Expression of N-SF-TAP-Rp1 and N-LAP-Rp1 Transgenes.

<p>A. Diagram of <i>Rp1</i> gene, N-SF-TAP-<i>Rp1</i> and N-LAP-<i>Rp1</i> transgenes. The TAP and LAP tags were introduced into the beginning of the <i>Rp1</i> coding sequence in exon 2 in BAC 314, which contains 140 kb of mouse genomic DNA surrounding the <i>Rp1</i> locus. B-C. Western blot analyses of Rp1 proteins in N-SF-TAP-<i>Rp1</i> and N-LAP-<i>Rp1</i> mice. Equal amounts of protein from retinal extracts of wild-type, N-SF-TAP-<i>Rp1</i> and N-LAP-<i>Rp1</i> mice were analyzed by Western blotting using anti-Rp1 antibodies. The blots were also probed with antibodies to ATPase as a loading control. The Rp1 levels for the different transgenic lines were quantified and normalized to the ATPase signals. B. N-SF-TAP-<i>Rp1</i> mice. The total level of Rp1 protein in line T1 was 144% of that observed in non-transgenic littermate controls, indicating that the transgene increased expression approximately 44%, or nearly the amount expected from a third <i>Rp1</i> allele. The N-SF-TAP-<i>Rp1</i> transgene in line T2 is over-expressed relative to the wild-type protein, as it increased the total Rp1 protein level to ∼300% of normal. The total level of Rp1 protein in line T3 was only slightly elevated, but the retinas in these mice were also significantly degenerated, with 40% of the photoreceptor nuclei remaining in the outer nuclear layer, suggesting that the N-SF-TAP-Rp1 protein in this transgenic line is also 2–3 fold greater than wild-type. C. N-LAP-<i>Rp1</i> mice. The levels of N-LAP-<i>Rp1</i> fusion protein in lines L1 and L2 mice were approximately half of that observed for the wild-type Rp1 protein, again indicating that the transgene increased expression approximately the amount expected from a third <i>Rp1</i> allele. In contrast, N-LAP-<i>Rp1</i> transgene in line L3 is over-expressed, and increased the total Rp1 protein level to ∼250% of normal. D. Immunofluorescence analyses of wild-type Rp1 protein (anti-Rp1 antibodies; red) and N-SF-TAP-Rp1 protein (anti-FLAG antibodies; green) in three N-SF-TAP-<i>Rp1</i> transgenic lines and wild-type littermate control. Note that the wild-type Rp1 protein is located in the axoneme of photoreceptor outer segments. The N-SF-TAP-Rp1 protein in transgene line T1 shares the same location, as indicated by the overlap of the two signals in the merged image panel (bottom left). There is also some N-SF-TAP-Rp1 signal in the synaptic region of photoreceptor cells that is not present in wild-type retinas. The N-SF-TAP-<i>Rp1</i> transgenes in T2 and T3 are over-expressed relative to the wild-type protein. The over-expressed N-SF-TAP-Rp1 protein localizes correctly to PSC axonemes, but also mis-localizes to photoreceptor inner segments. The N-SF-TAP-Rp1 signal in the synaptic region is also increased, especially in the line T3 retinas. Note that the outer nuclear layer is thinner in the line T3 sample, consistent with the photoreceptor degeneration observed in this transgene line. The ONL is also slightly thinner in the line T2 samples as well. E. Immunofluorescence analyses of wild-type Rp1 protein (anti-Rp1 antibodies; red) and N-LAP-Rp1 protein (EGFP; green) in three N-LAP-<i>Rp1</i> transgenic lines and wild-type littermate control. The N-LAP-Rp1 protein in transgene line L1 is located in the axoneme of PSCs, like the wild-type protein. In addition, there is EGFP signal from N-LAP-Rp1 protein in the inner segments and cell bodies of the photoreceptors. Since this was not detected by the anti-Rp1 antibodies, it must be due to truncated versions of the N-LAP-Rp1 protein that retain the N-terminal EGFP tag, but have lost the C-terminal antibody binding domain. The N-LAP-<i>Rp1</i> transgenes in line L3 is over-expressed relative to the wild-type protein. The over-expressed N-SF-TAP-Rp1 protein localizes correctly to PSC axonemes, but also mis-localizes to photoreceptor inner segments and cell bodies. As for the N-SF-TAP-Rp1 line T3, there is photoreceptor degeneration in the L3 line. The N-LAP-Rp1 transgene expression in line L2 is not completely uniform, with some cells that do not express the transgene evident. In addition, there are red signals in OPL in line L1 and L2, which could represent the non-specific signaling from the anti-c-Rp1 antibody. It is also possible that this immunoreactivity of Rp1 could correspond to the C-terminal fragments of Rp1. (IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; 400X magnification for all images).</p

<i>RP1</i> Gene, Clinical and Sequence Data for Family W04-348.

