Introduction

Carpenter syndrome (CRPTS) is an autosomal recessive disorder characterised by the core features of craniosynostosis and polysyndactyly of the hands and feet, with other reported features including obesity, congenital heart disease and cryptorchidism [1]. Originally described in 1901 in a family with craniosynostosis and polysyndactyly [2], the most common form of CRPTS (CRPT1; OMIM 201000) was shown in 2007 to be caused by biallelic pathogenic variants in RAB23 [3, 4]. RAB23 encodes a small GTPase that is a negative regulator of hedgehog signalling and loss of RAB23 function causes excessive hedgehog signalling resulting in craniosynostosis and polydactyly. Although the precise mechanism by which RAB23 represses hedgehog signalling remains unclear, there is evidence for differential regulation by RAB23 of downstream GLI transcription factors (repression of GLI2, activation of GLI3 repressor) [3, 5,6,7,8]. Since the original description of RAB23-associated CRPTS in 17 affected individuals [3], a further 22 cases have been described with common additional features including facial dysmorphism, short stature with obesity, cryptorchidism, congenital heart disease and variable developmental delay [4, 9,10,11,12,13,14,15]. Rarely, patients heterozygous for megabase-sized deletions of chromosome 7p14.1, including GLI3 and multiple contiguous genes, may present with a phenotype that overlaps with CRPTS [16, 17].

In 2012 a second genetic type of CRPTS (CRPT2; OMIM 614976) was delineated, caused by biallelic alterations in the gene encoding multiple epidermal-growth-factor-like-domains 8 (MEGF8). The initial report featured five affected individuals from four families, who harboured biallelic variants (missense, nonsense or splicing) in MEGF8. Analysis of the missense variants in a zebrafish embryo assay supported that these were deleterious for protein function. Although substantial clinical overlap was observed with RAB23-associated CRPTS, abnormal left-right patterning was more frequent (3/5 cases) [18] and mirrored observations in mice. Mice with alterations in Megf8 have polydactyly and altered left-right patterning, similar to those found in humans [19,20,21], whilst morpholino knockdown of megf8 in zebrafish also results in alterations in left-right patterning [19]. Mechanisms underlying the phenotype are unclear, although similar to RAB23, evidence supports MEGF8 as a negative regulator of hedgehog signalling [22]. In contrast to RAB23, MEGF8 appears to act by promoting degradation of the hedgehog pathway signal transduction factor Smoothened [6, 22].

Since the original description of MEGF8-associated CRPTS in 2012, we are only aware of one other family, comprising two affected siblings, formally reported in the literature [23]. Owing to the paucity of reported cases, evidence for CRPT2 was classified as being Moderate using the ClinGen Craniofacial Malformations gene-disease curation process [24, 25]. Here we describe a further eight affected individuals (thus more than doubling the number of documented cases of this rare pathological entity) and compare these to previously reported cases of MEGF8- and RAB23-associated CRPTS, emphasising that the core clinical features are similar between RAB23 and MEGF8 patients, but demonstrating that laterality defects are more common, and craniosynostosis appears less severe, in the MEGF8-associated form of the condition.

Patients and methods

A single family (Family 6) was formally enrolled into a research study (Genetic Basis of Craniofacial Malformation; London Riverside NHS Research Ethics Committee ref 09/H0706/20). For the remaining families, clinicians who had either previously contacted the Clinical Genetics Group at the MRC Weatherall Institute of Molecular Medicine Oxford, regarding patients with MEGF8 alterations, or presented unpublished data at specialist meetings, were re-contacted to ascertain whether the patient or parents would be willing to participate in the study. Following agreement, the clinician was sent a proforma to complete providing clinical details including MEGF8 alteration, details of craniosynostosis, hand and foot abnormalities, and cardiac, gastrointestinal and developmental findings. All participants or their legal guardians consented to inclusion in this study.

MEGF8 has two major splice forms, which differ depending upon the inclusion or exclusion of exon 12A, without altering the reading frame [18]. For comparison with the previous publication all variant details are numbered with respect to the shorter 2778 amino acid reference transcript (RefSeq accession NM_001410.3, Ensembl ENST00000334370.8); note that the MANE select transcript is the longer isoform encoding 2845 amino acids, which, however, shows low expression in most or all tissues (Genotype-tissue expression project analysis release V8 [26]).

