Mutations in DDHD2, encoding an intracellular phospholipase A(1), cause a recessive form of complex hereditary spastic paraplegia

Abstract

We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein's DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA(1) family to human neurologic disease.

Figure 1

Pedigrees of Families 1–4, Photographs of the Face, and Chromatograms Showing Sanger Confirmation of the DDHD2 Mutations Arrows indicate the individuals on whom exome sequencing was performed. The following abbreviations are used: M, mutant allele; −, wild-type allele; and C, control. (A) Family 1 (family identifier: W10-1338) has an affected sister and brother and compound heterozygous frameshift mutations c.1804_1805insT (p.Thr602Ilefs^∗18) and c.2057delA (p.Glu686Glyfs^∗35). (B) Family 2 has an affected sister and brother and compound heterozygous frameshift and missense mutations c.1386dupC (p.Ile463Hisfs^∗6) and c.1978G>C (p.Asp660His). (C) Consanguineous family 3 has seven affected individuals and homozygous mutation c.1546C>T (p.Arg516^∗). Pedigree numbering is according to the original pedigree by Al-Yahyaee et al. (D) Consanguineous family 4 (family identifier: W12-0041) has one affected male individual and homozygous mutation c.859C>T (p.Arg287^∗). (E) The protein structure of DDHD2 includes its four domains (WWE, lipase, SAM, and DDHD), and the position of all identified mutations are indicated.

Figure 2

CNS Imaging of Individual II-2 of Family 1 (A) Midsagittal T1-weighted MRI of the brain shows a marked thin corpus callosum (arrow). (B) Transverse T2-weighted MRI of the brain shows subtle white-matter hyperintensities (arrows). (C) Proton MRS obtained at a magnetic field of 1.5 tesla. Voxel was fixed just cranial of the basal-ganglia and thalamus area, of which proton MRS at long echo time (144 ms) was obtained. It shows the prominent pathologic lipid peak at 1.3 ppm (arrow), apart from the common spectral peaks of choline (Cho), creatine (Cr), and N-acetylaspartate (NAA). (D) Sagittal T2-weighted MRI of the spine shows a spinal syrinx (arrow). Similar brain abnormalities and proton MRS were found in the other families (Figures S3–S7).

Figure 3

Synapse Morphology and Organization at the Drosophila NMJ of Control and Ddhd-Knockdown Flies Three different RNAi lines, vdrcGD35956, vdrcGD35957, and vdrcKK108121, from the Vienna Drosophila Research Center, were used and compared to their genetic background lines, vdrcGD60000 (for vdrcGD35956 and vdrcGD35957) and vdrcKK60100 (for vdrcKK108121). RNAi was induced with the pan-neuronal UAS-dicer2; elav-Gal4 driver. Drosophila muscle 4 type 1b NMJs were analyzed as previously described. (A) Anti-dlg1 (upper panel) and anti-brp immunolabeling (middle panel) at the NMJ of control (vdrcGD60000) and Ddhd-knockdown (vdrcGD35956) larvae, as well as output of computer-assisted analysis with an in house-developed macro (bottom panel). Each white dot represents one active zone. (B) Quantification of active zones shows a significant reduction in all three RNAi lines compared to their genetic-background controls. The p values are from two-sided t tests. Error bars indicate the SEM. The following abbreviation is used: n, number of quantified synaptic terminals.