Autosomal dominant frontometaphyseal dysplasia: Delineation of the clinical phenotype

2.1 Patient ascertainment

Patients were recruited based on clinician-initiated referral and consented under approved protocols MEC/08/08/094 and 13/STH/56 (Health and Disability Ethics Committee, New Zealand). The diagnosis of FMD was confirmed based on published clinical and radiological criteria (Robertson et al., 2006). FLNA mutations were sought using Sanger sequencing to examine the exons and exon-intron boundaries of that gene. Copy number variants of FLNA were excluded by use of a customized multiplex ligation-dependent probe amplification (MLPA) probeset. Cases with a firm clinical diagnosis of FMD and no detectable mutation in the exons or intron exon boundaries of FLNA formed a cohort of 20 individuals. Thirteen individuals have been clinically reported before (Table 1) (Basart et al., 2015; Morava et al., 2003; Robertson et al., 2006). Nineteen individuals (all except individual 09) were described as part of a biochemical study of AD-FMD (Wade et al., 2016). We are including all known instances of AD-FMD here for a clarity.

Table 1. Clinical and radiological findings in AD-FMD

• U, unknown; na, not applicable; D, deceased; presented previously in *(Robertson et al., 2006).
• All individuals, except for 09, were described in (Wade et al., 2016). Where possible, individuals have been clinically re-evaluated therefore this table is this most up-to-date phenotypic spectrum.
• Protein substitution. see Supplementary Table SI for full mutation information.
• + Described in Present.
• − Described inAbsent.
• ^†(Basart et al., 2015).
• ^‡(Morava et al., 2003).
• ¹Cervical spinal cord compression.
• ²Aortic dilatation.

2.2 Mutation discovery

Previously we utilized whole exome sequencing and targeted Sanger sequencing (Wade et al., 2016) to undertake mutation discovery on 19 participants. Subsequently a sample from individual 09 (Table 1) was subjected to exome sequencing using the Illumina HiSeq (BGI-Europe, Denmark) platform after enrichment of the exome with the Agilent SureSelectXT Human All Exon 50 Mb capture kit. After read alignment with BWA and variant calling with GATK, variants were annotated by the Genetics Department of the Radboud UMC using an in-house analytical pipeline. Candidate variants were confirmed with Sanger sequencing.

3.1 Craniofacial findings

The 20 subjects with FMD described here all manifested the three mandatory diagnostic facial features of FMD; prominent supraorbital ridges, downslanting palpebral fissures, and hypertelorism (Robertson et al., 2006) (Figure 1, Table 1). A broad nasal bridge was a ubiquitous finding (20/20). Despite manifesting similar facial characteristics as seen in XL-FMD, patients with the recurrent C-terminal substitution in TAK1 (p.Pro485Leu), have coarser facial features than those individuals with XL-FMD (Figure 1). However those individuals with mutations in the 5′ region of MAP3K7, which encodes the kinase domain of TAK1 (patients 07, 19, and 20; Table 1 and Supplementary Table S1) manifest notably milder facial features (Figure 1, Table 1). Micrognathia, a mandatory diagnostic feature of XL-FMD, was only present in 16/20 (80%) of the individuals with TAB2 and MAP3K7 mutations (Table 1). Other features which may set AD-FMD apart from XL-FMD include the lack of bias toward affected males and the presence of hearing loss, which can be conductive, sensorineural or mixed. Some kind of deafness is observed in 6/8 (75%) males and 11/11(100%) females in this cohort compared to 67% of males and 27% of females with XL-FMD (Robertson et al., 2006) (Table 1). Cleft palate or a bifid uvula is more common in AD-FMD, occurring in 6/19 (32%) patients presented here compared to 5% of XL-FMD patients (Robertson et al., 2006) (Table 1). Radiological findings in the skull in those with AD-FMD were indistinguishable from those seen in XL-FMD for the majority of individuals, the exception being two individuals (07 and 19) with the milder presentation of AD-FMD due to substitutions in the kinase domain of TAK1: p.Gly168Arg (MAP3K7 c.502G>C) and p.Glu70Gln (MAP3K7 c.208G>C), who lacked skull-base sclerosis (Table 1 and Supplementary Table S1).

3.2 Limb and digit manifestations

Contractures of the fingers, wrists, and elbows are an important diagnostic feature of both XL-FMD and AD-FMD. In this AD-FMD cohort, 19/20 had digital (Figure 2a,c) and wrist contractures and 19/19 had either a contracture of the elbow joint or a dislocated radial head (Table 1). Ulna deviation of the hands, which is a mandatory diagnostic criterion of XL-FMD, was seen in 13/18 in this cohort (Table 1). There are other notable differences in the hands and feet of AD-FMD patients; the fingers and thumbs are long (as they also are in XL-FMD; Figure 2a,b, Table 1) but the spatulate finger and thumb tips seen in XL-FMD are not as prevalent. Additionally a valgus deformity of the feet was seen in 8/15 subjects (Table 1), a sign not previously associated with XL-FMD. Radiologically, undermodeling of the phalanges is a distinct finding (Figures 2b,d). The long bones can be slightly bowed, with dense cortices. Furthermore the femur and tibia display abnormal modeling of the diaphysis and metaphysis giving the appearance of an Erlenmeyer-flask (Figure 2e, Table 1).

