Marquez J et al. (2021), DLG5 variants are associated with multiple cong...

XB-ART-57158

J Med Genet 2021 Jul 01;587:453-464. doi: 10.1136/jmedgenet-2019-106805.

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DLG5 variants are associated with multiple congenital anomalies including ciliopathy phenotypes.

Marquez J , Mann N , Arana K , Deniz E , Ji W , Konstantino M , Mis EK , Deshpande C , Jeffries L , McGlynn J , Hugo H , Widmeier E , Konrad M , Tasic V , Morotti R , Baptista J , Ellard S , Lakhani SA , Hildebrandt F , Khokha MK .

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BACKGROUND: Cilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina. METHODS: Among individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model. RESULTS: Patients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function. CONCLUSION: These findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.

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Species referenced: Xenopus tropicalis
Genes referenced: dlg5 dnah9 foxf1 mcc pax6 ptch1 shh
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gRNAs referenced: dlg5 gRNA1

???displayArticle.disOnts??? ciliopathy [+]
Phenotypes: Xtr Wt + dlg5 CRISPR (Fig. S3 A) [+]

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	Figure 1 Whole exome sequencing identifies discs large 5 (DLG5) variants in patients. (A) Radiograph of patient I 2â€“1 fetal upper extremities reveals bilateral ectrodactyly. (B) I 2â€“1 fetal kidneys were largely cystic and dysplastic. (C) Ultrasound of II 2â€“1 kidneys show hydronephrosis. Yellow line depicts span of kidney and red arrows indicate dilated renal pelvis. (D) Pedigrees depict families in which the DLG5 variants were identified. (E) Sanger sequencing confirming variant allele presence and amino conservation through phylogeny for the mutated allele of DLG5.
	Figure 2 Knockdown of discs large 5 (dlg5) in Xenopus embryos causes kidney and brain ventricle dysmorphology. (A) Representative images of stage 45 embryos injected with either a standard control MO or dlg5 MO show oedema (black arrow head) and kidney dysplasia (outlined in yellow) resulting from dlg5 knockdown. (B) Representative images of control MO and dlg5 MO-injected sides of stage 45 embryos along with quantitation reveal a loss of cilia and increased proximal tubule diameter in the pronephroi. Each data point is the ratio within a single embryo of either cilia detected or maximal tubule diameter in the proximal tubule between two sides either both treated with control MO (Control MO/Control MO) or one treated with dlg5 MO and the other treated with control MO (dlg5 MO/Control MO). (C) Representative images and quantitation of stage 45 embryos injected with either a standard control MO or dlg5 MO imaged via optical coherence tomography demonstrate the ventricular dysmorphology that results from dlg5 knockdown. The arrow designates the typical division between ventricles that can be found lateral to the brain midline. Embryonic axes are labelled for reference. A, anterior; D, dorsal; P, posterior; V, ventral; L, left; R, right. Ventricles are labelled according to the adjacent subdivisions of the embryonic brain. T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon. Statistical tests carried out as two-tailed t-tests with **p<0.0001 (A) or an unpaired t-test with **p<0.0001 (C). Bars indicate mean and SD of individual values (B) or replicate means (C).
	Figure 3 Xenopus model of discs large 5 (dlg5) loss of function provides a platform for testing variant pathogenicity. (A) Schematic diagram of DLG5 protein with domain annotations and locations and effects of uncovered patient variants. CARD, caspase activation and recruitment domain; DUF, domain of unknown function; CC, coiled-coil domain; PDZ, postsynaptic density protein disc large tumour suppressor and zonula occludens-1 domain; SH3, src homology 3 domain; GUK, membrane-associated guanylate kinase domain. (B) Representative images of proximal pronephric tubule ciliation and morphology. Dotted lines indicate bounds of tubule assessed in the image. (C) Quantitation of cilia and tubule morphology rescue efficiency of wildtype and variant DLG5 mRNA demonstrate the differing functionality of DLG5 variants. Ratios are calculated as values from sides with mRNA along with MO divided by values from MO only sides. Red indicates ratios suggesting an exacerbation of the phenotype while green indicates ratios suggesting rescue of the phenotype. (D) Representative images and quantitation of brain ventricle morphology rescue efficiency of wildtype and p.Arg821Ter variant DLG5 mRNA demonstrate the loss of functionality of the p.Arg821Ter DLG5 variant. Statistical tests carried out as analysis of variance (ANOVA) followed by Dunnettâ€™s multiple comparison test with **p<0.0001, p<0.005 and p<0.05 (C) or an unpaired t-test test with p<0.05 (D). Bars indicate mean and SD of individual values (C) or replicate means (D).
	Figure 4 Knockdown of discs large 5 (dlg5) in Xenopus embryos causes cell polarity and Hedgehog signalling dysfunction. (A) Representative images of dnah9 in situ hybridisation in control MO and dlg5 MO sides of a stage 28 embryo depict fainter expression signal in the dlg5 depleted side. (B) Representative transverse sections of control MO and dlg5 MO sides of a stage 28 embryo reveal the more basal location of cells expressing the dnah9 marker of MCC cell fate. (C) Representative immunofluorescence images and quantitation of AcTub-positive ciliated cells/area and Lectin-positive goblet cells/area in control MO and dlg5 MO sides of a stage 28 embryo demonstrate the decreased density of MCCs and concomitant increased density of goblet cells in dlg5 depleted Xenopus epidermis. (D) Representative WISH for foxf1 Hedgehog target expression in control MO and dlg5 MO stage 45 pronephroi demonstrate the decreased Hedgehog signalling activity in dlg5 depleted pronephroi. (E) Representative expression and quantitation of ptch1 in control MO and dlg5 MO stage 45 brain ventricles as well as representative expression and quantitation of Pax6 in control MO and dlg5 MO stage 45 fourth ventricle demonstrate a decrease in ptch1 expression and expansion of Pax6. Black dotted line delineates bounds of the brain and white dotted line delineates bounds of fourth ventricle floor. A, anterior; D, dorsal; P, posterior; V, ventral. Statistical tests carried out as two-tailed t-tests for cell density (C) and Pax6 expression (E) and unpaired t-tests for foxf1 (D) and ptch1 (E) expression with *p<0.005â€‰and **p<0.0001. Bars indicate mean and SD of individual values (Eâ€”Pax6 expression) or replicate means (D and Eâ€”foxf1 or ptch1 expression).

	Supplemental Figure 1
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	Supplemental Figure 5
	dlg5 (discs large homolog 5) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.
	Figure 1. Whole exome sequencing identifies discs large 5 (DLG5) variants in patients. (A) Radiograph of patient I 2â€“1 fetal upper extremities reveals bilateral ectrodactyly. (B) I 2â€“1 fetal kidneys were largely cystic and dysplastic. (C) Ultrasound of II 2â€“1 kidneys show hydronephrosis. Yellow line depicts span of kidney and red arrows indicate dilated renal pelvis. (D) Pedigrees depict families in which the DLG5 variants were identified. (E) Sanger sequencing confirming variant allele presence and amino conservation through phylogeny for the mutated allele of DLG5.

References [+] :

Abdelhamed, A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice. 2018, Pubmed