HM Luk, IFM Lo, Y Aoki, TMF Tong, DHC Chan, STS Lam

Abstract

Cardio-facio-cutaneous (CFC) syndrome is a rare disorder of RASopathy that is caused by dysregulation of the RAS/MAPK signaling pathway. Germline mutations of BRAF, MEK1, MEK2 and KRAS genes account for 70-90% of the cases. We have performed mutational analysis for the clinically diagnosed CFC patients under the care of Clinical Genetics Service, Department of Health, Hong Kong Special Administrative Region between 2000 to 2011. Totally 7 unrelated clinically diagnosed Chinese CFC patients were studied. Mutations have been identified in 6 (85.7%) of our patients, including 5 (71.4%) BRAF mutations and 1 (14.2%) MEK2 mutation. Their clinical features were summarised. It can be concluded that the pattern of molecular defects and clinical characteristics of Chinese CFC patients are comparable with other populations without ethnic-specific difference.

Introduction

Cardio-facio-cutaneous (CFC) syndrome (OMIM 115150) is a rare sporadic multiple congenital anomaly syndrome characterised by distinctive craniofacial dysmorphism, cardiac malformation, ectodermal anomalies and neurodevelopmental delay. The first three cases were reported by Blumberg et al in 1979.¹ The name "CFC syndrome" was first coined by Reynold et al² in 1986 when five other cases were reported.Up to now more than two hundred cases have been reported, but the exact incidence of this rare syndrome is still unknown.

With more understanding of underlying molecular pathophysiology, CFC syndrome is now categorised as one of the RASopathy syndromes.³ The other well known RASopathy syndromes included Noonan syndrome, Costello syndrome, neurofibromatosis type 1 and LEOPARD syndrome. These disorders are caused by dysregulation of the RAS/mitogen-activated protein kinases (MAPK) signaling pathway,⁴ which plays an important role in regulating cellular growth and proliferation. Although each of them has its own unique characteristics, they have many overlapping features. Now four genes are known to cause CFC syndrome, namely BRAF, MEK1, MEK2 and KRAS. The mutations result in a gain of function. However, the genetic defects are still elusive in approximately 10-30% of cases.⁵ Therefore, stringent diagnostic criteria still have a role to play in diagnosis and management of CFC patients. Two commonly used CFC diagnostic criteria included Grebe and Clericuzio criteria and the CFC index.⁶ Both show high positive predictive values to a positive mutational analysis⁷ and concordance with the diagnosis.

In this study, we reviewed and summarised the clinical characteristics of the local Chinese CFC patients, and performed molecular analysis of the four known CFC-causing genes for these patients. The aim was to study the clinical and mutational spectrum in those established cases.

Materials and Methods

Patients

The Clinical Genetic Service (CGS) of the Department of Health is the only tertiary referral center for genetic diagnosis and counseling in Hong Kong. All patient records with the clinical diagnosis of CFC syndrome between 2000 to 2011 were retrieved from the computer database system of CGS. The records and clinical photos were reviewed by clinical geneticists, and the clinical features were analysed retrospectively with clinical diagnostic criteria. Informed consents were obtained from parents and/or patients for study and publication (included the clinical photos).

Patients with abnormal karyotype, PTPN11 or HRAS gene mutations were excluded from this study. Mutational analysis of the four CFC-causing genes was performed by the Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan.

Clinical Diagnostic Criteria for CFC Syndrome

The clinical diagnosis was based on the Grebe and Clericuzio criteria and the CFC index. For the Grebe and Clericuzio criteria,⁸ at least seven out of ten clinical features have to be present, which include macrocephaly, characteristic facial features, growth retardation, neurological impairment/developmental delay, gastrointestinal dysfunction, cardiac defect, sparse/curly hair, ocular abnormalities/dysfunction, history of polyhydramnios and hyperkeratotic skin lesions.

The CFC index is based on 82 clinical traits and their frequencies in patients with CFC syndrome.⁹ By adding the defined values of each trait, a CFC index score is calculated. Ninety-five percent of the patients with CFC syndrome have a score between 9.5 and 19.9.

CFC Syndrome Molecular Analysis

Genomic DNA was extracted from peripheral blood using QIAmp DNA minikit (Qiagen, Germany) according to instruction of manufacturer. The coding sequence of exons 1-5 of the KRAS gene{NM_004985.3}; exons 6, 11-15 of the BRAF gene{NM_004333.4}; exons 2 and 3 of the MEK1 {NM_002755.3} and MEK2 genes{NM_030662.3} were amplified by standard polymerase chain reaction (PCR). The primer pairs and experimental conditions used were as described by Narumi.⁷ The PCR products are gel-purified and sequenced on an ABI PRISM 310 automated DNA sequencer (Applied Biosystems, Foster City, CA). Parental samples were tested when a mutation was identified in the patients.

Results

Total of 9 patients with the clinical diagnosis of CFC syndrome were retrieved from the CGS registry. One patient had a HRAS mutation and the diagnosis was changed to Costello syndrome. Therefore, 8 unrelated patients with CFC syndrome were left for further analysis. Seven of them were Chinese and one was Nepalese. For those 7 Chinese CFC patients, the female to male ratio was 6:1. Their age ranged from 7 years to 22 years and 2 months with median of 11 years and 10 months. The clinical features of these 7 patients are summarised as Table 1.

