ABSTRACT
RASopathies comprise a group of congenital malformation syndromes with predominant neuro‐cardio‐facial‐cutaneous involvement resulting from pathogenic variants in RAS/mitogen‐activated protein kinase (MAPK) signaling pathway genes. In this study 33 patients are presented with their clinical and molecular findings as an RASopathy cohort including a family with an AMMECR1‐related disorder. The diagnostic distribution of the cohort included Noonan syndrome (n = 18), neurofibromatosis type 1 (n = 8), and single cases of cardiofaciocutaneous syndrome, Costello syndrome, neurofibromatosis‐Noonan syndrome, NF1 microdeletion syndrome, Noonan syndrome‐like disorder with loose anagen hair, Noonan syndrome with multiple lentigines and AMMECR1‐related midface hypoplasia, hearing impairment, elliptocytosis, and nephrocalcinosis (MIM# 300990). The most prevalent clinical manifestations were dermatological findings (90.9%), skeletal features (84.4%), cardiovascular involvement (75.8%), and typical craniofacial dysmorphism suggestive of RASopathy (72.7%). Variants were most frequently identified in PTPN11 and NF1, followed by single cases involving the BRAF, HRAS, LZTR1, RAF1, RIT1, SHOC2, and SOS1. Notably, one patient harbored a variant in AMMECR1, which is not involved in the RAS/MAPK pathway. Overall, this study delineates the clinical and molecular landscape of a cohort from Türkiye and underscores that the AMMECR1‐related phenotype represents a distinct entity that closely mimics Noonan syndrome.
Keywords: AMMECR1, exome sequencing, MFHIEN, Noonan syndrome, RAS/MAPK pathway, RASopathy
This study delineates the clinical and molecular landscape of a cohort from Türkiye and underscores that the AMMECR1‐related phenotype represents a distinct entity that closely mimics Noonan syndrome.
1. Introduction
The RAS/mitogen‐activated protein kinase (MAPK) pathway is a signaling cascade that plays a role in processes like cell proliferation, survival, differentiation, and metabolism. RASopathies constitute an umbrella term for a group of syndromes caused by pathogenic variants in genes encoding components of this pathway. This group includes phenotypes like Noonan syndrome (NS), neurofibromatosis type 1 (NF1), neurofibromatosis‐Noonan syndrome (NFNS), Costello syndrome (CS), cardiofaciocutaneous syndrome (CFCS), Noonan syndrome with multiple lentigines (NSML), Noonan syndrome‐like disorder with loose anagen hair (NSLAH), and Legius syndrome [1]. Common clinical features across these phenotypes include short stature, characteristic facial dysmorphism (triangular face, downslanting palpebral fissures, ptosis, and low‐set ears), intellectual disability, lymphatic dysfunction, and congenital heart defects. The overall prevalence of RASopathies is estimated to be approximately 1 in 1000 [2]. Although inheritance in RASopathies is predominantly autosomal dominant, autosomal recessive inheritance patterns have also been reported for the LZTR1‐ and SPRED2‐related NS [3, 4].
In exome‐sequencing (ES) analyses, pathogenic variants in genes not involved in the RAS/MAPK pathway have been identified in some patients presenting with a RASopathy‐like phenotype. Baraitser–Winter syndrome caused by ACTB variants and Aarskog–Scott syndrome associated with FGD1 mutations represent known examples of this phenomenon [5]. Furthermore, the use of ES facilitates the identification of phenotypes that share common clinical findings with RASopathies but are not classically included in the differential diagnosis.
In the present study, we aimed to delineate the molecular etiology and clinical landscape of patients presenting with phenotypic features suggestive of RASopathies. By utilizing ES, we sought to determine the diagnostic yield within this cohort and to illustrate the capability of ES in expanding the differential diagnosis of atypical presentations, including a novel AMMECR1 variant responsible for a RASopathy‐like phenotype.
