Loss of Protocadherin-12 Leads to Diencephalic-Mesencephalic Junction Dysplasia Syndrome

Abstract

Objective: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome.

Methods: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression.

Results: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth.

Interpretation: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655.

FIGURE 1.

Bi-allelic pathogenic variants in PCDH12 lead to microcephaly and spasticity. (A) Exons of PCDH12 with location of the patient pathogenic variants (top). Location of pathogenic variants relative to predicted protein (bottom). Cadherin domains (circle), Transmembrane domain (TM). (B) Pedigrees of consanguineous Families 1, 2, 3, 4, 5, 6, 7, and non-consanguineous Family 8. (C) Impaired PCDH12 expression in 293T cells transfected with vectors encoding pathogenic variants c.2511delG, c.2765delCT, and c.452C>T. (D) Pictures of affected members from Families 3, 4 and 5 showing the characteristic facial dysmorphism. Reproduced with permission of their parents.

FIGURE 2.

Bi-allelic pathogenic variants in PCDH12 lead to brainstem malformations. Head computed tomography (CT) or brain magnetic resonance imaging (MRI) of 12 affected individuals at the level of midbrain compared with control. All affected individuals showed brainstem malformations (circle). Butterfly sign was most obvious in the originally described families 1 and 2 (top row), consisting of an anterior midbrain cleft contiguous with the 3^rd ventricle. In other families in which PCDH12 pathogenic variants were identified from sequencing, the butterfly sign was not obvious (for example 5 and 8). PCDH12 pathogenic variant in cDNA (c.) and protein (p.) corresponding to NM_016580 and NP 057664 Entrez IDs.

FIGURE 3.

Bi-allelic pathogenic variants in PCDH12 lead to defects in white matter tracts. Midline sagittal brain MRI showing thinned corpus callosum (arrowheads) in many of the affected individuals.

FIGURE 4.

PCDH12 is expressed in several cell populations during human brain development. (A) RT-PCR of PCDH12 showing ubiquitous expression across human tissues and in fibroblasts. (B) Neural precursor cells (NPCs) and endothelial cells (ECs) derived from unaffected (U) and affected (A1 and A2) members of Family 1 are indistinguishable in differentiation. RT-PCR from NPCs and ECs showing PCDH12 expression in control and unaffected but not in affected cells. Differential expression of cell-specific markers is not dependent on the genotype. Isolated NPCs are PAX6 and nestin co-positive. Isolated ECs are calponin and vWF co-positive. No differences in immunostaining with neural and endothelial markers were observed. Scale bar, 400μm. (C) Single GFP-labeled NPCs showing reduced neurite length in cells from affected cells lacking PCDH12 (A1 and A2) compared with unaffected (U), at high cell density. Neurite length averaged 0.38 in unaffected and 0.15 or 0.21 μm in affected (A1 and A2, respectively). Scale bar 10 μm. Red: nestin. Green: GFP-labeled neurite, Yellow arrowhead: cell body. White arrowheads: neurite. Mean +/– SD. N≥30 per genotype, ≥5 cells assessed in duplicates in 3 independent experiments. ^** p<0.05, one-way ANOVA.