Blackburn ATM et al. (2019), DYRK1A-related intellectual disability: a syndr...

XB-ART-56082

Genet Med 2019 Dec 01;2112:2755-2764. doi: 10.1038/s41436-019-0576-0.

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DYRK1A-related intellectual disability: a syndrome associated with congenital anomalies of the kidney and urinary tract.

Blackburn ATM , Bekheirnia N , Uma VC , Corkins ME , Xu Y , Rosenfeld JA , Bainbridge MN , Yang Y , Liu P , Madan-Khetarpal S , Delgado MR , Hudgins L , Krantz I , Rodriguez-Buritica D , Wheeler PG , Al-Gazali L , Mohamed Saeed Mohamed Al Shamsi A , Gomez-Ospina N , Chao HT , Mirzaa GM , Scheuerle AE , Kukolich MK , Scaglia F , Eng C , Willsey HR , Braun MC , Lamb DJ , Miller RK , Bekheirnia MR .

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PURPOSE: Haploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A variants. METHODS: A large database of clinical exome sequencing (ES) was queried for de novo DYRK1A variants and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1a's role in renal development. RESULTS: Phenotypic details and variants of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic variant in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom presented with CAKUT/GD. Studies in Xenopus embryos demonstrated that knockdown of Dyrk1a, which is expressed in forming nephrons, disrupts the development of segments of embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by coinjecting wild-type human DYRK1A RNA, but not with DYRK1AR205* or DYRK1AL245R RNA. CONCLUSION: Evidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss-of-function studies in Xenopus substantiate a novel role for DYRK1A in GU development.

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Species referenced: Xenopus Xenopus tropicalis
Genes referenced: dyrk1a dyrk1a.2 tbl1x
???displayArticle.antibodies??? Kidney Ab1 Kidney Ab2
???displayArticle.morpholinos??? dyrk1a MO1

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Phenotypes: Xla Wt + dyrk1a MO (Fig. 3 B) [+]