2. Materials and Methods
2.1. Patient Cohort and Clinical Evaluation
This retrospective and prospective study included 33 patients with clinical features of RASopathy. All patients were evaluated at the Department of Medical Genetics, Istanbul University, Istanbul Faculty of Medicine, between 2000 and 2024. Demographic data, pedigrees, and family histories, as well as clinical, laboratory, radiological, and genetic findings from the patients' initial presentations and follow‐up visits, were collected from medical records. For patients evaluated during the study period, the clinical records were prospectively updated. The cases were evaluated using the NS Diagnostic Scoring System and the revised NF1 diagnostic criteria [6, 7]. The study was approved by the Clinical Research Ethics Committee of Istanbul University, Istanbul Faculty of Medicine (Date: October 20, 2023; File no.: 2023/1877; Approval no.: E‐29624016‐050.99‐2227523) and was conducted in accordance with the Declaration of Helsinki and its later amendments. Written informed consent was obtained from all patients or their legal guardians before their inclusion in the study.
2.2. Molecular Analysis and Bioinformatics
Molecular analyses were performed using targeted gene panels, clinical exome, ES, and Sanger sequencing, following standard protocols. Genomic DNA was isolated from peripheral venous blood samples collected in EDTA tubes using semi‐automated systems (QIAGEN and MagPurix) in accordance with the manufacturers' protocols. Targeted gene panel sequencing was performed on the Ion Torrent platform using the Ion PGM system. A panel was designed with Ion AmpliSeq Designer to cover the coding regions, exon–intron boundaries, and previously reported mutation sites of 17 genes (BRAF, CBL, HRAS, KAT6B, KRAS, MAP2K1, MAP2K2, NF1, NF2, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1, SPRED1, RRAS) associated with RASopathy phenotypes. Library preparation and sequencing procedures were conducted according to the manufacturer's instructions. Clinical exome and ES were performed using the SOPHiA DDM Clinical Exome Solution v3 and Twist HCExome v2 kits by Sophia Genetics, respectively. Sequencing was carried out on Illumina NextSeq 2000 and NovaSeq 6000 platforms. Data analyses were performed using IonReporter software for targeted panels and Sophia DDM (v4.4.6.0) for ES datasets. The reference genome used was NCBI Build 37 (hg19). Variant filtering criteria included minor allele frequency (< 0.05), genomic location (exonic and ±20 bp of splice sites), variant allele fraction (> 20%), sequencing depth (> 10×), and in‐house variant frequency. The variant reads were visually evaluated using the IGV tool. Variant pathogenicity was classified according to the criteria recommended by the American College of Medical Genetics and Genomics (ACMG) and the ClinGen RASopathy Expert Panel for the ACMG/AMP Variant Interpretation Guidelines [8, 9]. Sanger sequencing analyses were performed using specifically designed forward and reverse primers targeting the identified variants for confirmation and segregation studies.
3. Results
The study cohort consisted of 33 patients, comprising 18 males and 15 females (male‐to‐female ratio: 1.2). The age at initial examination ranged from 16 gestational weeks to 231 months, with a mean of 53.1 months. The age at molecular diagnosis ranged from 4 to 300 months, with a mean of 96.6 months. Parental consanguinity was present in 6 families (18.2%; n = 6/33). Advanced paternal age was observed in 9 families (27.3%; n = 9/33). Craniofacial features compatible with a typical RASopathy phenotype were observed in 24 patients (72.7%; n = 24/33). Skeletal findings were present in 27 patients (84.4%; n = 27/32). Short stature was detected in 14 patients (42.4%; n = 14/33). Endocrinological evaluation revealed growth hormone (GH) deficiency in three patients and hypothyroidism in three patients (30%; n = 6/20). The prevalence of dermatological findings was 90.9% (n = 30/33). Cardiovascular involvement was identified in 25 patients (75.7%; n = 25/33), with pulmonary stenosis being the most common finding (n = 16), followed by septal defects (n = 5). Lymphatic abnormalities were observed in 23.3% of the cases evaluated (n = 7/30). Hematological involvement was present in 28% of the patients (n = 7/25). Neoplasia was identified in seven patients (21.9%; n = 7/32) and all these patients had NF1 variants (63.6%; n = 7/11). Urogenital anomalies were detected in 8 of 32 patients (25%), with renal involvement (n = 5) and cryptorchidism (n = 4) being the most frequent findings. Neurological involvement was present in 16 patients (48.4%; n = 16/33). Hearing loss was identified in four patients (12.1%; n = 4/33) (Table S1).
