ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy

Abstract

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.

Fig 1. Loss-of-function mutations in ITPA identified in Martsolf-like syndrome with infantile cardiomyopathy.

(A) Pedigree for Family 4911 showing the transmission of the ITPA c.452G>A, p.Trp151* allele. (B) Pedigree for Family 5196 showing the transmission of the ITPA c.456_488+7del allele. Electropherograms from sequencing of the affected individual (lower) and his mother (upper) from Family 4911 (C) and the affected individual (lower) and her mother (upper) from Family 5196 (D) show the sequence changes. The mutation nomenclature is based on the reference sequences NM_033453 and NP_258412. (E) Western blotting shows that ITPA protein is absent in a lymphoblastoid cell line derived from the affected individual 5196 III:3 and reduced in a line derived from her mother. Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample.

Fig 2. Phenotype associated with complete loss of ITPase function.

(A) Photograph of the dissected heart from the affected individual (III:3) in Family 5196 showing increased trabeculation and fibroelastosis of the endocardium. (B,C) Photomicrographs (x200) showing fatty infiltration and fibroelastosis of the heart respectively (D) Photograph of the fixed brain showing microcephaly/micrencephaly. (E) Coronal slice through brain showing dilatation of the lateral and third ventricles with reduced volume of white matter. (F) Image (x100) showing atrophy of the cerebellar cortex. (G) Photomicrograph (x600) shows degenerate Purkinje cells in the cerebellar cortex with spheroids (white arrows) in the B-APP immunostain (H, x400). (I) Macroscope (x4) image of the medulla showing hypoplastic pyramidal tracts. (J) Photomicrograph (x100) showing vacuolation of the pyramidal tracts.

Fig 3. Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase.

(A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa-null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa-null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa-null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa-null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa-null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa-null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa-null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table. (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa-null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table. (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.

Fig 4. Creation, morphology and biochemical analysis of Itpa null mouse embryos.

(A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa-null cells upon probing the blot with polyclonal antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa-null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa-null embryonic heart. Sections were subjected to H&E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa-null heart was identified. Data are representative of duplicate experiments.

Fig 5. Transcriptomic and proteomic analyses of Itpa null heart.

(A) Heatmap.2 based clustering of per-transcript genome wide log₂ ratios from biological triplicates of RNAseq from Itpa-null embryonic hearts and littermate controls. (B,C) Plot of per-gene log₂ signal from Affymetrix MTA1.0 microarray (same data as A) and quantitative protein mass spectrometry (same data as D) on samples from wild-type and Itpa-null embryonic hearts. (D) Heatmap.2 based clustering of genome wide per-protein intensity from six biological replicates of quantitative mass spectrometry from Itpa-null embryonic hearts and littermate controls. (E, F) Subset of data from A & D focussed on transcripts and proteins that are known to be involved in mendialian causes of dilated cardiomyopathy. No major differences are detectable in any of the heatmaps. (G) Quantitative RT-PCR (qPCR) of selected transcripts in Itpa-null embryonic hearts and littermate controls. The data shown are derived from analysis of 10 individual cDNA preparations per genotype, each analysed in triplicate.

Fig 6. Summary of ITPA-related disease mechanisms tested.

The left hand panel shows a cartoon of the ITPase reactions involving dITP and ITP to create dIMP and IMP respectively. The top half of the right hand panels summarise RNA-based mechanisms and the bottom half DNA-based mechanisms. The blue panel summarises the molecular basis of the mechanism. The orange panel the prior evidence and the hypothesis and the green panel the data that is presented in this paper that is relevant to each of the hypotheses. Under the text in each of the right hand panels there are square brackets which indicate which reference, table or data figure are relevant to the preceding text.