHMC) Institutional Review Board.Parental consent was obtained for use of photographs.Clinical data was abstracted from medical records and entered in a secure database for analysis using de-identified information.Demographic information (e.g., age and sex), phenotypic information (e.g., prenatal findings, extent and location of craniosynostosis, other health problems), radiology images/reports, and genetic test results were collected.

| RESULTS
Five individuals with confirmed molecular diagnosis of a RASopathy were identified to have craniosynostosis.Two of these participants have Noonan syndrome with pathogenic variants in PTPN11 (n = 1) and KRAS (n = 1).The remaining three individuals have a diagnosis of CFC syndrome with pathogenic variants in KRAS (n = 3).Clinical imaging and molecular characteristics of all individuals are reviewed in Table 1.
Individual 1 was a male infant diagnosed prenatally with multiple congenital anomalies including asymmetric lambdoid suture initially detected on fetal MRI at 34 weeks 2 days gestation (Figure 1a,b).
Additional anomalies detected prenatally were right lung agenesis, tracheoesophageal fistula, and possible right congenital diaphragmatic hernia versus large eventration.Individual 1 was born at 35 4/7 weeks gestation, the fourth child to a 36-year-old mother.
Birth weight was 1.72 kg (0.4% percentile) and head circumference was 31.3 cm (2.6% percentile).Apgar scores were 6 and 9 at 1 and 5 min, respectively.A head ultrasound at Day of Life (DOL) 1 found a small cyst in the right choroid plexus, as well as an ovoid cystic area T A B L E 1 Description of patients with craniosynotosis and RASopathy.Individual 4 is an 11-year-old male born at 33 weeks gestation after pregnancy complicated by hydrops fetalis and polyhydramnios.

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His birthweight was 3.45 kg (43.3% percentile).He was intubated at delivery for respiratory distress and remained intubated for 3 weeks.
He stayed in the NICU for 10 weeks due to chronic lung disease, bilateral subdural hemorrhages, sinus tachycardia, and pulmonary hypertension.He was re-admitted to the hospital at 15 months old due to respiratory distress, hydrocephalus, ventriculitis, and cardiomyopathy.
Genetics was consulted and individual was noted to have coarse facial features, dolichocephaly with prominent sagittal ridge, and dry skin.