Results

We describe eight individuals from six unrelated families with clinical features compatible with CRPTS. Each family presented to a different clinical centre, and following diagnostic exome or genome sequencing, was found to harbour two variants in MEGF8 consistent with a causal role. The major molecular and clinical features in these individuals are summarised in Table 1 and their clinical presentations are outlined below. A more detailed clinical description of each patient is provided in the Supplementary Case Descriptions and Supplementary Table 1. To provide a comparison with previously reported cases of MEGF8- and RAB23-associated CRPTS, a summary of all cases is presented in Supplementary Table 2.

Table 1 Summary of clinical and molecular features of the newly reported MEGF8 Carpenter syndrome cases.
Full size table

The first patient (Subject 1) was referred to a clinical genetics department for reproductive counselling at the age of 37 years (Fig. 1A–D). He had presented during infancy with polysyndactyly and craniosynostosis, both treated surgically, and had a clinical diagnosis of CRPTS. Following negative clinical genetic testing, he was recruited to the 100,000 Genomes whole genome sequencing research project (100kGP). Initial analysis of the whole genome sequencing data did not reveal a diagnosis because the allele bearing the 3 nucleotide deletion was filtered out based on the quality threshold in operation [27], but further research investigation confirmed that he had two likely pathogenic alterations in MEGF8, which were shown to have been inherited biallelically. Brief details of this case were previously included in an analysis of patients with craniosynostosis in the 100kGP [27].

Fig. 1: Clinical features of individuals with MEGF8 variants.
figure 1

Hands (A, B; post-operative following multiple syndactyly releases) and feet (C, D) of subject 1 at the age of 37 years; facial features (E, F), hands (G, H) and feet (I) of subject 2.ii; facial features (J, K), hand (L) and foot (M) of subject 3; facial features (N, O), hands (P), feet (Q) and chest (R) of subject 6.1 at 11 months (N, Q) and 16 years (O, P, R); facial features (S), hands (T) and knee demonstrating broad patellae (U) of subject 6.ii at 10 years old.

Full size image

Family 2 comprises a foetus (Subject 2.i) terminated at 25 weeks’ gestation owing to multiple anomalies detected on ultrasound scanning, and a liveborn sibling (Subject 2.ii) (Fig. 1E-I). Post-mortem examination of the foetus documented dysmorphic facies, talipes equinovarus, hand syndactyly and abnormalities of left-right patterning. Her brother (Subject 2.ii) had antenatally-detected craniosynostosis. Post-natal findings included syndactyly, bowel malrotation with polysplenia, sagittal craniosynostosis, facial dysmorphism, and complex congenital heart disease; he required ventilation via a tracheostomy for pharyngomalacia and vocal cord paralysis. Aged two years old he has global developmental delay but is acquiring new skills. He is non-verbal but has good understanding and can communicate non-verbally, can sit and stand with support, but is not yet walking independently. Biallelically inherited MEGF8 variants were identified in a postnatal trio exome analysis of Subject 2.ii, and the same variants were subsequently identified in a stored sample from 2.i.

Subject 3 presented antenatally with increased nuchal translucency and oedema at 20 + 3 weeks’ gestation. Post-natal findings included metopic craniosynostosis, dysmorphic facies, polysyndactyly and talipes equinovarus (Fig. 1J–M). Reinterpretation of a trio foetal exome taking account of the post-natal findings revealed two MEGF8 variants, one maternally inherited and the other arising as a de novo variant. At the age of 20 months, he has evidence of mild speech delay and cannot walk independently, although he can walk with support and fine motor skills are unaffected.

Subject 4 presented with metopic craniosynostosis, cutaneous syndactyly of her hands and pre-axial polydactyly of her feet, together with a patent ductus arteriosus (PDA) and atrial septal defect (ASD). Aged 24 months she has delayed speech and gross motor skills. Targeted next generation sequencing with 300 gene capture and analysis of 50 genes associated with craniosynostosis identified two variants in MEGF8, consistent with the characteristic phenotype of Carpenter syndrome. One variant was maternally inherited, but no paternal sample was available.

Subject 5 is the 13th pregnancy of an unrelated couple with four living children, six early spontaneous miscarriages and two pregnancy losses at 20 and 23 weeks’ gestation owing to umbilical cord abnormalities. Antenatal ultrasound at 17 weeks of pregnancy demonstrated suspected craniosynostosis, short long bones, renal asymmetry and a horizontalized heart with four chambers. The couple requested a termination of pregnancy, which was performed at 20 weeks’ gestation. Post-mortem examination demonstrated dysmorphic facies, craniosynostosis of all sutures, polysyndactyly, left atrial isomerism and abdominal situs inversus, absent uterus and streak ovaries. Trio genome sequencing demonstrated biallelic alterations in MEGF8.