<p><b>A. </b><i>RP1</i> gene and identified mutations. The gene structure of <i>RP1</i> is depicted, with the locations of mutations that cause adRP and arRP indicated; the mutations above the <i>RP1</i> gene structure cause dominant RP, labeled as adRP in red; whereas mutations below the <i>RP1</i> gene structure cause recessive RP, labeled as arRP in blue; frameshift mutation p.P229QfsX35 reported to cause arRP in this study is in bold. The portion of the gene that encodes that DCX domains is also indicated. The arrow on the top indicates the location of the R677X (human) and Q662X (mouse) mutations. <b>B.</b> Pedigree for family W04-348. The c.686delC, p.P229QfsX35 mutation is designated by <i>M</i>. <b>C.</b> ERG traces from a normal control, the patient’s parents (I-1, I-2 (age 57), and the affected patient (II-1). The five standard ISCEV recordings are shown, from the top including: scotopic rod responses, scotopic combined rod-cone responses, oscillatory potentials, photopic single flash and photopic 30Hz responses. The amplitude and time scales are indicated. The ERG responses of the patient’s parents show normal amplitudes and implicit times; the patient had no recordable rod or cone responses. The deflections shown in the 30 Hz recordings for the patient are due to motion artifact. The thicker traces are from the right eye, the thinner from the left eye. <b>D.</b> Fundus photos (left) and fundus autofluorescence images (right) of the affected patient II-1(age 30) and his father I-1 (age 60). The patient has typical findings of RP, with optic disc pallor, attenuation of the retinal blood vessels, RPE atrophy and bone spicule pigmentation outside the macula. As shown in the autofluorescence image, the RPE in the macular region is relatively preserved. In contrast, the father’s fundi are normal. <b>E.</b> Sequence traces showing the homozygous c.686delC mutation in patient II-1, carrier status of this mutation in the patient’s father I-1, and the wild-type sequence in an unaffected Dutch control. Note that the sequence trace of the mutant allele in individual I-1 is shifted slightly.</p

Expression of the full-length N-SF-TAP-Rp1 and N-LAP-Rp1 proteins prevents photoreceptor degeneration in Rp1^Q662X/Q662X knock-in mice.

<p>A. Frozen sections of retina from 2-month-old mice of the genotypes indicated were stained with anti-C-Rp1 antibodies (red). The wild-type Rp1 protein is located in the axonemes of PSC; full-length Rp1 protein is not detected in the <i>Rp1</i>-Q662X knock-in mice. Full-length Rp1 protein is also detected in the PSC axonemes of the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> and <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. There is a mosaic pattern of full-length Rp1 protein expression in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. (INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; 400X magnification for all images). B. Retinal histology from 2-month-old mice of the genotypes indicated. The retinal structure of the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice is normal, in contrast to the early photoreceptor degeneration in the <i>Rp1</i>-Q662X mice. There is partial preservation of retina structure in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice (INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; 400× magnification for all images). C. Ultrastructure of PSCs in two-month old mice of the genotypes indicated. Note that the structure of the PSC and organization of the outer segment discs are normal in the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice, in contrast to the disorganized PSC observed in the <i>Rp1</i>^Q662X/Q662X mice. There is a mixture of cells with normal PSC and cells with disorganized PSC in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice (OS, outer segment; RPE, retinal pigment epithelium. Bars  = 2 µm). D. Amplitudes of ERG responses from 2 month-old and 12-month-old mice of the genotypes indicated. At 2 months not that the rod and cone ERG amplitudes are close to normal in the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice, in contrast to the reduction of photoreceptor function observed in the <i>Rp1</i>^Q662X/Q662X mice. There is partial restoration of photoreceptor function in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. At 12 months photoreceptor function is relatively well preserved in the <i>Rp1</i>-Q662X : N-SF-TAP-<i>Rp1</i> mice. There is also some residual photoreceptor function in the <i>Rp1</i>-Q662X : N-LAP-<i>Rp1</i> mice. Significant differences compared to controls are indicated by * (P<0.01) and ** (P<0.05).</p

Retinal phenotypes of N-SF-TAP-Rp1 and N-LAP-Rp1 transgenic mice.

<p><b>A.</b> Retinal histology from 1-year-old mice of the genotypes indicated. Note that the retinal structure of the N-SF-TAP-<i>Rp1</i> line T1 and N-LAP-<i>Rp1</i> lines L1 and L2 is normal. In contrast, there is photoreceptor degeneration evident in N-SF-TAP-<i>Rp1</i> lines T2 and T3, and N-LAP-<i>Rp1</i> line L3, with loss of photoreceptor nuclei and shortening of photoreceptor outer segments. (INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; 400× magnification for all images). <b>B.</b> Ultrastructure of photoreceptor sensory cilia in one-year old N-SF-TAP-<i>Rp1</i> line T1 and N-LAP-<i>Rp1</i> line L2 mice, compared to that of wild-type littermate control. Note that the structure of the PSC and organization of the outer segment discs are normal in both transgenic lines, consistent with the histology shown in A. <b>C.</b> Amplitudes of ERG responses from 1-year-old N-SF-TAP-<i>Rp1</i> line T1 and N-LAP-<i>Rp1</i> line L2 mice, compared to that of wild-type littermate controls. Note that the rod and cone ERG amplitudes are normal in both lines of transgenic mice.</p

Generation and Characterization of <i>Rp1</i>^Q662X/Q662X Knock-in Mice. A.