| DISCUSSION
This study describes five individuals with RASopathies and cooccurring craniosynostosis.Two individuals had abnormal head shape concerning for craniosynostosis noted prenatally (Table 1).These results are consistent with other recent case reports of individuals with RASopathies and craniosynostosis (Ueda et al., 2017;Weaver et al., 2022) adding to the growing literature that demonstrates craniosynostosis can occur in individuals with RASopathies.
In addition to these genes, recent studies have demonstrated that pathogenic variants in ERF, a molecule that interacts with the RAS/MAPK pathway, can cause craniosynostosis (ERF-related craniosynostosis) (Twigg et al., 2013).In studies of mice carrying a mutation within this gene, they initially showed delayed markers of osteogenesis and later craniosynostosis (Twigg et al., 2013;Twigg & Wilkie, 2015).A reduced dosage of ERF protein leading to craniosynostosis may suggest an interaction between the RAS and the FGFR signaling pathways as multiple independent authors have shown (Kim et al., 2003;Shukla et al., 2007).However, the role of ERF activation for the development of craniosynostosis is still not well understood and more studies are needed to elucidate these mechanisms.
All the individuals in this cohort exhibit typical features of RASopathies in addition to cranial deformities.However, three of the five individuals were noted to have abnormal head shape concerning for craniosynostosis prenatally or shortly after birth (Table 1).This early onset has not been described in prior reports of RASopathy-associated craniosynostosis.The majority of these individuals also had multi-organ involvement such as cardiac abnormalities and feeding concerns.
Three of these individuals had involvement of the sagittal sutures, two had involvement of the lambdoidal sutures, and three had involvement of the coronal sutures (Table 1).Other studies regarding RASopathies and craniosynostosis have found little involvement of coronal suture (Ueda et al., 2017).Three of these five individuals required surgical repair for the craniosynostosis.A few of these individuals also exhibit additional uncommon complications such as right lung agenesis and tracheoesophageal fistula (Individual 1), and aqueductal stenosis (Individuals 3 and 4).These results highlight the evolving phenotypic associations in the RASopathies.
A limitation of this retrospective study is that not all these individuals received exome sequencing so we cannot rule out the possibility of additional diagnoses related to the craniosynostosis.For the two individuals with exome trio sequencing data available there was no secondary diagnosis associated with craniosynostosis.This finding combined with the recently reported cases of craniosynostosis and RASopathies suggests a true association.Several individuals have limited information about prenatal medical history and limited imaging available.This is a single center experience and additional larger cohort studies will be important to define the spectrum of manifestations related to this underrecognized complication of the RASopathies.
Our results add to the growing clinical evidence that craniosynostosis is related to RAS activation.In the setting of a RASopathy diagnosis, there is likely increased risk for presentation of craniosynostosis.
Among this group of disorders, craniosynostosis appears to be T A B L E 2 Summary of RASopathy genes, type of craniosynostosis, and diagnosis in perinatal period.
Craniofacial features included down-slanting palpebral fissures, bilateral epicanthal folds, depressed nasal bridge, anteverted nares, low-set cupped ears, coarse eyebrows, facial hypotonia, square facial shape, wide mouth with downturned corners and hypertelorism.A brain MRI at 15 months of age demonstrated moderate enlargement of the lateral and third ventricles was observed.This brain MRI also indicated that individual 4 had congenital hydrocephalus secondary to aqueductal stenosis, previously treated with ventricular drainage.CT scan noted incidental sinus and temporal bone findings in addition to a new small left frontal subdural hematoma.Additionally, his medical history was notable for hypertrophic cardiomyopathy with mild left ventricular outlet tract obstruction and dysplastic aortic, mitral and tricuspid valves (Figure1f,g).Noonan spectrum chip testing was ordered and identified a pathogenic missense variant in KRAS (c.178G>C, p.Gly60Arg).He followed up with Genetics at 5 year old and was noted to have prominent sagittal suture concerning for craniosynostosis.He did not have further imaging or surgical intervention for repair of craniosynostosis.Individual 5 is a 3-year-old female born at 37 weeks 2 days gestation and weighed 3.54 kg (61.4% percentile), the third child of a 33 year old mother.Prenatal history noted possible renal concern on 20-week ultrasound but no further follow up was done during pregnancy.After birth, she was admitted to the NICU for 6 weeks due initially to meconium aspiration.During NICU stay, this individual also had abnormal echo noting a PFO, brain MRI which resulted unremarkable, modified barium swallow study showing delayed laryngeal closure and aspiration of breast milk, had a g-tube placed, and had an abdominal ultrasound which was unremarkable.Most recent echo now notes mild concentric left ventricular hypertrophy, mild hypoplastic transverse arch, and possible bicuspid valve.Sleep study showed obstructive sleep apnea; now post adenoidectomy.Skeletal survey resulted unremarkable.She also has short stature (1st percentile), hypotonia and hyperflexibility requiring use of SMOs, oral aversion, reflux, and nystagmus.On physical exam, she is noted to have distinct features including bilateral epicanthal folds, down-slanting palpebral fissures, coarse facial features, low-set ears with thick lobes and F I G U R E 1 Imaging demonstrating craniosynostosis in Individuals 1-5.(a, b, Individual 1) (a) sagittal view on prenatal MRI; (b) transverse view on prenatal MRI.(c, Individual 2) (c) sagittal view on x-ray.(d, e, Individual 3) (d) transverse view on CT; (e) coronal view of a model of skull.(f, g, Individual 4) (f) sagittal view on MRI; (g) transverse view on MRI.(h, i, Individual 5) (h) CT scan with 3D reconstruction with near complete fusion of sagittal suture, premature closure of lambdoid, coronal, and squamosal sutures; frontal bossing; anterior fontanelle remains patent; (i) frontal bossing, j-shaped sella.dimple in posterior side of each lobe.Also, she is noted to have always had a very large anterior fontanelle although it has finally begun closing and a hump is now developing at the anterior edge of the fontanelle.Genetics work-up included microarray, skeletal dysplasia panel, SNIP1 sequencing, and lysosomal storage disorder analysis.All resulted negative.Around 15 months old, exome sequencing was ordered and resulted with a de novo pathogenic variant in KRAS (c.178G>C, p.-Gly60Arg).Of note, a maternally inherited likely pathogenic variant in MT-ATP6 (m.8717A>G) was also identified with approximately 2% heteroplasmy.After diagnosis, she followed up with Genetics to discuss further management.A CT of head with 3D reconstruction was ordered and noted mild to moderate third ventricular enlargement and premature closure of multiple cranial sutures (Figure1h,i).
(Ueda et al., 2017;Weaver et al., 2022;Zollino et al., 2017) most frequently reported in BRAF, HRAS, KRAS, and PTPN11 (Table2)(Ueda et al., 2017;Weaver et al., 2022;Zollino et al., 2017).More research is needed to determine how often craniosynostosis occurs in individuals with RASopathies, and what other factors may influence this relationship.These results further illustrate that when an individual presents with craniosynostosis, RASopathies should be included in the differential, especially when other abnormalities are present (e.g., congenital heart defects, aqueductal stenosis).Likewise, when an individual presents with a RASopathy, clinicians should monitor for craniosynostosis as a possible complication.Recognizing craniosynostosis as a possible manifestation of RASopathies could decrease time to diagnosis through informing testing utility.Future research could be directed toward gathering more quantitative information on craniosynostosis and RASopathies, specifically the incidence and prevalence in larger cohorts.