Family 6 comprises two male siblings now aged 16 (subject 6.i) and 10 (subject 6.ii) years (Fig. 1J–U). Both shared features including facial dysmorphism, cutaneous syndactyly, bilateral cryptorchidism, mild pectus deformity and developmental delay or learning difficulties. Subject 6.i additionally had metopic and sagittal synostosis and bilateral preaxial polydactyly of the feet, whilst subject 6.ii had a ventricular septal defect. Exome sequencing demonstrated biallelic variants in MEGF8.

Genetic variants

All individuals harboured two different heterozygous variants in MEGF8 (Table 1, Fig. 2). In four families, inheritance was demonstrated to be biallelic based upon analysis of both parents; this inheritance pattern is likely, but not formally proven in Subject 4, for whom only the maternal sample was available. Unusually for a recessive disorder, in Subject 3 one of the variants arose de novo, so the allelic relationship could not be deduced based on parental inheritance.

Fig. 2: Structure of MEGF8 and CRPT2-associated gene alterations reported in this study (red) and previously published (black).
figure 2

A Diagram of the 41 exons of MEGF8. The 2778 amino acid reference sequence (RefSeq accession NM_001410.3, Ensembl ENST00000334370.8) omits exon 12A, shown in red. Genetic variants are shown, previously described are shown in black, those identified in this work are shown in red. B Domain organisation of MEGF8 protein with previously described variants shown in black and those identified in this work shown in red.

Full size image

The twelve variants described here (Table 1, Fig. 2) comprise four missense, three in-frame deletions and five likely truncating variants (two affecting canonical splice donor sequences, one at position +5 of a splice donor sequence, and two frameshifts). Only one of these variants, (p.(Arg1499His)), has previously been reported in the literature independently of the patients in this study, being present in a homozygous state in a patient with MEGF8-associated CRPT2 [18]. All of the variants are either absent, or present at an extremely low frequency in the heterozygous state in the gnomAD v4.0.0 database [28]. Whilst we did not perform functional assays on any variants, the pattern of variants observed, in combination with the very distinct clinical features of CRPTS and failure to identify other plausible pathogenic variants, suggest that the phenotypes described are likely to be caused by the associated combinations of MEGF8 variants. Regarding the three novel missense variants ((p.(Leu293Pro), p.(Arg2376Cys) and p.(Val2713Met)), all are located at extremely conserved amino acids (Supplementary Fig. 1), including invertebrates, have CADD scores >25 and are considered likely pathogenic by AlphaMissense (all three scoring 0.96 out of a maximum of 1) [29]. At position 293, vertebrate and invertebrate organisms all have a leucine or the chemically similar isoleucine. By alignment with human KEAP1, this variant locates within a β–strand of the second of six blades of a predicted Kelch domain [30] where substitution to proline is expected to be highly disruptive. The arginine at position 2376 is predicted to lie in a laminin EGF-like domain and is highly conserved across evolutionary time. Similarly, the valine at position 2713 lies within a predicted intracellular region that is very highly conserved (Supplementary Fig. 1), but does not correspond to an identified domain. It is predicted with moderate confidence by AlphaFold [31] to reside in a region of β–strand (Uniprot accession Q05BT0), but the consequence of substitution to methionine is unclear. Interestingly the three in-frame deletions, although each is distinct, all affect the same stretch of four conserved amino acids located in the single predicted helical transmembrane region (Fig. 2). No genotype-phenotype correlations are apparent. No individual harboured two loss-of-function alleles.

Craniofacial features

Six of eight individuals had craniosynostosis, the remaining two individuals without craniosynostosis comprising a foetus terminated at 25 weeks’ gestation and a ten year old. These six cases demonstrated a spectrum of phenotypic severity, from Subject 5 who had craniosynostosis affecting all sutures detected prenatally at 17 weeks’ gestation, to Subjects 2.ii, 3 and 4 who had single suture fusion, of either the metopic or sagittal suture (Table 1). Considering all patients with MEGF8-associated CRPT2 to date, six out of fifteen cases have had involvement of a single midline suture, comprising five with metopic and one with sagittal craniosynostosis, and only four individuals had multiple suture craniosynostosis. In contrast, 32 out of 39 reported cases of RAB23 CRPTS had synostosis of more than one suture and involvement of both coronal or all sutures is common [3, 4, 9,10,11,12,13,14,15] (Supplementary Table 2). A relatively consistent pattern of dysmorphic facial features was apparent in the series, including micrognathia, hypertelorism (including in the presence of metopic synostosis), a broad depressed nasal bridge and small low-set posteriorly rotated ears (Fig. 1).

