Brain calcifications and PCDH12 variants

DISCUSSION

We show here that a homozygous nonsense PCDH12 variant, detected in patients with severe developmental delay and microcephaly,¹ is associated with brain calcifications. This feature should therefore be added to the phenotypic spectrum of this rare disorder. The pattern of calcifications is, however, different from the typical findings in PFBC, where calcifications always affect at least both pallidum,³ and resembled to those observed in various neuroinfectious prenatal conditions, such as TORCH infections.¹⁸ Brain calcification is a highly informative feature on brain imaging of children with neurodevelopmental disorders.¹⁸ Although CT is the reference imaging tool for detecting and assessing calcifications, MRI is the primary imaging tool for the detection of all other brain abnormalities in the absence of radiation. T2* or susceptibility-weighted images increase the diagnostic performance of MRI for calcification compared with the other sequences. However, they can sometimes miss small calcifications, and they are still complementary with CT to describe precise shape and intensity and to definitely conclude on the differential identification with iron deposits.^19,20 In our patient, neither T2* nor susceptibility-weighted images were available.

In the original report, the efficiency of nonsense-mediated decay has been measured as 84%, suggesting a strong loss of function. The patients carrying the nonsense PCDH12 variant in a homozygous state may still express little amount of the truncated protein, but no full-length PCDH12. This supports the hypothesis that loss of function of PCDH12 is the mechanism leading to the patient's phenotype, including brain calcification.

In a candidate gene approach, we searched for rare PCDH12 variants in PFBC and BCUC patients and found no protein-truncating variants. Three heterozygous missense variants, predicted damaging by at least one of the tools, were identified in 2 patients with PFBC and 1 patient with BCUC. Given the fact that biallelic loss of PCDH12 function leads to a severe neurodevelopmental phenotype, it is unlikely that these variants have a dominant-negative effect. However, as they are missense variants, their putative effect on protein function is hard to predict, and it remains possible that they are responsible for loss of function, gain of function, or have a neutral effect on protein function. The frequencies of theses variants in the patients' respective populations as estimated in ExAC are not inconsistent with a causative effect, as they are in the same frequency ranges as other disease-causing variants in SLC20A2.⁸ Because neither segregation nor functional data are available, it is not possible to conclude about their pathogenicity at this stage.

Besides PFBC, brain calcifications can be detected in other numerous distinct conditions, such as systemic phosphocalcic metabolism disorders of inherited or acquired cause, in utero or postnatal infections, interferonopathies, inborn errors of metabolism, and other rare inherited diseases.²¹ Calcifications are believed to be related to increased type-I interferon response in both in utero viral infections and interferonopathies.²² Several of these clinical presentations, including TORCH in utero infections and typical Aicardi-Goutières syndrome, are similar to the ones observed in the PCDH12 homozygous carriers. In other conditions, mutations in OCLN and JAM3 genes, encoding endothelial cell adhesion proteins, result in microangiopathy associated with calcifications.^18,23,24 Given the known function of PCDH12, we postulate that similar mechanisms could be associated with the calcifications observed in the PCDH12 homozygous loss-of-function carriers.

PCDH12 is a protocadherin associated with membrane physical stability and adhesion.²⁵ A Pcdh12 knockout mouse model revealed several age-independent vessel impairments, such as ramifications of medial elastic lamellae and increased inner diameter and circumferential mid-wall stress.²⁶ PCDH12 has been widely studied as a key-player cadherin involved in placental maintenance and also a preeclampsia biomarker; however, little is known about its involvement in brain physiology. It is conceivable that mutations in PCDH12 and SLC20A2, which share similar expression patterns in the brain, might lead to similar phenotypes. Of interest, Slc20a2 knockout mice developed not only brain calcifications but also fetal growth restriction, lower birth viability, and placental calcification associated with thickened basement membranes.²⁷ In both mouse models, the placental phenotype and the vascular impairment are additional putative links between SLC20A2 and PCDH12, which deserve additional studies on mouse models.

PCDH12 biallelic loss of function causes a severe neurodevelopmental phenotype associated with brain calcifications. Rare predicted damaging heterozygous PCDH12 variants were identified in patients with PFBC or BCUC here, but whether they are associated with brain calcification or not remains to be determined. To address this question, follow-up studies will be necessary including screening other series, assessing the segregation of rare variants and functional consequences.