Variants were most frequently identified in the PTPN11 (n = 14/33; 42.4%) and NF1 (n = 11/33; 33.3%). BRAF, HRAS, LZTR1, RAF1, RIT1, SHOC2, and SOS1 variants were identified in single patients each. In one patient, a variant was detected in the AMMECR1, which is not associated with the RAS/MAPK pathway. The de novo variant rate was determined to be 57.1% (n = 8/14). Four of the 29 identified variants were novel (13.8%). Three of the novel variants were identified in the NF1 gene. Two of these cases (P#20; c.246dup and P#26; c.5338_5342del) had a clinical diagnosis of NF1, while the other was consistent with NS (P#21; c.3611G>A). The other novel variant was identified in the AMMECR1 gene (P#18; c.578_581del). Based on clinical and molecular findings, the diagnostic distribution of the cohort was determined as follows: NS in 18 patients (n = 17, PTPN11; n = 1, NF1), NF1 in 8 patients, NF1 microdeletion syndrome in 1 patient, NFNS in 1 patient, CFCS in 1 patient (n = 1, BRAF), CS in 1 patient (n = 1, HRAS), NSML in 1 patient (n = 1, PTPN11), NSLAH in 1 patient (n = 1, SHOC2), and MFHIEN in 1 patient (n = 1, AMMECR1). Table 1 summarizes the molecular characteristics of the identified variants, including the implicated genes, functional domains and zygosity and segregation data, as well as dbSNP/ClinVar IDs, ACMG classifications, and PubMed IDs for previously reported variants with clinical diagnoses.
TABLE 1.
Molecular findings and clinical diagnosis of the patients in this study.
| Patient ID | Clinical diagnosis | Gene (transcript) | Variation | Domain | Zygosity | Segregation | dbSNP ID (ClinVar ID) | ACMG classification | Publication |
|---|---|---|---|---|---|---|---|---|---|
| P#1 | NS | PTPN11 (NM_002834.5) |
c.124A>G p.(Thr42Ala) |
N‐SH2 | Heterozygous | NA | rs397507501 (ID: 40482) | Pathogenic (PS2_VS, PS3, PS4, PM2_P, PM5, PP2, PP3) | PMID: 11992261 [10] |
| P#2 | |||||||||
| P#3 | |||||||||
| P#4 | |||||||||
| P#5 | NS | PTPN11 (NM_002834.5) |
c.179G>C p.(Gly60Ala) |
N‐SH2 | Heterozygous | NA | rs397507509 (ID: 40493) | Pathogenic (PS2_VS, PS3, PS4, PM5_S, PP2, PP3) | PMID: 11992261 [10] |
| P#6 | NS | PTPN11 (NM_002834.5) |
c.188A>G p.(Tyr63Cys) |
N‐SH2 | Heterozygous | de novo | rs121918459 (ID: 13333) | Pathogenic (PS3, PS4, PM1, PP1_S, PP2, PP3) | PMID: 11704759 [11] |
| P#7 | NS | PTPN11 (NM_002834.5) |
c.218C>T p.(Thr73Ile) |
N‐SH2 | Heterozygous | de novo | rs121918462 (ID: 13334) | Pathogenic (PS2_VS, PS3, PS4, PM2_P, PM5_S, PP2, PP3) | PMID: 11992261 [10] |
| P#8 | NS | PTPN11 (NM_002834.5) |
c.236A>G p.(Gln79Arg) |
N‐SH2 | Heterozygous | de novo | rs121918466 (ID: 13340) | Pathogenic (PS2_VS, PS3, PS4, PM5_S, PP1_S, PP2, PP3) | PMID: 11704759 [11] |
| P#9 | NS | PTPN11 (NM_002834.5) |
c.854 T>C p.(Phe285Ser) |
PTPase | Heterozygous | NA | rs121918463 (ID: 13335) | Pathogenic (PS2_VS, PS3, PS4, PM2_P, PM5_S, PP2, PP3) | PMID: 11992261 [10] |
| P#10 | NS | PTPN11 (NM_002834.5) |
c.922A>G p.(Asn308Asp) |
PTPase | Heterozygous | de novo | rs28933386 (ID: 13326) | Pathogenic (PS2_VS, PS3, PM2, PP1_S, PP2, PP3) | PMID: 11704759 [11] |
