Diverse Clinical Manifestations of Cardiofaciocutaneous Syndrome Type 3 in Two Patients from South East Asia

Cardiofaciocutaneous syndrome (CFCS) is a RASopathy disorder caused by germline pathogenic variants in genes belonging to the Ras/Mitogen-Activated Protein Kinase (RAS/MAPK) pathway which regulates cell differentiation, proliferation, and apoptosis. CFCS is characterized by multiple congenital anomalies including cardiac defects, ectodermal abnormalities, distinctive craniofacial features, and varying degrees of intellectual disability [Rauen, 2016; Grant et al., 2018]. It shares many clinical features with other RASopathies, a group of neurodevelopmental disorders with a collective prevalence of 1:1,000, and overlaps the most with Costello syndrome (CS; OMIM #214080) and Noonan syndrome (NS; OMIM #163950) [Pierpont et al., 2014].

CFCS is genetically and phenotypically heterogeneous with 4 subtypes. While craniofacial features, hair abnormalities, and intellectual disability occur similarly across all CFCS subtypes, CFC1 (OMIM #115150) has a higher incidence of benign nevi, malignant melanoma, and pulmonary stenosis [Niihori et al., 2006] and is associated with mutations in BRAF (OMIM *164757). CFC2 (OMIM #615278) is associated with mutations in KRAS (OMIM *190070) and has a lower incidence of skin manifestations but the phenotype is the most variable [Niihori et al., 2006]. CFC3 (OMIM #615279) and CFC4 (OMIM #615280) are both associated with variants in genes encoding the Mitogen-Activated Protein Kinases (MAP2K1: OMIM *176872 and MAP2K2: OMIM *601263), previously known as Map/Erk kinase-1 and 2 (MEK1 and MEK2). As CFC1 makes up about 75% of reported cases with identified mutations, phenotypic comparison among the 4 subtypes is limited by the smaller number of patients with the other 3 subtypes. In addition to the genetic heterogeneity, shared features like growth retardation and sparse hair that are found in most CFCS patients are all clinical presentations that might be mistaken for other syndromes or CS, making a definitive clinical diagnosis of CFCS challenging [Gripp et al., 2007; Ciara et al., 2015].

The spectrum of mutations for CFCS has been reported in European and American populations as well as several countries in South America and Northern Asia, but reports on the genetic variants found in patients from South East Asia are few. In this study, we demonstrate the confirmation of the clinical diagnosis of CFCS for 2 patients through molecular testing and compare their features with other CFCS patients who also had mutations in MAP2K1.

The 2 patients were referred to the clinical geneticists in our hospital for evaluation of syndromic disorders. They were recruited for genomic sequencing after written informed consent from the parents.

Venous blood samples were collected in EDTA tubes from the patients and their parents. Genomic DNA was isolated using Gentra Puregene Blood Kit (Qiagen, USA). Gene panel sequencing was performed using the TruSight One panel and the MiSeq Sequencer (Illumina, San Diego, USA). Sequence output was processed using the MiSeq Reporter pipeline (Illumina) and aligned to human genome reference GRCh37/hg19. Variant annotation and prioritization were performed using wANNOVAR and Illumina Variant Studio (Illumina). Potential variants were confirmed by Sanger sequencing in the index patients, followed by testing on parental samples for inheritance status.

Patient 1 (LL) is the second of 3 children born to a non-consanguineous Chinese-Indonesian family. There was no significant family history. She was delivered via emergency lower segment Caesarean section (LSCS) at 31 weeks gestation in another hospital due to maternal chorioangioma. The patient was born well with a birth weight of 1.880 kg (50th–90th percentile), length of 44.3 cm (90th–97th percentile), and head circumference of 30.5 cm (90th–97th percentile). She was referred to our hospital at 6 months of age for evaluation of failure to thrive (FTT). Her weight at presentation was 3.965 kg (<3rd percentile, −3.0 SD), height was 53.7 cm (<3rd percentile, −3.1 SD), and head circumference was 39 cm (<3rd percentile, −2.8 SD). Her poor growth was associated with feeding difficulties with poor suck complicated by abdominal distension with intermittent vomiting and constipation. Her developmental milestones were appropriate for her age, and there was no known significant family history of growth failure and developmental delay. Physical examination showed abdominal distension but it felt soft on palpation. Her full blood count showed microcytic hypochromic anemia. Other investigations on her blood including thyroid function test, liver function test, renal function test, and other electrolytes (serum calcium, magnesium, and phosphate) were unremarkable. As she had a history of prolonged passage of meconium at 2 weeks of life, a rectal suction biopsy was performed, which ruled out Hirschsprung disease.

