Genetic heterogeneity of heritable ectopic mineralization disorders in a large international cohort

Abstract

Purpose: Heritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization.

Methods: Sequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo.

Results: A total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity.

Conclusion: The results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.

Keywords: Ectopic mineralization; Functional assessment; Genetic heterogeneity; Multigene next-generation sequencing panel; Pseudoxanthoma elasticum; Variant interpretation.

Figure 1.. Genetic spectrum of 478 patients with ectopic mineralization.

(a) Left panel: Among 478 patients, 327 had biallelic variants in ABCC6 (68.6%, 327/478), 96 had monoallelic ABCC6 variant (20.1%, 96/478), 40 did not have variants in any of the 29 genes (8.4%, 40/478), 15 had variants in other genes (3.1%, 15/478). In addition to ABCC6, variants were also identified in other genes in 60 and 19 patients (numbers are boxed), accounting for 18.5% and 19.8% of patients with biallelic and monoallelic ABCC6 variants, respectively. Right panel: Variant distribution in the 15 patients who carry biallelic variants in GGCX, ENPP1, SAMD9, ENTPD1, GALNT3, and PLG, or monoallelic variant in GGCX or ENPP1. Among 106 previously unreported variants in genes other than ABCC6, 5 were Class 4 (4 LP and 1 P), 10 were Class 5 (10 P), and 93 were Class 3 (1 LB, 12 LP, and 80 VUS) (for details, see Table S1). (b) Signature of distinct variants in ABCC6. Among 159 distinct variants in ABCC6, missense variants were the most frequent (58.5%, 93/159), followed by indel (18.8%, 30/159), nonsense (11.3%, 18/159), and splicing variants (10.7%, 17/159). A total of 73 distinct variants were not previously reported (45.9%, 73/159); these included 40 missense, 8 nonsense, 18 indels, and 7 putative splicing variants. Among the 73 novel variants, 43 were Class 4 (10 P and 33 LP), 27 were Class 5 (27 P), and 3 were Class 3 (2 LP and 1 VUS) (for details, see Table S1). (c) Recurrent ABCC6 variants identified in 10 or more patients.

Figure 2.. ABCC6 gene/protein structure and sequence variants found in this study.

The 3D structure of human ABCC6 protein was visualized by PyMol v.2 (Schrödinger, Inc., New York, NY). Variants in red represent those that were not previously reported. Variants in blue boxes were analyzed in functional assays. Variants with a red asterisk are those that do not meet our variant classification criteria but nonetheless included in our functional study. (a) Missense variants in the TMD0 (light blue), TMD1 (light blue), L1 (light purple), and TMD2 (light blue) domains as well as N’ and C’ terminus. Missense variants in the L0 (green), NBD1 (purple), and NBD2 (blue) domains were shown in panel b. (b) Missense variants in L0, NBD1, and NBD2 domains. (c) Different types of variants, including insertion, deletion, splicing variants, and premature termination variants, were shown in the ABCC6 gene structure consisting of 31 exons.

Figure 3.. Illustration of mini-gene constructs and evaluation of splicing events of seven ABCC6 variants.

NTC, no template control. (a) The pCMV-3Tag-8 or pCMV-3Tag-3a was used as expression vector for mini-gene splicing assay in HepG2 cells. Amplification of the cDNA utilizes a forward primer in the first exon in the mini-gene and the reverse primer in the Flag-tag region in the vector, thus abrogates concerns of amplification of endogenous ABCC6 transcript in HepG2 cells. (b) Schematic illustration of the mini-gene structure consisting of the c.999–52C>T variant, gel electrophoresis of the RT-PCR products (arrows) and normal splicing. (c) Schematic illustration of the mini-gene structure consisting of c.1868–5T>G and c.2070+5G>A variants and gel electrophoresis of the RT-PCR products (arrows). Sanger sequencing of the products revealed that c.1868–5T>G and c.2070+5G>A caused skipping of exon 15 and 16, respectively. (d) Schematic illustration of the mini-gene structure consisting of the c.3735G>A variant (arrow, the last nucleotide in exon 26). RT-PCR showed that this variant resulted in a smaller product (arrowhead) than that of the WT mini-gene (arrow). Sanger sequencing of the products revealed that c.3735G>A caused skipping of exon 26. (e) Schematic illustration of the mini-gene structure consisting of c.3883–6G>A, c.3883–24G>A, and c.3883–46A>G variants. Two separate RT-PCR reactions were performed due to the difficulty of amplification of both large and small products in the same reaction. Small products up to 1 Kb (possibility of exon skipping) and large products up to 4 Kb (possibility of intron retention) were separated by gel electrophoresis. Sanger sequencing of the products (indicated by arrows) revealed that the transcript of the WT and c.3883–24G>A mini-gene resulted in normal splicing. In contrast, c.3883–6G>A introduced a new splice acceptor site “AG” two nucleotides before the canonical 3’ splicing acceptor site in intron 27, therefore, resulting in the addition of four nucleotides “gcAG” into the mRNA. The c.3883–46A>G variant resulted in skipping of exon 28.

Figure 4.. ABCC6 protein expression in the liver of mice administered with recombinant adenoviruses containing human ABCC6 transgenes.

The Abcc6^−/−Rag1^−/− mice, at six weeks of age, were administered a single injection of 4×10⁸ IFU of adenovirus carrying either WT human ABCC6 cDNA or a missense variant. ABCC6 expression in the mouse liver was analyzed one and four weeks after injection. Immunofluorescent labeling of human ABCC6 (Green) revealed that the WT protein was expressed at one week and its level was maintained at four weeks. Dual labeling with Na,K-ATPase (Red) revealed their co-localization on the basolateral side of the plasma membrane of hepatocytes. Compared with the WT protein, p.R518Q, p.R760W, and p.R807Q resulted in reduced ABCC6 abundance and stability, with mixed plasma membrane and intracellular localization. Mutants p.T364R and p.R1138Q had exclusive intracellular expression with reduced protein expression. In contrast, p.R391G and p.G1302R had ABCC6 abundance and expression pattern similar to the WT protein. Scale bar, 200 μm (solid line) and 24 μm (dashed line). Blue, DAPI staining of nuclei. n = 6 – 8 mice per group.

Figure 5.. Histopathology, plasma PPi levels, and the degree of ectopic mineralization in the Abcc6^−/−Rag1^−/− mice.

The Abcc6^−/−Rag1^−/− mice, at 6 weeks of age, were administered a single injection of 4×10⁸ IFU of adenovirus carrying either WT human ABCC6 cDNA or a missense variant. The mice were analyzed four weeks after injection, at ten weeks of age. (a) Ectopic mineralization in the dermal sheath of vibrissae was analyzed by von Kossa stains (arrows). Scale bar, 400 μm. (b) Plasma PPi levels in Abcc6^−/−Rag1^−/− mice administered with recombinant adenoviruses. (c) The calcium content in the muzzle skin containing vibrissae. The data were presented as mean ± SD. n = 6 – 8 mice per group. *P < 0.01, **P < 0.001 compared with C57BL/6J WT mice; ^#P < 0.01, ^##P < 0.001 compared with Abcc6^−/−Rag1^−/− control mice; ns, not significant. WT, wild-type; KO, Abcc6^−/− knockout.