Late-Onset Autosomal Dominant Macular Degeneration Caused by Deletion of the CRX Gene.

PURPOSE: To characterize the phenotype observed in a case series with macular disease and determine the cause. DESIGN: Multicenter case series. PARTICIPANTS: Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. METHODS: Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. MAIN OUTCOME MEASURES: Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. RESULTS: All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. CONCLUSIONS: Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.

The Magnitude and Determinants of Tinnitus among Health Science Students at King Khalid University.

BACKGROUND: Tinnitus is a symptom that is defined as a subjective perception of noise in an absence of external sound. It is an indicator of auditory system abnormalities. It can also be present in individuals without any hearing abnormalities. Difficulty to consternate, insomnia, and decreased speech discrimination are the most common symptoms related to tinnitus. AIM: To assess the magnitude and pattern with determinants of tinnitus among health science students at King Khalid University. Methodology. A descriptive cross-sectional survey was conducted targeting all accessible students in health science colleges in King Khalid University which is the main university in the Aseer region, south of Saudi Arabia. Students were included consecutively from different faculties and different grades. Data were collected through a self-administered prestructured questionnaire, which was distributed and recollected the next day. Tinnitus was screened using an adapted form of the National Health and Nutrition Examination Survey (NHANES). RESULTS: A total sample of 400 students have been included with their ages ranging from 18 to 30 years with a mean age of 22 ± 1.8 years), and 28.5% of the students recorded positive findings. Tinnitus was bilateral among 51.8% of students, and 44.7% of tinnitus students hear buzzing sound while 21.1% have hissing sound and 10.5% had pulsating sound. Among 46.5% of students with tinnitus, the heard sound was of moderate loudness and intermittent among 64.9% of them. Conclusions and Recommendations. In conclusion, the study revealed that just more than a quarter of students complained of tinnitus which was bilateral among half of them. Tinnitus frequency was mainly moderate in intensity and intermittent. Having ear problems, loud sounds, and allergy were the most important predictors of having Tinnitus.

Long-Read Nanopore Sequencing of RPGR ORF15 is Enhanced Following DNase I Treatment of MinION Flow Cells.

INTRODUCTION: RPGR ORF15 is an exon present almost exclusively in the retinal transcript of RPGR. It is purine-rich, repetitive and notoriously hard to sequence, but is a hotspot for mutations causing X-linked retinitis pigmentosa. METHODS: Long-read nanopore sequencing on MinION and Flongle flow cells was used to sequence RPGR ORF15 in genomic DNA from patients with inherited retinal dystrophy. A flow cell wash kit was used on a MinION flow cell to increase yield. Findings were confirmed by PacBio SMRT long-read sequencing. RESULTS: We showed that long-read nanopore sequencing successfully reads through a 2 kb PCR-amplified fragment containing ORF15. We generated reads of sufficient quality and cumulative read-depth to detect pathogenic RP-causing variants. However, we observed that this G-rich, repetitive DNA segment rapidly blocks the available pores, resulting in sequence yields less than 5% of the expected output. This limited the extent to which samples could be pooled, increasing cost. We tested the utility of a MinION wash kit containing DNase I to digest DNA fragments remaining on the flow cell, regenerating the pores. Use of the DNase I treatment allowed repeated re-loading, increasing the sequence reads obtained. Our customised workflow was used to screen pooled amplification products from previously unsolved inherited retinal disease (IRD) in patients, identifying two new cases with pathogenic ORF15 variants. DISCUSSION: We report the novel finding that long-read nanopore sequencing can read through RPGR-ORF15, a DNA sequence not captured by short-read next-generation sequencing (NGS), but with a more reduced yield. Use of a flow cell wash kit containing DNase I unblocks the pores, allowing reloading of further library aliquots over a 72-h period, increasing yield. The workflow we describe provides a novel solution to the need for a rapid, robust, scalable, cost-effective ORF15 screening protocol.