An echocardiogram revealed mild tethering of the pulmonary valve and a small (hemodynamically insignificant) atrial septal defect (ASD), and these were determined not to be the cause of her FTT. Her swallowing was assessed to be normal by the speech therapist with no oropharyngeal dysphagia. Her FTT was attributed to poor nutritional intake, and she was given appropriate feeding and dietary advice. While she started to grow, she remained below the 3rd centile for weight and height (Fig. 1). Endocrine investigation revealed low insulin-like growth factor-1 (IGF-1) at 35 µg/L with low to normal growth hormone (GH) level at 7 µg/L. She was started on a trial of GH with minimal improvement. In the meantime, cytogenetic investigation revealed a normal karyotype which ruled out Turner syndrome. She was then referred to the genetics clinic to rule out other syndromic causes for her poor growth.

At presentation to our genetics clinic at 2 years 3 months, weight of Patient 1 was 9.8 kg (<3rd percentile, −2.3 SD), height was 80 cm (<3rd percentile, −2.2 SD), and head circumference was 48.2 cm (50th–75th percentile, +0.5 SD). She was noted to have distinctive craniofacial features including relative macrocephaly, sparse hair, hypertelorism, downslanting palpebral fissures, low set ears, short neck, and crowded teeth. Examination of other systems revealed no significant findings. After obtaining written informed consent, targeted next-generation sequencing was performed with the TruSight One panel which revealed a de novo mutation in MAP2K1 (NM_002755.3:c.370C>T; p.P124S). This variant has been documented and classified as pathogenic in ClinVar (VCV000375981.6). The variant is also classified as pathogenic (PS2+PM2+PS3) for our patient according to the guidelines from ACMG/AMP [Richards et al., 2015]. The identification of this variant confirmed a diagnosis of CFCS which was consistent with her clinical phenotype.

Her growth continued to be below the 3rd percentile for her age. In view of her low IGF-1 levels, she was continued on GH until 5 years 1 month; thereafter, her height had been consistently between the 3rd and 10th percentiles (Fig. 1). Dermatological examination revealed 2 café-au-lait spots (on the back and trunk) at 2 years 8 months, which increased by 2 more (on the back and leg) when examined at 6 years 1 month. A follow-up echocardiogram showed spontaneous closure of the ASD and stable (mild) tethering of the pulmonary valve. An ophthalmological review showed mild intermittent bilateral exotropia, bilateral astigmatism, and mild bilateral hyperopia.

The patient had mild developmental delay in the fine motor skills and also speech and language domains. When she was evaluated at our hospital at 2 years 8 months, her neurodevelopment was normal and she was able to walk, climb stairs, speak in 2–3-word sentences, play with her siblings, and feed herself. When assessed at 5 years, she had mild difficulty with articulation and fine motor skills and required interventions with speech and language and occupational therapies. She displayed mild learning issues and the commencement of her primary school education was deferred by 1 year. A formal neuropsychological assessment was not performed.

Patient 2 (NA) is the first of 2 children born to a non-consanguineous Malay family. There was no significant family history. The pregnancy was complicated by mild maternal pre-eclampsia and anti-Ro positive, anti-La negative systemic lupus erythematosus. While a fetal anatomy scan at 19 weeks gestation was normal, a growth scan at 28 weeks gestation showed bilateral ventriculomegaly (right 11.4 mm, left 9.2 mm) with no obvious 3rd or 4th ventricular dilatation. An emergency LSCS for non-reassuring fetal status was carried out at 34 weeks and 4 days. The patient was born well with measurements appropriate for gestational age, with a birth weight of 2.240 kg (10th–50th percentile), length of 42 cm (10th–50th percentile), and head circumference of 32 cm (50th–90th percentile). She was also noted to have bilateral single palmar crease and low-set ears.

She had transient tachypnea of the newborn and required supplemental oxygen support for her first 6 h of life. Follow-up postnatal cranial ultrasound showed mild prominence of lateral ventricles. A cardiac murmur was detected during screening, and an echocardiogram showed a small patent oval foramen. At 4 months, she was hospitalized for severe FTT (Fig. 1) associated with feeding difficulties. She was investigated for metabolic, endocrine, malabsorptive, rheumatological, and genetic causes of FTT. Assessment by the speech therapist did not find any evidence of oropharyngeal dysphagia, and the parents were advised by the dietician to concentrate her milk feeds to increase caloric intake. She needed top-up feeding via a nasogastric (NG) tube to complete the feed volume.