| P#11 | |||||||||
| P#12 | NS |
PTPN11 |
c.923A>G p.(Asn308Ser) |
PTPase | Heterozygous | NA |
rs121918455 (ID: 13327) |
Pathogenic (PS2_VS, PS3, PS4, PM2_P, PM5_S, PP1_S, PP2) | PMID: 11992261 [10] |
| P#13 | NS | PTPN11 (NM_002834.5) |
c.1510A>G p.(Met504Val) |
PTPase | Heterozygous | NA | rs397507547 (ID: 40562) | Pathogenic (PS3, PS4_M, PM6_S, PP2, PP3) | PMID: 11704759 [11] |
| P#14 | NS | RAF1 (NM_002880.3) |
c.782C>T p.(Pro261Leu) |
CR2 | Heterozygous | NA | rs397516828 (ID: 120246) | Pathogenic (PS2_VS, PS3, PS4, PM2_P, PM5_S, PP3) | PMID: 17603482 [12] |
| P#15 | NS | RIT1 (NM_006912.5) |
c.242A>G p.(Glu81Gly) |
Switch II | Heterozygous | Paternal | rs869025193 (ID: 183405) | Pathogenic (PS2_VS, PS3, PS4, PM1, PM2_P, PM5, PP3) | PMID: 23791108 [13] |
| P#16 | NS | SOS1 (NM_005633.4) |
c.508A>G p.(Lys170Glu) |
HD | Heterozygous | de novo | rs397517172 (ID: 40651) | Pathogenic (PS3, PM1, PM2, PM6, PP2, PP3) | PMID: 19020799 [14] |
| P#17 | NS | LZTR1 (NM_006767.4) |
c.2326‐5 T>C p.(?) |
— | Homozygous | rs1412999901 (ID: 1789650) | VUS (PM2_P) | — | |
| P#18 | MFHIEN | AMMECR1 (NM_015365.3) |
c.578_581del p.(Leu193ProfsTer12) |
— | Hemizygous | Maternal | — | Pathogenic (PVS1, PM2_P, PP1) | Novel |
| P#19 | NF1 | NF1 (NM_000267.3) |
c.70A>T p.(Lys24Ter) |
— | Heterozygous | NA |
rs2143625250 (ID: 1385992) |
Likely Pathogenic (PVS1, PM2_P) | — |
| P#20 | NF1 | NF1 (NM_000267.3) |
c.246dup p.(Gln83SerfsTer24) |
— | Heterozygous | Paternal | — | Pathogenic (PVS1, PM2_P, PP1, PP4) | Novel |
| P#21 | NS | NF1 (NM_000267.3) |
c.3611G>A p.(Arg1204Gln) |
GRD | Heterozygous | Maternal | — | Likely pathogenic (PM1, PM2_P, PM5, PP2) | Novel |
| P#22 | NF1 | NF1 (NM_000267.3) |
c.3763C>T p.(Gln1255Ter) |
GRD | Heterozygous | NA | rs1060500308 (ID: 404504) | Pathogenic (PVS1, PS4, PM2_P) | PMID: 23913538 [15] |
| P#23 | NF1 | NF1 (NM_000267.3) |
c.4585G>T p.(Glu1529Ter) |
— | Heterozygous | NA | rs2151466355 (ID: 3236836) | Pathogenic (PVS1, PS4, PM2_P) | PMID: 29618358 [16] |
| P#24 | NF1 | NF1 (NM_000267.3) |
c.4867G>T p.(Asp1623Tyr) |
Sec14‐PH | Heterozygous | de novo | rs1131691123 (ID: 429003) | Likely Pathogenic (PM1, PM2_P, PM5, PM6, PP2, PP3) | PMID: 23656349 [17] |
| P#25 | NF1 | NF1 (NM_000267.3) |
c.4986C>A p.(Asn1662Lys) |
Sec14‐PH | Heterozygous | NA | — | Pathogenic (PS1, PS4, PM2_P, PP2, PP3) | PMID: 24676943 [18] |
| P#26 | NF1 |
NF1 |
c.5338_5342del p.(Leu1780HisfsTer16) |
Sec14‐PH | Heterozygous | Paternal | — |
Pathogenic (PVS1, PM2_P, PP1, PP4) |
Novel |
| P#27 | NFNS | NF1 (NM_000267.3) |
c.5606G>T p.(Gly1869Val) |
HLR | Heterozygous | de novo | rs1567613630 (ID: 578661) | Pathogenic (PS2, PS4, PM1, PM2_P, PP2, PP3) | PMID: 26275891 [19] |
| P#28 | NF1 | NF1 (NM_000267.3) |
c.6792C>A p.(Tyr2264Ter) |
HLR | Heterozygous | NA | rs772295894 (ID: 439973) | Pathogenic (PVS1, PS2, PS3, PS4, PM2_P, PP1) | PMID: 8837715 [20] |
| P#29 | NF1 microdeletion syndrome | NC_000017.10 | g.28525332_30321750del | Heterozygous | NA | ‐(ID: 153736) | Pathogenic (2A, 4L) | PMID: 11468690 [21] | |