Limb abnormalities

All eight individuals had cutaneous syndactyly involving multiple digits of the hands, and in six individuals (Subjects 1, 2.ii, 3, 5, 6.i, 6.ii) the feet were also affected. Five individuals had polydactyly of the hands or feet and three had talipes equinovarus (Table 1, Fig. 2). Brachydactyly with missing or uncalcified middle phalanges was also present (Table 1, Supplementary Table 1). Cutaneous syndactyly is a universal feature in all cases of MEGF8-associated CRPT2 to date (15/15 individuals, Supplementary Table 2) and is also near-universal in RAB23-associated CRPT1 (38/39 individuals), with polydactyly, abnormalities of the middle phalanges and talipes also being common in both forms (Supplementary Table 2).

Left-right patterning abnormalities

Three of eight individuals demonstrated a laterality defect, comprising both siblings from family 2, and Subject 5. In Family 2, foetus 2.i demonstrated left atrial isomerism, abdominal situs inversus, bi-lobed lungs bilaterally, midline liver and polysplenia and her surviving brother (2.ii) demonstrated complex congenital heart disease, polysplenia and bowel malrotation; whilst Subject 5 demonstrated heterotaxy with polysplenia. None of the eight individuals had dextrocardia and five did not have a left-right patterning abnormality. Considering all reported cases of MEGF8- and RAB23-associated CRPTS, laterality defects were significantly more common in CRPT2 (7/15 cases) than in CRPT1 (4/39 reported cases, Fisher’s exact test, P = 0.006, Supplementary Table 2).

Prenatal features

Six of eight individuals in this series (Subjects 2.i, 2.ii, 3, 5, 6.i, and 6.ii) presented prenatally, with features of abnormal tissue fluid accumulation combined with other congenital abnormalities. These included increased nuchal translucency, polyhydramnios and congenital heart disease (Family 2); prenatal non-immune hydrops (Subject 3), increased nuchal translucency/cystic hygroma with craniosynostosis and renal asymmetry (Subject 5) and in family 6, ventriculomegaly (subject 6.i) or enlarged cisterna magna with congenital heart disease (subject 6.ii). Two pregnancies (Subjects 2.i and 5) were terminated owing to the severity of the foetal anomalies. Subject 3 had amniocentesis and prenatal exome testing, but the diagnosis was not made until the genetic data were re-examined in light of the post-natal findings. The parents of Subject 5 had six early spontaneous miscarriages and two pregnancy losses at 20 and 23 weeks’ gestation possibly owing to umbilical cord abnormalities. It is uncertain whether these foetal losses were similarly affected by CRPTS, but there was additionally a maternal history of a pituitary adenoma about which no further information is available.

Additional clinical features

All five males in this series had cryptorchidism (Subjects 1 (unilateral), 2.ii, 3, 6.i and 6.ii, Table 1). Combined with previously reported males with MEGF8-associated CRPT2, all males with MEGF8-associated CRPTS have demonstrated cryptorchidism, which is also common but not universally reported in RAB23-associated CRPT1 (MEGF8: 9/9 males; RAB23 14/23 males; Fisher’s exact P = 0.035). Developmental delay is common, but of variable severity, ranging in this series from Subjects 2, 3 and 4 who have speech and gross motor delay, to Subject 1 who attended mainstream school with some childhood speech and language therapy and is university educated. This spectrum of severity is also reflected in the previously reported cases of both CRPT1 and CRPT2, with no clear differences depending upon causal gene (Supplementary Table 2). Congenital heart disease is present in 6/8 cases reported here, 4/7 previously reported cases of MEGF8-associated CRPTS and 10/39 previously reported cases of RAB23-associated CRPTS (Supplementary Table 2). Subject 2.ii has required long-term ventilation via a tracheostomy for respiratory difficulties, pharyngomalacia and vocal cord paralysis. Some individuals demonstrated additional skeletal features including two individuals with short long bones and three with pectus deformities or chest asymmetry. In family 6 broad patellae, camptodactyly and delayed dentition were additionally noted features and subject 6.i had a stable s-shaped scoliosis of the thoracic spine (Supplementary Table 1).

Discussion

We describe eight individuals from six different families, each of whom harbours two different variants in the MEGF8 gene, more than doubling the number of reported families from 5 to 11 and increasing the total number of affected individuals reported with MEGF8-associated CRPT2 to fifteen.