3.3 Other radiological characteristics

Other radiological features which may help distinguish XL-FMD from AD-FMD include scoliosis, present in 16/20 (80%) with AD-FMD (Figure 3a, Table 1) denoting it as more prevalent in AD-FMD than in the X-linked condition (56% of males and 18% of females (Robertson et al., 2006)), an observation not previously noted in studies of FLNA mutation-negative FMD (Morava et al., 2003; Robertson et al., 2006). Another radiological finding which may be more prevalent in AD-FMD is the presence of cervical vertebral fusions, seen in 12/19 (63%) individuals in this cohort (Figure 3b, Table 1). Cervical fusions are only seen in 33% of FLNA-mutation positive males, and have never been reported in FLNA mutation-positive females (Robertson et al., 2006). Notably one individual with a MAP3K7 mutation developed a cervical spine myelopathy secondary to cervical vertebral anomalies.

3.4 Non-skeletal findings

The extraskeletal manifestations of FMD result in significant morbidity and are often the cause of early mortality. Individuals with AD-FMD manifest many of the same extraskeletal malformations as the patients with XL-FMD without the bias toward an increased prevalence in males. Subglottic stenosis or congenital stridor was seen in 6/16 (38%) individuals studied here (Table 1) compared to 11% of males and 9% of females with XL-FMD (Robertson et al., 2006). Genitourinary tract defects, usually in the form of urethral obstruction, were seen in 3/8 (38%) of the male patients described here (Table 1) and have previously been reported in 22% of males with XL-FMD. There are no previous reports of such defects in females with XL-FMD (Robertson et al., 2006), however 2/11 (18%) female patients in this cohort have some form of genitourinary tract defect (Table 1). Structural heart defects which are sometimes present in XL-FMD males (22%) were found in 2/6 (33%) males and 4/10 (40%) females with AD-FMD. A small number of patients with dilation of the aorta, that was slowly progressive in one instance, were noted (Table 1). Finally, and significantly, intellectual disability was observed in 3/20 individuals with AD-FMD (Table 1) but has never been previously associated with FMD of any description (Robertson et al., 2006). Intellectual disability is only seen in the individuals with the TAK1 p.Pro485Leu substitution, and a further individual with intellectual disability has been reported with this substitution in another study (Martinez et al., 2016).

3.5 Predisposition to keloid scarring

Keloid scar formation is not a reported manifestation of XL-FMD but has been connected with another FLNA-associated phenotype, which manifests with cardiac and kidney malformations and joint contractures (Atwal et al., 2015; Lah et al., 2015). Individuals with the skeletal manifestations of FMD, together with an increased tendency to form keloid scars generally do not have a mutation in FLNA (Basart et al., 2015; Wade et al., 2016). In the AD-FMD cohort studied here 10/20 patients have some form of keloid scarring (Table 1) and this finding is more prevalent in females (7/10). The severity of the scarring varies considerably between individuals with one individual developing florid keloid after relatively trivial procedures such as venepuncture (Basart et al., 2015).

3.6 Genotype-phenotype correlation in AD-FMD

The most common FMD-causing mutation found in MAP3K7 Leads to the substitution p.Pro485Leu, and was observed in 15/20 (75%) individuals in this cohort. The other three mutations found in MAP3K7 predict substitutions in the N-terminal kinase domain of TAK1 (Supplementary Table S1) (Wade et al., 2016). In one case the mutation was de novo, in the other two unrelated cases the mutation was transmitted from the affected mother (Wade et al., 2016). Substitutions in the kinase domain lead to a notably milder presentation than the recurrent mutation (Figure 4, Table 1). The three individuals with kinase domain substitutions have less dramatic undermodeling of the phalanges, minimal splaying of the metaphyses and can lack skull base sclerosis (Figure 4, Table 1). None of these three individuals have developed keloid.

FMD has overlapping features with the recently described dominant condition cardiospondylocarpofacial syndrome (CSCF) which is associated with missense mutations, or small deletions, affecting the N-terminal region of TAK1 (Le Goff et al., 2016). These include facial characteristics such as hypertelorism and full cheeks, as well as deafness, cervical fusions, and cardiac defects (Le Goff et al., 2016); however clear clinical distinctions can be made between the two conditions. CSCF is characterized by brachydactyly, short extremities and joint laxity (Le Goff et al., 2016), in direct contrast to the FMD phenotype which, in contradistinction, exhibits arachnodactyly and joint contractures (Table 1). Furthermore the three patients with kinase domain substitutions have the characteristic prominent brow ridges which are absent in individuals with CSCF.

3.7 Individuals with mutations in TAB2

Here we describe two individuals with TAB2 substitutions p.Gln540Arg (TAB2 c.1619A>G; NM_ NM_015093.5) and p.Glu569Lys (TAB2 c.1705G>A), caused by variants in the same exon of TAB2. The p.Gln540Arg variant is newly described, not present in the Exome Aggregation Consortium (ExAC) database and the affected residue is phylogenetically conserved (Supplementary Figure S1). Variant effect prediction software predicted the p.Gln540Arg substitution as pathogenic: SIFT score of 0 (damaging), PolyPhen2 score of 0.954 (probably damaging), and predicted to be disease causing by MutationTaster. Both of the described variants in TAB2 had arisen de novo. The subject with this variant has a remarkably similar presentation to the original female we described with a TAB2 mutation (Figure 1), and both resemble the individuals with the recurrent MAP3K7 mutation more than those subjects with kinase domain substitutions in TAK1 (Figure 1). Neither subject with a TAB2 variant has keloid scars.