| P#30 | CFCS | BRAF (NM_004333.4) |
c.1403 T>C p.(Phe468Ser) |
KD | Heterozygous | NA | rs397507473 (ID: 40366) | Pathogenic (PS2_VS, PS4, PM1, PM2_P, PM5, PP2, PP3) | PMID: 16439621 [22] |
| P#31 | CS | HRAS (NM_005343.2) |
c.175G>A p.(Ala59Thr) |
Switch II | Heterozygous | NA | rs727503093 (ID: 40435) | Likely Pathogenic (PS4,M, PM1, PM2,P, PP1, PP3) | — |
| P#32 | NSLAH | SHOC2 (NM_007373.4) |
c.4A>G p.(Ser2Gly) |
N‐terminal | Heterozygous | de novo | rs267607048 | Pathogenic (PS2_VS, PS3, PS4, PM2, PP2) | PMID: 19684605 [23] |
| P#33 | NSML | PTPN11 (NM_002834.5) |
c.836A>G p.(Tyr279Cys) |
PTPase | Heterozygous | NA | rs121918456 | Pathogenic (PS4, PM1, PM2_P, PM6, PP1, PP2, PP3) | PMID: 11992261 [10] |
Abbreviations: CFCS, cardiofaciocutaneous syndrome; CS, Costello syndrome; MFHIEN, midface hypoplasia, hearing impairment, elliptocytosis, and nephrocalcinosis; NA, not available; NF1, neurofibromatosis 1; NFNS, NF1‐Noonan syndrome; NS, Noonan syndrome; NSLAH, Noonan syndrome‐like disorder with loose anagen hair; NSML, Noonan syndrome with multiple lentigines.
4. Discussion
RASopathies are a group of congenital malformation syndromes characterized by neurological, cardiac, craniofacial, and cutaneous features. The considerable phenotypic overlap among these syndromes complicates their clinical distinction. In addition, some of these conditions are considered to be allelic disorders, as they arise from pathogenic variants in the same genes [2].
In our study, three novel variants were identified in the NF1. Notably, one of these variants was detected in P#21, who presented exclusively with the clinical features of NS. Based on the diagnostic scoring system for NS, P#21 met the diagnostic criteria by fulfilling three major (facial, cardiac, and chest wall) and two minor (height and other) criteria while completely lacking NF1‐related cutaneous findings such as café‐au‐lait (CAL) spots. Furthermore, no clinical involvement was observed in the patient's mother, who harbored the same variant, highlighting a significant degree of intrafamilial phenotypic variability as a phenomenon previously reported among individuals with identical NF1 variants [24]. Historically, following the initial description of the NF1 phenotype, Watson syndrome and NFNS were introduced to the literature and subsequently demonstrated to be allelic disorders [25, 26]. Due to this shared genetic etiology, these conditions exhibit substantial phenotypic overlap and can often be clinically indistinguishable. Supporting our findings, Witkowski et al. reported two cases clinically diagnosed with NS who harbored pathogenic/likely pathogenic NF1 variants but lacked classical NF1‐associated cutaneous signs, including CAL spots [27]. Collectively, the literature and our present findings indicate that the clinical spectrum of NF1‐related disorders is highly variable. Therefore, NF1 variants should be considered in the etiology of NS cases, even in the absolute absence of NF1‐related cutaneous manifestations.
