DYNC2H1 hypomorphic or retina-predominant variants cause nonsyndromic retinal degeneration

Abstract

Purpose: Determining the role of DYNC2H1 variants in nonsyndromic inherited retinal disease (IRD).

Methods: Genome and exome sequencing were performed for five unrelated cases of IRD with no identified variant. In vitro assays were developed to validate the variants identified (fibroblast assay, induced pluripotent stem cell [iPSC] derived retinal organoids, and a dynein motility assay).

Results: Four novel DYNC2H1 variants (V1, g.103327020_103327021dup; V2, g.103055779A>T; V3, g.103112272C>G; V4, g.103070104A>C) and one previously reported variant (V5, g.103339363T>G) were identified. In proband 1 (V1/V2), V1 was predicted to introduce a premature termination codon (PTC), whereas V2 disrupted the exon 41 splice donor site causing incomplete skipping of exon 41. V1 and V2 impaired dynein-2 motility in vitro and perturbed IFT88 distribution within cilia. V3, homozygous in probands 2-4, is predicted to cause a PTC in a retina-predominant transcript. Analysis of retinal organoids showed that this new transcript expression increased with organoid differentiation. V4, a novel missense variant, was in trans with V5, previously associated with Jeune asphyxiating thoracic dystrophy (JATD).

Conclusion: The DYNC2H1 variants discussed herein were either hypomorphic or affecting a retina-predominant transcript and caused nonsyndromic IRD. Dynein variants, specifically DYNC2H1 variants are reported as a cause of non syndromic IRD.

Keywords: DYNC2H1; inherited retinal disease (IRD); intraflagellar transport (IFT); primary cilia; retinitis pigmentosa (RP).

Fig. 1. Phenotype documentation and family pedigrees.

(I) Proband 1: (a) Retinal imaging of the right and left eye showing some retinal pigmentary changes and narrowing of the vessels. The optic nerve is overexposed and only had mild pallor. (b) Optical coherence tomography (OCT) centered on the macula showing thinning of the outer retina. (c) MAIA™ microperimetry showing decreased retinal sensitivity (black dots) despite decent vision. This leads to fixation instability (turquoise blur from fixation points). (d) Chest X-ray, lateral and anterior–posterior (AP) view showing normal size and chest structure. The pedigree shows segregation of the variants with the phenotype. Filled symbol: affected, arrow: proband. (II) Proband 2: (a) Wide field imaging of the retina of proband 2 showing equatorial bone spiculing changes characteristic of retinitis pigmentosa (RP). (b) Wide field fundus autofluorescence showing a hyperfluorescent area that represents the limits of the functional retina. (c) OCT imaging showing thinning of the outer retina. (d) Left: normal chest X-ray (AP) and right: normal hand X-ray. This pedigree shows consanguinity with very little information about family members who were not available for segregation analysis. Diamond shape symbol with no number: uncertain number of relatives, unknown gender. (III) Proband 3: (a) Fundus autofluorescence showing retinal pigment epithelium atrophy (dark spots throughout). The right eye has a hyperfluorescent ring outlining viable retina. (b) Below is a retina photograph showing attenuated vessels, a marker of photoreceptor degeneration. (c) The OCT shows retinal thinning. Pedigree of the family is also showing an autosomal recessive inheritance pattern and consanguinity. This proband was genotyped through a large genome sequencing initiative, and family members were not available for segregation analysis. Diamond shape symbol with number: number of children, unknown gender. (IV) Proband 4: (a) Wide field fundus autofluorescence showing pigmentary changes and a hyperfluorescent ring larger than for proband 2 though they are the same age. This suggests a larger field of vision. (b) OCT view showing thinning of the outer retina. The pedigree shows pseudodominant (autosomal recessive) inheritance, seen in consanguineous families though consanguinity is not documented. The family was not available for segregation analysis. (V) Proband 5: (a) Imaging of the retina showing retinal atrophy outside the central area (macula) but the vessels were not severely constricted. bone spicules were visible only in the periphery (not shown). (b) OCT imaging showing thinning of the outer retina. c) Normal chest X-ray (AP view). The family pedigree is shown with segregation of variants with the phenotype. (VI) Normal OCT showing the different layers of the retina. Important to this work is the outer retina, which includes CL connecting cilia layer, ISL inner segment layer, OSL outer segment layer, RPE/BM retinal pigment epithelium/Bruch’s membrane, WT wild type.

