A mutation in KIF7 is responsible for the autosomal recessive syndrome of macrocephaly, multiple epiphyseal dysplasia and distinctive facial appearance

The study was approved by the UCSD and Al-Ain District Human Research Ethics Committees and the family provided informed consent for study. Part of the family has been reported previously[10]. In brief, the family is inbred of Omani origin living in the United Arab Emirates (UAE). There are a total of five children affected in two branches (Figure1A). All children presented with macrocephaly with frontal bossing, hypertelorism, flat malar regions, low set ears, and short neck (see Figure1B, C, D and E for examples). In addition, they all have some degree of pectus excavatum, spindle shaped fingers, clinodactyly, prominent joints and genu valgum. Lymphedema was present in two children only. Skeletal survey showed generalized epiphyseal dysplasia in all affected (see Figure2A, B, and C for examples). Neuroimaging in all except 1 (case 2, not done) showed macrocephaly with hypoplasia of corpus callosum and fronto-temporal brain atrophy (Figure3). All except 1 had very mild developmental delay (case 2 had normal developmental history) (Table1).

In order to identify the gene for this condition, we performed whole exome sequencing on two affected siblings. Blood DNA was extracted using Qiagen reagents (Qiagen Inc., USA), then subjected to exome capture with the Agilent SureSelect Human All Exome 50 Mb kit (Agilent Technologies, Inc., USA), sequenced on an Illumina HiSeq2000 instrument (Illumina, Inc., USA), resulting in ~94% recovery at >10× coverage. GATK[12] was used for variant identification and intersected with identity-by-descent blocks identified by HomozygosityMapper[13, 14], and then filtered for homozygous variants shared between the two affected. In one affected we found 4519 homozygous variants of which 4190 were in the UTRs and therefore probably not functional. In addition, 292 of the remaining 329 variants were seen in homozygous state in families with non-overlapping phenotypes from our in-house 1000 exomes from Middle Eastern patients. Furthermore, 9 of the remaining 38 variants were present in the heterozygous state in 1% or more of the cohort (i.e. out of Hardy Weinberg equilibrium with the disease frequency). Finally, of the remaining 29 variants, only the KIF7 variant fell within the previously established linkage interval15q26[11].

We identified a mutation at base position chr15:90173657 (according to the hg19 assembly[15], corresponding to a c.3179A > G base transervsion in the KIF7 gene (accession NM_198525.2). The mutation was found in a homozygous state in all the affected members of this family and is heterozygous in the parents (Figure4A) and some siblings. It segregated according to a recessive mode of inheritance, as predicted. This mutation was absent from 188 mixed Omani/UAE ethnically matched controls. This single base substitution leads to a change of an asparagine residue in position 1060 of KIF7 to serine (p.N1060S) located after the structural maintenance of chromosomes (SMC) domain of KIF7 (Figure4B). The asparagine in this position is absolutely conserved in KIF7 family members and related proteins (Figure4C).

The phenotype in acrocallosal syndrome is very broad but Courtens et al. 1997[16] suggested minimal diagnostic criteria to help confirm clinical diagnosis. Three out of the 4 following criteria should be present: total or partial absence of the corpus callosum, minor craniofacial anomalies, moderate-severe mental retardation and polydactyly. Although the features in our family do not satisfy these criteria, there are many similarities in the phenotype but there are also some major differences. Similarities include corpus callosum anomalies, macrocephaly with frontal bossing and depressed nasal bridge with widely spaced eyes. Polydactyly is considered a major feature of ACLS. All patients reported with ACLS had polydactyly with or without syndactyly (either of hands or feet or both and either preaxial or postaxial) except patient 4 of the family reported by Gelman-Kohan et al.[17]. Polydactyly or syndactyly was absent in all the affected children in our family. One of our cases had mild webbing of the fingers. All the children, however, had spindle shaped fingers which has been reported in some patients with ACLS. Another difference between our family and ACLS is the degree of mental retardation which is usually moderate-severe in ACLS and was very mild or absent in our cases. However, there is 1 report of 2 patients with ACLS with normal development at 7 and 8 years of age[18].

Skeletal abnormalities were major manifestations of the syndrome we reported. These included genu valgum, pectus excavatum and prominent joints[10]. Radiological examination revealed generalized epiphyseal dysplasia in all affected children. Although major skeletal abnormalities were reported in some patients with ACLS syndrome, they are typically restricted to short metacarpals and phalanges[17], osseous defect in parieto-occipital area of the skull, hip dysplasia, bipartite clavicle[8], and metatarsus adductus[16]. MED has not been reported previously in ACLS or other syndromes associated with KIF7 mutations including hydrolethalis and Joubert syndrome (Table1).

The presence of MED in a phenotype associated with KIF7 mutation is not surprising since it has been shown recently that KIF7 has a major role in the growth plate where it regulates hedgehog signaling activity[19]. Chondrocytes proliferation and differentiation in the growth plate requires a proper regulation of the Indian hedgehog (Ihh) signaling which is the major hedgehog ligand in chondrocytes[19]. Ihh signaling in growth plate is mediated by three Gli zinc-finger proteins (Gli1-Gli3)[20]. Studying knockout models indicates that Gli2 and Gli3 are involved in Ihh-dependent chondrocyte development[19]. Mice lacking Ihh do not show ossification in endochondral bones and their chondrocytes exhibit reduced proliferation with an expanded hypertrophic zone in the growth plate[21]. Similarly, Gli2 knockout mice have an expanded hypertrophic zone and reduced bone formation[22]. This similarity indicated that the Ihh mutant phenotype is partially due to the loss of the Gli2 transcription activation function[22]. On the other hand, loss of Gli3 in the Ihh knockout mice reduces the defects in the proliferation and differentiation of chondrocytes[23]. These data demonstrated the importance of Ihh in controlling the transcription repression function of Gli3 in growth plate chondrocytes[23].

Kif7 and another evolutionary conserved protein called Sufu regulate the transcriptional activity of Gli proteins[24]. Hsu et al.[19] demonstrated experimentally that Sufu is a major negative regulator of the Ihh pathway in the growth plate while Kif7 plays dual roles in controlling Ihh signaling and chondrocytes development (Figure5). They showed that in a wild type chondrocyte, Kif7 positively modulates Ihh pathway activity during development through downregulation of Sufu. These authors showed that in the absence of Sufu, Kif7 negatively regulates Ihh signaling by inhibiting Gli-mediated transcription but the underlying mechanism is not defined. Consistently, Sufu-deficient chondrocytes have augmented Ihh pathway activity, increased proliferation and delayed differentiation while Kif7-deficient chondrocytes exhibited lower Hh pathway activity, a decrease in proliferation and expansion of the hypertrophic zone.

It would be interesting to test KIF7 gene in other patients with epiphyseal dysplasia and corpus callosum abnormalities in order to further delineate the phenotype of KIF7 mutations.