CS is a RASopathy caused by pathogenic variants in the HRAS gene, characterized by hypotonia, feeding difficulties, short stature, developmental delay or intellectual disability, macrocephaly, coarse facial features, curly or sparse/fine hair, deep palmar and plantar creases, loose and soft skin, facial and perianal papillomas, joint laxity with ulnar deviation of the wrists and fingers, Achilles tendon tightness, and cardiac involvement. HRAS variants associated with CS most frequently affect the p.Gly12 amino acid within the P‐loop domain. Following p.Gly12, the p.(Gly13Asp) variant, the second most frequently involved residue, has been reported in the literature to present with a milder phenotype and an absence of malignancy. Various studies have demonstrated that p.(Thr58Ile), p.(Gly60Val), p.(Ala146Val), and p.(Glu37dup) variants also manifest with a milder phenotype compared to the typical CS phenotype [28]. In our case, P#31, coarse facial features, intellectual disability, and several other classic CS findings were absent. Before molecular testing, CFCS was considered as a preliminary diagnosis due to the predominance of ectodermal and cardiac findings. Frey et al. demonstrated that substitutions affecting HRAS amino acids 58, 59, and 60 are associated with prominent ectodermal features and a significantly milder phenotype compared to classic CS. These residues are located within the Switch II/G3 domain and the DXXGQ motif, and variants in this region are proposed to result in a distinct entity termed HRAS‐related RASopathy [29]. The phenotype of P#31 was consistent with this emerging clinical entity, and the diagnosis was therefore evaluated as HRAS‐related RASopathy.
In our cohort, P#32 harbored the recurrent c.4A>G variant in the SHOC2 and presented with systemic lupus erythematosus (SLE) alongside the classical features of SHOC2‐related NSLAH [23]. To date, including our patient previously described by Menentoğlu et al., SLE has been documented in seven cases harboring this specific variant [30, 31, 32, 33, 34, 35]. The predisposition to autoimmune manifestations in these patients is attributed to the dysregulation of the RAS/MAPK pathway [36]. RAS/MAPK signaling has also been shown to be impaired in SLE. This results in diminished ERK activation in T lymphocytes, DNA hypomethylation, and increased CD40L expression. These alterations lead to heightened B‐cell activation, antibody production, and tissue inflammation [31].
In this study, the targeted gene panel performed with a preliminary diagnosis of NS for P#18 yielded normal results, despite the patient meeting one major (height) and three minor (facial, cardiac, and chest wall) criteria in the diagnostic scoring system. ES analysis was performed on the patient, similarly affected brother, and his healthy parents. A novel hemizygous variant, AMMECR1 (NM_015365.3) c.578_581del, was identified in the patient and his brother, while their mother was found to be a heterozygous carrier. In P#18 and his brother, findings consistent with the MFHIEN phenotype, including elliptocytosis, hearing loss, and renal involvement, were observed.
While the function of the nuclear‐localized AMMECR1 protein in humans has not yet been elucidated, it has been reported to be incorporated into protein complexes involved in RNA transport and processing in yeast [37, 38]. The AMME complex was first reported by Jonsson et al. as an X‐linked contiguous gene deletion syndrome [39]. In 2016, Andreoletti et al. identified a hemizygous missense variant, c.530G>A, p.(Gly177Asp), in the AMMECR1 gene located within this deletion region in two brothers; they demonstrated that this substitution occurs within the highly conserved LRGCIG motif [40]. Subsequently, in 2017, Basel‐Vanagaite et al. reported a male patient and his similarly affected maternal uncle both harboring a hemizygous c.133C>T, p.(Arg45*) variant [41]. These individuals shared clinical features such as cleft palate, club feet, a flat nasal bridge with a flat facial profile, thin lips, micrognathia, hearing loss, and elliptocytosis, consistent with the cases described by Andreoletti et al. [40, 41]. Furthermore, in 2017, Moysés‐Oliveira et al. reported that the AMMECR1 gene was responsible for the phenotype in four males presenting with short stature, hearing loss, cardiac and skeletal anomalies, and a female patient with a 46,X,t(X;9)(q23;q11.2) karyotype [42]. AMMECR1 variants have also been reported in prenatal cases within the studies of Wang et al. and Koene et al. [43, 44]. Clinical findings of 12 previously reported cases from seven families in the literature [40, 41, 42, 43, 44] and the clinical features of the two siblings in our study with AMMECR1 variants suggest that MFHIEN cases exhibit a significant phenotypic overlap with NS. In the combined cohort (n = 14), certain systemic findings stood out with remarkably high rates, specifically short stature (8/12), cardiac involvement (8/14), and renal involvement (6/13). In addition, although less consistently observed than other findings, a subset of patients exhibited facial features reminiscent of the NS phenotype, including triangular face (3/12), downslanting palpebral fissures (5/12), and low‐set, posteriorly rotated ears (4/12). Comprehensive clinical data for the MFHIEN cohort are summarized in Figure 1A. To further quantify this phenotypic mimicry, we compared the frequencies of features from the NS diagnostic scoring system between the 14 cases with AMMECR1 variants (MFHIEN cohort) and the 17 NS patients in our cohort, as illustrated in Figure 1B [40, 41, 42, 43, 44]. Notably, cardiac involvement was a prominent shared feature between the two groups (100% in the NS cohort vs. 57.1% in the MFHIEN cohort). Furthermore, short stature was more frequent in the MFHIEN cohort (66.7%) compared to the NS cohort (41.2%). Despite this substantial overlap in growth and cardiac features, distinct clinical differences were present. Hallmark NS features, such as pectus excavatum and low‐set, posteriorly rotated ears, were markedly less frequent in MFHIEN patients. More importantly, certain features, including midface hypoplasia, elliptocytosis, and nephrocalcinosis, were entirely absent in the NS cohort.