Fig. 2. Inclusion of DYNC2H1 retinal microexon in human retinal organoid development.

(a) Reverse-transcription polymerase chain reaction (RT-PCR) showing the expression of DYNC2H1 in BJ fibroblasts (fibs), induced pluripotent stem cells (iPSCs), and retinal organoids at 40, 90, 100, 120, 150, and 200 days of differentiation. The shorter PCR product on the gel corresponds to the canonical DYNC2H1 transcript from exons 63 and 65. The longer amplicon (*) includes the retinal-specific microexon 64 (human exon ID GT_21385 on http://ascot.cs.jhu.edu/). (b) Sequencing electropherograms of the canonical and retinal (*) isoforms with alignment to the DYNC2H1 reference sequence. The retinal microexon is boxed in red. (c) Retinal organoid differentiation was confirmed by RT-PCR for markers of retinal differentiation (PAX6, VSX2, CRX, NRL, NR2E3) at 40, 60, 80, 100, 150, and 200 days of differentiation.

Fig. 3. Analysis of the different DYNC2H1 transcripts of proband 1.

Sanger sequencing (a), Agena MassARRAY experiments (b), and in vitro microtubule gliding assays (c–f) show that proband 1 variably expresses three DYNC2H1 transcripts with impaired in vitro microtubule gliding. Variants are numbered according to the DYNC2H1 transcript NM_001080463.1. (a) The top right electropherogram shows that transcript 1 is expressed even though V1 (orange) causes a frameshift and premature termination codon (PTC) in exon 88 (brown). Top left and bottom electropherograms show that V2 (red) causes incomplete in-frame exon skipping of exon 41 (blue), resulting in the expression of transcript 2a with V2 and transcript 2b missing exon 41. Exon 40: pink. Exon 42: green. Rest of the DYNC2H1 exons and introns: black dashed lines. Location of transcript-specific identifier nucleotides used for Agena MassARRAY experiments: roman numerals. (b) MassARRAY shows product peaks for identifier nucleotides I–IV and shows that 2b is the dominant transcript. Allelotype frequency = (peak allele 1 – peak area variance allele 1) / (peak allele 1 – area variance allele 1) + (peak allele 2 – area variance allele 2). (c) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of purified wild type (WT), V2, and V1 dynein-2 motor domains. (d) Fluorescently labeled (Alexa-488) microtubules (MTs) are added and translocated by dynein in the presence of adenosine triphosphate (ATP). The movement is monitored using total internal reflection fluorescence (TIRF) microscopy. Time sequence of microtubule translocation by immobilized wild type, V2, and V1 dynein motor domains at 1.6 nM input concentration. (e) Plot of microtubule gliding velocity. Lines show mean values (± s.d.). n = 312 microtubule gliding events (WT), n = 157 (V2), n = 91 (V1). Experiments were carried out in duplicate with two different protein preparations. cDNA complementary DNA, mRNA messenger RNA, SNP single-nucleotide polymorphism.

Fig. 4. Ciliated proband 1 fibroblasts accumulate IFT88 (green) in ciliary axonemes.

(a) Wild-type (WT) (top) and proband 1 V1/V2 mutant (bottom) fibroblasts are stained with acetylated alpha tubulin (cilia axonemes, red), gamma tubulin (basal body, red), and Hoescht (nuclei, blue). Scale bars are 18 µm. Panels on the right show enlargements of boxed ciliary IFT88 staining. Scale bars are 2 µm for enlargements. (b) Boxplot represents the distribution of IFT88 fluorescence intensity in proband 1 V1/V2 mutant and WT fibroblast cells. Data points are overlaid onto the boxplot to highlight the overlapping fluorescence intensity measurements in patient and WT cells. IFT88 fluorescence intensity was measured in 100 patient’s? and 100 WT cilia. ***P < 0.0001 (Student’s unpaired t-test).