<p>Gene targeting strategy to introduce Q662X mutation into the endogenous <i>Rp1</i> locus. The gene targeting vector was produced by introducing the Neo-Zeo selection cassette and Q662X mutation into the <i>Rp1</i> gene in a BAC which contains 140 kb of mouse genomic DNA including the <i>Rp1</i> locus, using homologous recombination techniques. The gene targeting vector was then retrieved from the modified BAC, and used to transfect mouse ES cells. Correctly targeted ES cells were injected into blastocysts to generate <i>Rp1</i>-Neo-Q662X mice. Crosses to Flpe deleter mice were used to remove the Frt-flanked Neo-Zeo selection cassette and generate the final <i>Rp1</i>-Q662X allele. B, <i>Bam</i>HI restriction sites; P, <i>Pme</i>I restriction sites. <b>B.</b> Southern blots using the 5′ and 3′ probes indicated in A demonstrating correct targeting of the <i>Rp1</i> locus in mouse ES cells. The 12.6 and 18 kb bands in the 5′ and 3′ blots, respectively, indicate correct recombination to generate the <i>Rp1</i>-Neo-Q662X allele. The large size of the wild-type Pme1 restriction fragment (60 kb) in the 3′ blot makes it difficult to resolve this band with standard electrophoresis techniques. <b>C.</b> Sequence traces from PCR products amplified from wild-type, heterozygous (wt/ki) and homozygous (ki/ki) mice. <b>D.</b> Example PCR-based genotyping of <i>Rp1</i>-Q662X (ki) knock-in mice. Using PCR primers that amplify across the residual Frt-LoxP sites, the wild-type and ki alleles can be readily distinguished. <b>E.</b> RT-PCR of retinal RNA from mice of indicated genotypes showing the correct splicing of mutant Rp1-Q662X allele as that of wild type Rp1 allele, using primer sets bridging exon 2 to 3 and exon 3 to 4. <b>F.</b> Predicted proteins produced by the wild-type (wt) and knock-in (ki) <i>Rp1</i> alleles, showing locations of the anti-N-Rp1 and anti-C-Rp1 antibodies used in panels G and H. <b>G.</b> Western blot of retinal extracts from mice of the indicated genotypes showing production of the 74 kD truncated protein by the knock-in (ki) allele. Note that there is no full-length Rp1 protein detected in the retinas of the homozygous ki/ki mice by either the anti-N-Rp1 or anti-C-Rp1 antibodies. <b>H.</b> Immunofluorescence analysis of Rp1 proteins (red) produced in the retinas of wild-type (wt/wt) vs. homozygous knock-in (ki/ki) mice using the anti-N-Rp1 and anti-C-Rp1 antibodies indicated in panel E. Note that the truncated Rp1 protein produced in the retinas of the ki/ki mice locates correctly to the axonemes of PSC, just like the full-length protein in the wt/wt retinas. Note also the lack of full-length Rp1 protein production in the retinas of the ki/ki mice (AX, axoneme; IS, inner segment; ONL, outer nuclear layer; OS, outer segment).</p

Retinal degeneration phenotype in the Rp1-Q662X mice. A.

<p>Light microscopic images of semi-thin sections of retinas from wild-type (wt/wt) and homozygous <i>Rp1</i>-Q662X knock-in mice (ki/ki) of the ages indicated. Note that thinning of the outer nuclear layer in the ki/ki mice is evident at 1 month, and progresses so that by 6 months of age only 2–3 rows of photoreceptor nuclei remain. Note also the disorganization and shortening of the photoreceptor outer segments in the ki/ki mice. (GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium; 400× magnification for all images). <b>B, C.</b> Electron micrographs of retinas from wt/wt and ki/ki mice at 10 days (B) and 1 month of age (C). At 10 days of age small packets of enlarged disc membranes parallel to the axis of the axonemes replaced the normal perpendicularly orientated stacks of discs observed in control animals. These defects of outer segments were even more evident at 1 month of age, with disorganized membranous whirls in place of outer segments, compared to the well aligned, uniform discs in the control retina (IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium; bars  = 2 µm). <b>D–F.</b> Average rod and cone ERG amplitudes for wild-type (wt/wt), heterozygous (wt/ki) and homozygous (ki/ki) <i>Rp1</i>-Q662X knock-in mice. The amplitudes + SD are shown for mice of the three genotypes at 1 month (D), 6 months (E) and 30 months of age (F). Significant differences (P<0.05) indicated by *. Note that the rod-a waves of the ki/ki mice were significantly decreased at 1 month of age. By 6 months of age, all measures of rod and cone function were decreased in the ki/ki mice. By 30 months of age, no recordable ERG responses were detected.</p

A novel <i>HSD17B10</i> mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

<p>We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene <i>HSD17B10</i> for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in <i>HSD17B10</i> cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5′-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.</p