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	Figure 2. In situ hybridization of dyrk1a across developmental stages demonstrates kidney expression in X. laevis and X. tropicalis.To demonstrate spatial-temporal expression of dyrk1a in the kidney, in situ hybridization was performed. Given that the RNA probe was designed against the X. tropicalis sequence, both species were analyzed. Pronephric kidney development occurs between stages 12.5-40. Expression of dyrk1a can be visualized in stage 31-40 embryos suggesting Dyrk1a may be important for kidney development. For clarity, insets (D’ and J’) for stage 30/31 tadpole kidneys with 200μm scale bars have been added. All other scale bars represent 1000μm.
	Figure 3. Loss of Dyrk1a affects kidney development in Xenopus laevis.(A-E’) Embryos were unilaterally injected at the 8-cell stage with 10ng of Dyrk1a MO or Standard MO (Std MO) along with 50 pg β-gal, wild-type, DYRK1AR205, or DYRK1AL245R RNA. Stage 40 tadpoles were stained with kidney antibodies 3G8, which label the proximal tubules and 4A6, which labels the distal and connecting tubules. Letters without apostrophes (A-E) represent the injected side, whereas letters with apostrophes (A’-E’) represent the uninjected side. (B) Knockdown with a translation-blocking Dyrk1a MO disrupts kidney development which can be partially rescued (C) by co-injecting with wild-type human DYRK1A RNA but not (D-E) DYRK1AR205 or DYRK1AL245R RNA. (A) Co-injection of a Standard MO and β-gal serve as a negative control. Scale bars represent 100 μm. (F) The graph demonstrates a significant difference between embryos injected with either Dyrk1a MO + β-gal or Dyrk1a MO + DYRK1AR205* versus with Dyrk1a MO + DYRK1A suggesting successful rescue with human DYRK1A but not with the nonsense RNA. (G) The second graph demonstrates a significant difference between embryos injected with Dyrk1a MO + DYRK1AL245R versus with Dyrk1a MO + DYRK1A, which suggests that the missense RNA also fails to rescue. Significance was established against embryos that had a moderate or severe kidney phenotype (orange bar) and excluded embryos that had a weak phenotype (yellow bar). (F) * (asterisk) = p<0.001 comparing individual experimental groups to Standard MO + β-gal. # (pound sign) = p<0.006 comparing Dyrk1a MO + DYRK1A to Dyrk1a MO + DYRK1AR205. (G) (asterisk) = p<0.001 comparing Standard MO + β-gal to Dyrk1a MO + β-gal or Dyrk1a MO + DYRK1AL245R. # (pound sign) = p<0.05 comparing Dyrk1a MO + DYRK1A to Dyrk1a MO + DYRK1AL245R. For edema assays, embryos were injected at the 4-cell stage in both ventral cells to target both kidneys while avoiding the dorsal cells fated to become the heart and liver, which can also lead to edema. (H) Embryos injected with the Standard MO did not develop edema while embryos injected (I) with the Dyrk1a MO did develop edema and also suffered from abnormal kidney formation. (J) The graph demonstrates a significant difference in edema and kidney abnormalities in embryos injected with either Standard MO or Dyrk1a MO. * (asterisk) = p<0.008 comparing Standard MO to Dyrk1a MO embryos with edema, defects in one, and defects in both kidneys. Error bars represent standard error. For ease of comparison of (n) and p-values across conditions, please refer to Table S1-3.
	Supplementary Figure 1. Human and Xenopus laevis protein alignment and short and long Xenopus homeolog alignment for dyrk1a 5’UTR. (A) Human DYRK1A mRNA transcript variant 1 (NM_001396.4) and Xenopus laevis dyrk1a short homeolog mRNA (NM_001163197.1) were translated and aligned using Clustal Omega. The overall identity between the protein sequences is 91.3% while the kinase domain has 97.5% identity demonstrating DYRK1A is highly conserved. Grey shading represents the kinase domain of DYRK1A. Symbols reflect Clustal Omega’s analysis of the residues and examples of specific residue groups that reflect each symbol can be found at their website https://www.ebi.ac.uk/Tools/msa/clustalo/. An * (asterisk) indicates positions with a fully conserved residue. A : (colon) indicates conservation between groups of strongly similar properties. A . (period) indicates conservation between groups of weakly similar properties. DYRK1A encodes a protein of 763 or 754 amino acid residues which results from alternative splicing with the longer isoform representing the canonical sequence shown above. Xenopus is missing residues 70-78 which are the same residues the short isoform of human DYRK1A (754 amino acids) is missing. This suggests that Xenopus most likely expresses an isoform of DYRK1A that is orthologous to the short isoform (NM_001347721.2) in human. (B) Alignment of dyrk1a’s 5’UTR demonstrate in grey shading where the morpholino targets and that it hits both long and short homeologs in Xenopus. Underlined text represents the coding sequence of dyrk1a. The Dyrk1a morpholino was designed against the short homeolog of dyrk1a (top sequence), however, here it is shown it targets both the short and long (bottom sequence) (identified via a BLAT search of Crick http://genomes.crick.ac.uk/). The morpholino does not target dyrk1a.2 which appears to be a duplication of dyrk1a with amino acid conservation only maintained in the kinase domain.
	Supplementary Figure 2. The Dyrk1a MO correctly targets Xenopus dyrk1a RNA and wild-type, missense, and truncated human DYRK1A proteins are expressed in neurula stage Xenopus embryos. Two constructs were generated with a GFP tag to demonstrate that the MO targets the endogenous 5’ untranslated region (UTR) of dyrk1a’s transcript. (A, bottom) Schematic demonstrates that the 5’ UTR construct contains part of dyrk1a’s endogenous (endo) 5’ UTR that is recognized by the Dyrk1a MO (A, top) while the control construct does not. (B) Single-cell embryos were injected with 10 ng of either Dyrk1a MO or Standard MO (Std MO) and co-injected with 1 ng RNA (either Xenopus dyrk1a control, or Xenopus dyrk1a + 5’ UTR). Western blot demonstrates complete reduction of GFP protein levels, indicative of loss of exogenous Dyrk1a in neurula stage embryos injected with Dyrk1a MO and dyrk1a + 5’ UTR RNA (lane 5) compared to the Standard MO and dyrk1a + 5’ UTR (lane 4) and Standard MO or Dyrk1a MO and dyrk1a control RNA (lane 2, lane 3). GAPDH was used as a loading control. (C) Wild-type human DYRK1A cDNA was inserted into a pCS2-HA vector. DYRK1AR205* and DYRK1AL245R variants were generated from the wild-type human pCS2-HA-DYRK1A vector. Western blot demonstrates HA protein is present in wild-type, missense (lanes 2 and 3 ~95 kDa), and truncated DYRK1AR205* (lane 4 ~25 kDa) lanes, demonstrating Xenopus neurula stage embryos can successfully translate human DYRK1A RNA. GAPDH was used as a loading control. Bar under the molecular weight indicates its position on the blot.
	Supplementary Figure 3. Overexpressing DYRK1AR205* or DYRK1AL245R does not cause a gain-of-function phenotype. (A) Xenopus embryos were injected at the 8-cell stage with the rescue dose (50pg) of either -gal, wild-type DYRK1A, DYRK1AR205, or DYRK1AL245R RNA. (B) No significant difference (p>0.7) was found between either -gal and wild-type DYRK1A, DYRK1AR205, or DYRK1AL245R suggesting neither variant elicits a gain-of-function phenotype. It should be noted that some embryos injected with wild-type human DYRK1A RNA had slightly underdeveloped kidneys suggesting DYRK1A overexpression may also affect kidney development. Experiments were repeated three times with error bars representing standard error. The total number of Xenopus embryos are as follows: β-gal: 67, DYRK1A: 76, DYRK1AR205*: 88, DYRK1AL245R: 65. Scale bar represents 100 µm.
	Supplementary Figure 4. Scoring system for Xenopus embryonic kidneys. Kidneys were scored at Nieuwkoop and Faber stage 40.12 (A) A normal kidney consists of three branches in the proximal region with a “trunk” connecting to the convoluted looping of the early distal region, and strong immunostaining of the late distal region. (B) A weak phenotype consists of loss of one of the branches in the proximal region and possibly less early distal looping and/or partial loss of late distal immunostaining. (C) A moderate phenotype consists of loss of two of the branches in the proximal region with moderate loss of early distal looping and possible loss of late distal immunostaining. (D) A severe phenotype consists of complete loss of proximal tubules with severe or complete loss of early distal looping and the late distal region. (E) The schematic represents a stereotypical Xenopus embryonic kidney with proximal tubules in light blue, distal tubules in blue, and connecting tubules in dark blue. Scale bar represents 100 µm.

References [+] :

Aranda, DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles. 2011, Pubmed