FIGURE 1.
Clinical characterization of the midface hypoplasia, hearing impairment, elliptocytosis, and nephrocalcinosis (MFHIEN) cohort and phenotypic overlap with Noonan syndrome (NS). (A) Comprehensive clinical features of 14 patients with AMMECR1 variants (the MFHIEN cohort), encompassing 12 previously reported cases and two novel siblings from the present study. (B) The radar chart illustrates the relative frequencies (%) of seven specific clinical findings across two patient groups. The blue‐shaded area represents the NS cohort in this study (n = 17), while the red‐shaded area corresponds to the MFHIEN cohort (n = 14). Concentric dashed circles indicate the percentage of patients exhibiting each trait. The chart highlights significant phenotypic overlap in specific areas, such as cardiac involvement and short stature, while demonstrating that hallmark NS features, particularly pectus excavatum, developmental delay, and specific facial dysmorphism (e.g., triangular face, downslanting palpebral fissures) are less frequent in the MFHIEN group. [Colour figure can be viewed at wileyonlinelibrary.com]
These findings suggest that although MFHIEN mimics the craniofacial profile of NS in some patients, the absence of features such as pectus excavatum, coupled with the presence of unique MFHIEN traits like elliptocytosis and hearing impairment, underscores a distinct phenotypic demarcation between the two phenotypes.
Consequently, this study demonstrates that genes not known to be associated with the RAS/MAPK pathway may also contribute to the differential diagnosis of RASopathies. Given that P#18 meets the clinical diagnostic criteria for NS, the overlapping clinical features shared between MFHIEN and NS suggest that this AMMECR1‐related phenotype should be considered in the differential diagnosis of NS.
5. Conclusion
By evaluating the clinical and molecular findings of a 33‐patient cohort, this study provides further insights into the phenotypic spectrum of RASopathies. Furthermore, our study underscores the critical role of ES in the differential diagnosis of RASopathies and RASopathy‐mimicking syndromes, as it enables the identification of pathogenic variants in genes beyond the RAS/MAPK pathway that may be overlooked by targeted panel‐based approaches. The clinical features shared between MFHIEN and NS suggest that this AMMECR1‐related phenotype should be considered in the differential diagnosis of NS. A limitation of our study is the relatively small number of MFHIEN cases identified to date; therefore, our observations regarding the phenotypic similarity between MFHIEN and NS require further validation in larger cohorts as additional cases are reported in the literature.
Author Contributions
E.N.K.A. designed the study content and drafted the manuscript, which was subsequently reviewed by all authors. E.N.K.A., U.A., Ş.A., T.K., G.Y.S., A.D.A., and H.K. performed clinical evaluation of the patients. E.N.K.A., G.T., and Z.O.U. analyzed the Targeted panel, ES, and Sanger sequencing data. A.D.A. and Z.O.U. provided project supervision.
Funding
This work was supported by the Istanbul University Scientific Research Projects Unit (TTU‐2024‐40461).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Table S1: Clinical findings of the patients.
Acknowledgments
This study was supported by the Istanbul University Scientific Research Projects Unit (Project ID TTU‐2024‐40461).
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: Clinical findings of the patients.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.