As noted above, no patient reported to date with CRPT2 has been biallelic for two loss-of function MEGF8 alleles. Mice completely lacking Megf8 are embryonic lethal and null mutations of the Drosophila homologue of MEGF8 die as larvae [20, 21, 32], suggesting some MEGF8 function is needed for viability. Most likely, the missense and in-frame deletion alleles allow some residual function, providing rescue from early lethality. A notable feature of MEGF8-associated CRPT2 is the wide range of clinical severity, from prenatal presentation with multiple foetal anomalies (Subjects 2i and 5) to graduate level education (Subject 1, who is the only individual compound heterozygous for missense and in-frame deletion variants). Variable residual function associated with diverse alleles and allele combinations identified in MEGF8-associated CRPT2 may explain the spectrum of phenotypes seen. Also of note is that in 7 of the 11 independent families documented to date, one of the two MEGF8 alleles has been predicted to cause complete loss of function (3 canonical splice site, 2 nonsense, and 2 frameshift substitutions). Given the rarity of MEGF8-related CRPT2 in the context of a very large protein target (2778 amino acids), we speculate that there may be a relatively narrow window of residual MEGF8 activity that is embryopathic, so that many homozygous or compound heterozygous combinations of missense alleles exceed a threshold of activity and are without pathogenic effect. Of interest, the gnomADv4.0.0 database lists 68 different amino acid substitutions, occurring at widely scattered locations throughout the protein, for which at least one homozygote exists. Hence we urge that great caution should be exerted in the pathogenic interpretation of biallelic MEGF8 variants, in the absence of the characteristic clinical features described here and in previous publications [18, 23]

The CRPT2 patients reported here and previously emphasise that the core features of CRPTS are similar whether caused by RAB23 or MEGF8 alterations [3, 4, 9,10,11,12,13,14,15, 18, 23]. In particular craniosynostosis with complete cutaneous syndactyly of multiple digits is a near universal presentation, with polydactyly, cryptorchidism, talipes equinovarus, congenital heart defects and variable developmental delay being common features in both CRPT1 and CRPT2. However, left-right patterning abnormalities are a more common finding in CRPT2 [18], being present in just under half of cases to date, but only around one tenth of RAB23-associated CRPT1 [3, 4, 9,10,11,12,13,14,15]. Where laterality defects have been demonstrated in CRPT1, these are generally milder than in CRPT2 comprising only one reported individual with true abdominal situs inversus, two individuals with accessory spleens but no other evidence of heterotaxy and one individual with a double outlet right ventricle [4, 10, 12]. The clinical findings in CRPT2 mirror the heterotaxy seen in mice with Megf8 alterations, and megf8 knockdown in zebrafish, which was attributed to abnormal propagation of asymmetric Nodal expression in the lateral plate mesoderm [19]. However, 8/15 individuals with MEGF8-associated CRPTS do not have any evidence of a laterality defect, indicating reduced penetrance of this phenotypic feature. Even considering mouse models lacking Megf8, left-right patterning abnormalities were variable, with only around a half having dextrocardia or transposition of the great arteries [21], suggesting that additional unidentified factors interact with the MEGF8 genotype to determine final left-right patterning in MEGF8-associated CRPTS.

Craniosynostosis is a nearly universal feature in cases of both RAB23- and MEGF8-associated CRPTS. However, the review presented here suggests that craniosynostosis in CRPT2 is often less severe. Whereas multi-suture synostosis is common in the reported RAB23 cases, six out of the fifteen MEGF8 cases have had documented fusion of only a single midline suture (metopic or sagittal) [18, 23], most commonly isolated metopic craniosynostosis. Nevertheless, there is clearly a spectrum of severity as one individual with CRPT2 had synostosis of all sutures and presented prenatally (case 5).

Dysmorphic facial features (hypertelorism, epicanthic folds, wide flat nasal bridge, high palate, low set posteriorly rotated ears) are shared by both types of CRPTS. These dysmorphic features appear a characteristic part of the syndrome, rather simply than a secondary effect of craniosynostosis, illustrated by the paradoxical finding of metopic synostosis with hypertelorism rather than the expected hypotelorism in cases of MEGF8 CRPTS reported here and elsewhere [1, 18].

In conclusion, we report a further eight cases of CRPTS caused by biallelic variants in MEGF8, strongly supporting that CRPT2 is a distinct clinical entity. These cases confirm that MEGF8-associated CRPT2 shares many core features of RAB23-associated CRPT1, but differences include a significantly higher frequency of left-right patterning abnormalities, and lower frequency of multi-suture craniosynostosis in the MEGF8-associated condition, refining the phenotypic spectrum of this rare condition.