CFC Mutational Spectrum

Mutations were identified in 6 out of 7 index Chinese patients with the detection rate of 85.7%. Among them 5 (71.4%) were located in the BRAF gene and 1 (14.3%) in the MEK2 gene. All are missense and de novo mutations. All mutations have been reported in the literature to be associated with CFC syndrome,^5,10-12 therefore expected to be pathogenic. The results are summarised in Table 2.

Figure 1 Features of Chinese patients with CFC syndrome. Note the characteristic facial gestalt of these patients: high forehead, relative macrocephaly, bitemporal narrowing, hypoplasia of the supraorbital ridges, ocular hypertelorism, epicanthal folds, ptosis (patient 4 and 5), short nose with depressed bridge, low-set ears. There were also sparse eyebrows and sparse and curly hair (patient 4 and 6).

Discussion

Although CFC syndrome was first described over 30 years ago,¹ the diagnosis was mainly based on clinical criteria until 2006 when the underlying genetic defects were identified.¹⁰ Since then, more and more CFC patients with molecular confirmation have been reported in the literature, but these are mainly Caucasian patients. This is the first reported series of Chinese CFC patients with detailed clinical and molecular analyses.

Totally 8 patients with clinically diagnosed CFC syndrome were included in this study. All have a CFC index score falling bewteen 13.5 and 17.8, and fulfill at least 7 of the 10 Grebe and Clericuzio criteria. Excluding the one patient who is non-Chinese, mutations were identified in 5 out of 6 Chinese patients with a detection rate of 85.7%. Five (71.4%) mutations were found in BRAF gene and 1 (14.3%) in MEK2 gene. No KRAS mutation was present in our patients. It is well reported that around 75%, 25% and <2-3% of CFC-causing mutations are located in the BRAF, MEK1/MEK2 and KRAS genes, respectively.^5,10 Therefore, the spectrum of mutations in our Chinese CFC patients is comparable to other populations.^5,11,12

Concerning the mutations identified in our Chinese CFC patients, all have been reported in the literature.^5,10-12 Despite the advancement in understanding the molecular pathophysiology of CFC syndrome, the molecular defect is still unknown in 10-30% of patients.⁵ For these patients, the genetic defects might reside in other regions of the four known genes that have not been analyzed, or in other genes that are yet to be identified in the RAS/MAPK pathway. For this reason, clinical diagnosis following stringent diagnostic criteria still has its essential role in the management of CFC patients before the molecular genetics of CFC syndrome is fully understood.⁶

The spectrum of clinical features of our Chinese cohort is consistent with studies in other populations.^13-16 The only exception is polyhydraminos and ventriculomegaly/hydrocephalus. It is reported that more than half of CFC syndrome patients are complicated by polyhydraminos antenatally⁵ and ventriculomegaly/hydrocephalus during early childhood.¹⁷ However, only two patients in our study had polyhydramnios and one patient with ventriculomegaly. The difference may be due to our small sample size and ascertainment bias. In view of the considerable phenotypic overlap among the RASopathy syndromes, particularly CFC syndrome, Costello syndrome and Noonan syndrome; PTPN11, SOS1 and HRAS mutational analyses are warranted for those CFC or CFC-like patients without mutations detected in the BRAF, MEK1/2, and KRAS genes.¹²

Currently, the management of CFC syndrome is mainly supportive with regular cardiac assessment for late onset cardiomyopathy.¹¹ With better understanding of underlying molecular defect, new drugs that target on inhibition of RAS/MAPK hyperactivation may provide therapeutic effect on CFC syndrome.¹⁸

Although somatic mutations in BRAF gene have been found in 7% of all neoplasias,⁵ germline mutations of BRAF gene in CFC syndrome are not associated with an increased risk of cancer, in contrast to other RASopathies. Regular cancer surveillance may not be needed.¹¹ Up till now, no genotype-phenotype correlation can be found in CFC patients.⁵ With more molecularly confirmed cases of CFC syndrome being diagnosed, genotype-phenotype correlation may become clear and provide guidance on management and prognostication of CFC patients in the future.

Finally, as CFC syndrome is phenotypically overlapping with other RASopathies, particularly Noonan syndrome and Costello syndrome and clinically distinction between them sometimes are difficult. It is recommended that all these group of patients should be referred to clinical geneticist for assessment and molecular confirmation if possible, so as to provide the optimal counseling and specific surveillance for different RASopathies.

Conclusions

Cardio-facio-cutaneous (CFC) syndrome is a rare, usually sporadic, RASopathy syndrome that is characterised by distinctive craniofacial dysmorphism, cardiac malformation, ectodermal anomalies and neurodevelopmental delay. We have performed the first clinical and molecular study for Chinese patients with CFC syndrome. Mutations have been identified in 85.7% of the clinically diagnosed CFC patients in our cohort, with 71.4% and 14.3% detected in the BRAF gene and the MEK2 gene, respectively. The spectrum of molecular defects is comparable to that of other populations. The pattern of clinical features is also similar to other ethnic groups. With the overlapping clinical features among the RASopathies, PTPN11, SOS1 and HRAS mutation analyses should also be considered for those CFC or CFC-like patients without mutations in the BRAF, MEK1/2 and KRAS genes. With more clinical and basic researches on CFC syndrome, we shall have better understanding on the natural course and the underlying pathophysiology, which in turn will improve management and prognostication of CFC patients.

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