Chromosomal microarray analysis showed no copy number abnormality. She was referred to the genetics clinic at 6 months of age for further genetic investigations. Her weight then was 2.71 kg (<3rd percentile, −4.7 SD), height was 52 cm (<3rd percentile, −3.6 SD), and head circumference was 37 cm (3rd–10th percentile). Craniofacial examination revealed relative macrocephaly, sparse hair and eyebrows, a short nose with a depressed nasal bridge, and posteriorly rotated and low set ears. Examination of the extremities showed deep palmar creases and overlapping 1st and 5th toes (Fig. 2). Neurological examination found hypertonia, hyperreflexia, limitations in elbow extension and right hip abduction, and ulnar deviation. Severe developmental delay was also noted with significant head lag and inability to roll over. Targeted next-generation sequencing with the TruSight One panel uncovered a de novo mutation in MAP2K1 (NM_002755.3:c.371C>A; p.P124Q) which has been reported in a patient with CFC3 [Gripp et al., 2007] and also in ClinVar (VCV000040743.2). The variant is also classified as pathogenic (PS2+PM2+PM5+PM1) for this patient based on the ACMG/AMP criteria [Richards et al., 2015], confirming her diagnosis of CFCS.

The patient continued to have poor growth and was placed on full NG tube bolus feeding at 6 months. Subsequent review at 1 year 2 months found oropharyngeal dysphagia associated with gastroesophageal reflux disease (GERD), for which she was started on continuous NG tube feeding and proton pump inhibitors. A follow-up echocardiogram showed spontaneous closure of the patent oval foramen, but revealed mild right ventricular hypertrophy (RVH) at 2 years 7 months, and asymmetrical septal hypertrophy was detected at 3 years 5 months. While her RVH has remained stable, her septal hypertrophy worsened over time and prompted the initiation of propranolol.

Follow-up neurological examinations found variable tone with contractures and persistent fixed flexion deformities in her knees and elbows which are being managed with Botulinum toxin injections, serial casting, and physical therapy. She also has femoral head subluxation which is being monitored and managed with a Sitting Walking And Standing Hip (SWASH) orthosis. At 2 years 6 months, she developed sudden-onset epileptic spasms which correlated with epileptiform activity on electroencephalography. She was started on Levetiracetam with good control. However, she developed breakthrough seizures precipitated by an episode of Clostridium difficile-positive enterocolitis causing gastrointestinal (GI) sepsis at 3 years 5 months. She subsequently developed hypoxic-ischaemic encephalopathy with non-epileptic anoxic myoclonus and dystonia as a result of cardiopulmonary collapse during the admission. Her dystonia is currently being managed on Baclofen. She was also referred for ophthalmological review to monitor for ocular involvement and was found to have left convergent strabismus and bilateral hyperopia.

The patient also has chronic lung disease (CLD) with mild tachypnea and mild subcostal retractions at baseline. This is likely multifactorial as a result of obstructive sleep apnea, microaspirations from presumed GERD, as well as recurrent respiratory tract infections. She had a history of hospitalizations: at age 7 months for acute laryngotracheobronchitis (ALTB), at 2 years 8 months for pneumonia with parapneumonic effusion, and at 2 years 5 months for an upper respiratory tract infection. Her episode of GI sepsis at 3 years 5 months was also complicated by left lung collapse. To manage her CLD, she was started on continuous positive airway pressure (CPAP) ventilation at 3 years 5 months. During her admission for ALTB, she was also noted to have inspiratory stridor and was referred for investigation. Microlaryngobronchoscopy was performed and showed posterior arytenoid swelling due to chronic inflammation which was non-obstructive.

Developmental assessment found severe global developmental delay, with her developmental age assessed to be between 3 and 6 months when she was 5 years 4 months old. It was observed that she could turn from left to right and use her legs to push herself to the back but not crawl or sit independently. She could understand simple instructions and make noises to communicate but could not babble. She could smile and look around to follow bright objects with her eyes but could not grab them. She is currently receiving early intervention to maximize her developmental growth including physiotherapy, occupational therapy, and speech and language therapy. A formal neuropsychological evaluation was not performed.

We report 2 patients with CFCS caused by MAP2K1 mutations and compare their phenotypic features with 37 cases comprising mostly European patients from published cases (Table 1) [Rodriguez-Viciana et al., 2006; Gripp et al., 2007; Yoon et al., 2007; Nyström et al., 2008; Dentici et al., 2009; Allanson et al., 2011; Papadopoulou et al., 2011; Siegel et al., 2011; Chen et al., 2014; Nishi et al., 2015; Bessis et al., 2019; Leach et al., 2019]. While both our patients presented with similar features in infancy and early childhood, one had a milder disease course. Patient 1 presented in infancy with ASD, pulmonary valve tethering, feeding difficulties, and postnatal onset growth retardation, then developed characteristic craniofacial features and mild developmental delay. In contrast, Patient 2 presented with ASD in infancy, characteristic craniofacial features, feeding difficulties necessitating NG tube feeding, and severe FTT which was further complicated by GERD. She also had right ventricular and septal hypertrophy, epileptic spasms, fixed flexion deformities, and severe global developmental delay.

