Whole Exome Analysis Identifies Frequent <i>CNGA1</i> Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

<div><p>Objective</p><p>The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.</p><p>Methods</p><p>In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all <i>CNGA1</i> exons of the other 69 RP patients were performed.</p><p>Results</p><p>Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of <i>CNGA1</i> (four patients), <i>EYS</i> (three patients) and <i>SAG</i> (one patient) in eight patients and potential disease-causing gene variants of <i>USH2A</i> (two patients), <i>EYS</i> (one patient), <i>TULP1</i> (one patient) and <i>C2orf71</i> (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the <i>CNGA1</i> gene mutation revealed one patient with a homozygous mutation.</p><p>Conclusions</p><p>This is the first identification of <i>CNGA1</i> mutations in arRP Japanese patients. The frequency of <i>CNGA1</i> gene mutation was 5.1% (5/99 patients). <i>CNGA1</i> mutations are one of the most frequent arRP-causing mutations in Japanese patients.</p></div

Sequence data of all six identified <i>CNGA1</i> mutations in this study.

<p>A-1 to F-1 show the normal sequence data for the <i>CNGA1</i> gene. A-2 to F-2 show the sequence data for heterozygous <i>CNGA1</i> mutations (c.191delG, c.265delC, c.G860A, c.G1271A, c.1429delG and c.G2042C, respectively). A-3 and B-3 show the sequence data for homozygous <i>CNGA1</i> mutations (c.191delG and c.265delC).</p

Ophthalmic findings in five patients with retinitis pigmentosa with compound heterozygous or homozygous <i>CNGA1</i> mutations.

<p>BCVA = decimal corrective visual acuity; ERG = electroretinography; M = male; F = female.</p><p>Ophthalmic findings in five patients with retinitis pigmentosa with compound heterozygous or homozygous <i>CNGA1</i> mutations.</p

Pedigrees identified with arRP-causing mutations or potential arRP-causing variants.

<p>The solid squares (male) and circles (female) represent affected patients. The proband of each family is indicated by a black arrow. Unaffected family members are represented by white icons. The slash symbol indicates deceased individuals. The doubled line indicates consanguineous marriage. The generation number is shown on the left.</p

A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems - Fig 6

<p><b>A</b>. Brains from wild type (left) and <i>frogleg</i> (right) were of similar size. Scale is in centimeters. <b>B</b>. Brain weights, however, differed in wild type (+/+) and homozygote (<i>frogleg</i>) rats of 4 weeks, 10 weeks and 7 months of age, being considerably smaller in the <i>frogleg</i> rats compared to their littermates at all ages tested (mean+SEM).</p

Electrophoresis and western analysis of protein lysates from the brains of <i>frogleg</i> and littermate rats.

<p>In panel A are shown the staining patterns for 5 individual rats: lane 1 is lysate from a wild type, lanes 2 and 3 are from rats heterozygous for the <i>Bckdk</i> mutation, and lanes 4 and 5 are from homozygotes. In panel B equal aliquots of the same 5 samples were electrophoresed, transferred to nitrocellulose and probed with an antibody to BCKDH subunit E1α. It is evident that similar amounts of the total enzyme are present in all 5 samples. In panel C the amount of BCKDH phosphorylated at Ser293 of the E1α subunit is determined by probing the same 5 lysates with an antibody specific for the phosphorylated form of the enzyme. While the wildtype and heterozygous animals have a clear band, no immunoreactivity is detected in the 2 samples homozygous for the mutation. To demonstrate that the bands present on panel C represent the phosphorylated enzyme, aliquots of the same lysates were treated with calf intestinal alkaline phosphatase before electrophoresis. The bands present in lanes 1–3 of panel C are no longer visible.</p

Appearance of <i>frogleg</i> homozygous rat at 20 days of age.

<p>The animal on the left shows the typical appearance of the <i>frogleg</i> pup, relative to a non-<i>frogleg</i> littermate on the right. Note the small size, the thin, rough coat, and the abnormal rotation of the hind limbs of the affected animal.</p

Linkage map of the <i>frogleg</i> locus on rat chromosome 1.

<p>Ideogram of rat chromosome 1, showing polymorphic markers in the region of the <i>Bckdk</i> gene. Nucleotide sequence positions were determined by locating original marker amplimer sequences to the RGSC 6.0 / rn6 July 2014 assembly of the rat genome. MLINK-derived maximum LOD scores and corresponding theta values indicate linkage distance to the disease locus. The analysis was based on 11 affected and 10 unaffected individuals. Significant linkage scores identifying the initial disease interval (> 3.0) are enclosed in the box.</p

Immunolabeling of neuromuscular junctions of soleus and extensor digitorum longus (EDL) muscles.

<p>Representative data for muscles isolated from 7 month old <i>frogleg</i> rat are shown in Panels A for the EDL and Panels B for the soleus. For the EDL, panel A shows staining (green) with antibodies to neurofilament (NF200) and synaptic vesicle protein (SV2), while panel A’ shows staining of the nicotinic acetylcholine receptors at the junction with α-bungarotoxin (red). A” shows the merged image, with yellow indicating normal innervation of each of the 5 junctions shown. In contrast, for the soleus, panel B indicates very little immunoreactivity with SV2 or NF200. Staining of the nicotinic acetylcholine receptors in B’ is normal, but in the merged image (B”) only 2 of the 7 numbered junctions show evidence of partial innervation (arrowheads at numbers 5 and 6), with the other 5 being fully denervated. Scale bar = 50 μm. Panels C and D show the cumulative data for soleus and EDL muscles respectively, for 7 month old animals. Black bars are for wild type and red bars for <i>frogleg</i> animals. In WT EDL and soleus muscles, 97.1% and 98.6% of NMJs are intact. In contrast, while the EDL NMJs were 89.8% intact in the <i>frogleg</i> mutant at 7M, only 4.4% of the NMJs for the soleus remained intact at that age. Signs of denervation were predominant in soleus muscles, with NMJs being either partially (42.6%) or completely (51.1%) denervated. Error bars = SD; * P< 0.05 (Relative to control). In panel E, the time course of denervation in the soleus muscle for <i>frogleg</i> and wild type rats aged 1–7 months is shown.</p

Transmission electron microscopy images of cross sections of hind limb nerves from a <i>frogleg</i> rat.

<p>A. Sciatic nerve showing evidence of rare active demyelination (arrow). In the ventral root (B) of the sciatic nerve (as well as in the nerve itself) there were axons that were thinly myelinated (arrows in B). In the sural nerve, there was evidence of rare Wallerian-like degeneration, intra-axonal inclusions, and denervated Schwann cells. Scale bar = 2 microns.</p