Sporadic hypothalamic hamartoma is a ciliopathy with somatic and bi-allelic contributions

Abstract

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.

Figure 1

Sporadic hypothalamic hamartoma cohort and genetic analyses. The 27 sporadic hypothalamic hamartoma patients derived from our previously published report (4) and new cases that have not previously been investigated. ES; exome sequencing, CMA; chromosomal microarray. Patients from our previous report with molecular diagnoses (n = 9) or insufficient DNA remaining (n = 7) for analysis were excluded. ^aNine patients had exome data re-analysed from our previous study (4). The remaining five underwent exome analysis as part of this study. ^bThree cases had previously identified CNVs that overlap with candidate SHH pathway genes.

Figure 2

The exome and Sanger sequencing data supporting a bi-allelic allelic phase in the three cases T1198, T25063 and T735. In all three cases, the corresponding germline variant is the most prevalent allele in hypothalamic hamartoma tissue indicating the somatic CNVs detected in these cases are on the alternate allele. (A) Integrative genomics viewer (IGV) shot of paired exome sequencing of T1198 showing the DYNC2I1 c.1703-1G > A variant, top panel within IGV is hypothalamic hamartoma and bottom panel is blood. (B) IGV shot of paired exome sequencing of T735 showing the IFT140 c.1901del; p.Lys634ArgfsTer10 variant, top panel within IGV is hypothalamic hamartoma and bottom panel is blood. (C) Sanger sequencing of DYNC2I1 c.2190_2191delAA; p. Arg731AlafsTer73 variant within T25063.

Figure 3

Exploring burden of protein truncating variants in hypothalamic hamartoma patients in two gene sets separated by minor allele frequency. The two gene sets listed are known cilia genes and ion channel genes. ‘Ultra-rare’ refers to variants absent in gnomAD, ‘Rare’ refers to variants present in gnomAD but with a population-specific minor allele frequency less than 0.1%. ‘PTV’ indicates protein truncating variant. Number of genes in set in parenthesis. Odds ratio, confidence intervals and FDR corrected p-value were generated from the Fisher’s exact test. Odds ratio is displayed for each test with FDR-adjusted p-values in parentheses. X-axis displays the log of the odds ratio and confidence intervals. Arrows indicate lower bound of confidence interval has been truncated.

Figure 4

The sonic hedgehog pathway and primary cilia. In response to the hedgehog ligand binding to PTCH1, SMO localizes to the axoneme of primary cilia (7). The GLI protein complexes including GLI3 localize to the tip of primary cilia (8) and are processed into the activated form, GLI^A. IFT mediated retrograde transport and specifically the IFT dynein-2 and IFT-A complexes facilitate the movement of the GLI^A complex to regulate downstream targets of the pathway. Red text; the genes in which bi-allelic variants have been identified within our sporadic hypothalamic hamartoma cohort. Blue text; the genes in which dominant somatic variants have previously been identified in sporadic hypothalamic hamartoma. OFD1 encodes a protein that localizes to the basal body of primary cilia and is required for ciliogenesis (12). Single somatic PRKACA variants have previously been identified in sporadic hypothalamic hamartoma (4) which encodes one of two catalytic subunits of the PKA protein complex that localizes to primary cilia (42). Figure adapted from (12).