The constellation of features in Patient 2 is severe and adversely affects her quality of life which is unusual for CFCS. It sets her apart from Patient 1 and also other reported CFCS patients, with severe features thought to be more commonly seen in CS. As many case reports have shown that there is significant phenotypic overlap between CS and CFCS including feeding difficulties and FTT, the use of genetic testing results in addition to phenotypic features would provide an additional tool for diagnostic clarity, which is necessary for differential clinical management. For instance, tumor surveillance is required in CS as there is an increased risk of malignant tumors, but the risk of malignancy is low for CFCS. However, in the case of the 2 variants identified in our 2 patients, both of which are classified as pathogenic by the ClinGen RASopathy Variant Curation Expert Panel, they have also appeared as somatic variants in multiple patients with various neoplasms.

Currently, there are only 17 damaging variants associated with CFCS in the Human Genome Mutation Database. Except for a 3-nucleotide in-frame deletion, the other 16 are missense substitutions and not loss-of-function or truncating mutations. The variants in our 2 patients are 1 nucleotide apart and affect the same codon in the kinase domain. Both involve the substitution of a nonpolar residue (proline) with a polar residue (serine for Patient 1 and glutamine for Patient 2). It is therefore intriguing that their disease course and severity are so different. In the case of Patient 2, the identified variant has been reported in another patient initially diagnosed with CS but who had no HRAS mutation. He was subsequently found to carry this MAP2K1 variant [Gripp et al., 2007].

Our description of the clinical progression of Patient 2 may elucidate the clinical trajectory of patients with this variant, as her severe disease course appears to correlate with that of the patient from Gripp et al. [2007], who died at 7 weeks from complications following cardiac surgical repair of his congenital heart disease. Both Patient 2 and that patient were delivered at 34 weeks via LSCS, had FTT, feeding difficulties necessitating NG tube feeding, congenital heart defects, and presented with Costello-like phenotypes. Functional studies have shown that this variant could cause RASopathy phenotype as well as cancer in zebrafish embryos [Jindal et al., 2017]. Characterization of additional patients with this variant will be necessary to establish a genotype-phenotype correlation.

The presentations of Patient 2 add to existing reports that support expanding the CFCS phenotype to include features previously thought to be more suggestive of CS and affirm the use of associated gene mutations to confirm the diagnosis of each syndrome. However, it is worth noting that Patient 2 presented with ulnar deviation, a feature found to be more common in HRAS-positive CS patients [Gripp et al., 2007]. It is not entirely surprising as HRAS encodes a GTPase that interacts upstream of MAP2K1 in the same pathway. Further analysis with additional patients carrying specific HRAS/MAP2K1 mutations would be necessary to evaluate the differential occurrence of this feature in CFCS and CS.

In summary, we report 2 CFCS patients with MAP2K1 mutations who have different disease severity. While Patient 1 has a mild disease course presenting with features commonly seen in CFCS, Patient 2 has a severe disease course, presenting with Costello-like features. With the significant phenotypic overlap between CFCS, CS, and other RASopathies, certain phenotypic features can become diagnostic red herrings. We demonstrate the utility of molecular confirmation to differentiate between closely-related syndromes to achieve a more accurate diagnosis and inform better management. The phenotype of Patient 2 also suggests that although there is a strong correlation between the mutated gene and each syndrome, conditions within the RASopathies spectrum may not be clinically distinct but is instead on a continuous spectrum.

The study was approved by the SingHealth Institutional Review Board (CIRB Ref 2014/571/F) which oversees all research activities in the hospital. As the 2 patients were minors, written informed consent was obtained from their parents for genetic investigation, publication, and accompanying images.

All authors declare no conflict of interest.

This work was supported by the National Research Foundation Singapore under its NMRC Centre Grant Program (NMRC Project No. NMRC/CG/M003/2017) administered by the Singapore Ministry of Health’s National Medical Research Council. Saumya Jamuar is supported by CSAINV21jun-003 from the National Medical Research Council, Republic of Singapore.

Elyn Yo-Lin Tzen and Ai Ling Koh: writing of first draft. Jiin Ying Lim and Sylvia Kam: genetic counseling. Sue Mei Cheah, Jonathan Tze Liang Choo, Zhi Min Ng, Biju Thomas: clinical investigations. Saumya Jamuar: clinical investigations and editing. Ene-Choo Tan: molecular investigations, funding support, and final draft. All authors read and approved the manuscript.

The data that support the findings of this study are not publicly available due to personal data protect Act. Details of sequencing results are available on request from the corresponding author.