Hereditary Eye Disease Enrichment Panel Research Articles

Screening copy number variations in 35 unsolved inherited retinal disease families

The purpose of this study was to screen Copy Number Variations (CNVs) in 35 unsolved Inherited Retinal Dystrophy (IRD) families. Initially, next generation sequencing, including a specific Hereditary Eye Disease Enrichment Panel or Whole exome sequencing, was employed to screen (likely) pathogenic Single-nucleotide Variants (SNVs) and small Insertions and Deletions (indels) for these cases. All available SNVs and indels were further validated and co-segregation analyses were performed in available family members by Sanger sequencing. If not, after excluding deep intronic variants, Multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescence PCR (QF-PCR) and Sanger sequencing were employed to screen CNVs. We determined that 18 probands who had heterozygous SNVs/indels or whose parents were not consanguineous but had homozygous SNVs/indels in autosomal recessive IRDs genes had CNVs in another allele of these genes, 11 families had disease-causing hemizygous CNVs in X-linked IRD genes, 6 families had (likely) pathogenic heterozygous CNVs in PRPF31 gene. Of 35 families, 33 different CNVs in 16 IRD-associated genes were detected, with PRPF31, EYS and USH2A the most common disease-causing gene in CNVs. Twenty-six and 7 of them were deletion and duplication CNVs, respectively. Among them, 14 CNVs were first reported in this study. Our research indicates that CNVs contribute a lot to IRDs, and screening of CNVs substantially increases the diagnostic rate of IRD. Our results emphasize that MLPA and QF-PCR are ideal methods to validate CNVs, and the novel CNVs reported herein expand the mutational spectrums of IRDs.

Analysis of RPGR gene mutations in 41 Chinese families affected by X-linked inherited retinal dystrophy.

Background: This study analyzed the phenotypes and genotypes of 41 Chinese families with inherited retinal dystrophy (IRD) and RPGR gene mutations. Methods: This retrospective analysis evaluated a cohort of 41 patients who were subjected to a specific Hereditary Eye Disease Enrichment Panel (HEDEP) analysis. All (likely) pathogenic variants were determined by Sanger sequencing, and co-segregation analyses were performed on the available family members. All cases were subjected to Sanger sequencing for RPGR open reading frame 15 (ORF15) mutations. Results: A total of 41 probands from different families with a clinical diagnosis of retinitis pigmentosa (RP; 34 cases) and cone-rod dystrophy (CORD; 7 cases) were included in this cohort. According to clinical information, 2, 18, and 21 cases were first assigned as autosomal dominant (AD), sporadic, and X-linked (XL) inheritance, respectively. Several cases of affected females who presented with a male phenotype have been described, posing challenges at diagnosis related to the apparent family history of AD. Mutations were located in RPGR exons or introns 1–14 and in ORF15 of 12 of 41 (29.3%) and 29 of 41 (70.7%) subjects, respectively. Thirty-four (likely) pathogenic mutations were identified. Frameshifts were the most frequently observed variants, followed by nonsense, splice, and missense mutations. Herein, a detailed description of four RP patients carrying RPGR intronic mutations is reported, and in vitro splice assays were performed to confirm the pathogenicity of these intronic mutations. Conclusion: Our findings provide useful insights for the genetic and clinical counseling of patients with XL IRD, which will be useful for ongoing and future gene therapy trials.

Novel variants in PDE6A and PDE6B genes and its phenotypes in patients with retinitis pigmentosa in Chinese families

BackgroundRetinitis pigmentosa (RP) is a genetically heterogeneous disease with 89 causative genes identified to date. However, only approximately 60% of RP cases genetically solved to date, predicating that many novel disease-causing variants are yet to be identified. The purpose of this study is to identify novel variants in PDE6A and PDE6B genes and present its phenotypes in patients with retinitis pigmentosa in Chinese families.MethodsFive retinitis pigmentosa patients with PDE6A variants and three with PDE6B variants were identified through a hereditary eye disease enrichment panel (HEDEP), all patients’ medical and ophthalmic histories were collected, and ophthalmological examinations were performed, followed by an analysis of the possible causative variants. Sanger sequencing was used to verify the variants.ResultsWe identified 20 variants in eight patients: 16 of them were identified in either PDE6A or PDE6B in a compound heterozygous state. Additional four heterozygous variants were identified in the genes ADGRA3, CA4, OPTN, RHO. Two novel genetic changes in PDE6A were identified (c.1246G > A and c.1747 T > A), three novel genetic changes in PDE6B were identified (c.401 T > C, c.2293G > C and c.1610-1612del), out of the novel identified variants one was most probably non-pathogenic (c.2293G > C), all other novel variants are pathogenic. Additional variant was identified in CA4 and RHO, which can cause ADRP (c.243G > A, c.688G > A). In addition, a novel variant in ADGRA3 was identified (c.921-1G > A).ConclusionsThis study reveals novel and known variants in PDE6A and PDE6B genes in Chinese families with autosomal recessive RP, and expands the clinical and genetic findings of photoreceptor-specific enzyme deficiencies.

Molecular diagnosis based on comprehensive genetic testing in 800 Chinese families with non-syndromic inherited retinal dystrophies.

Inherited retinal dystrophies (IRDs) are a group of monogenic diseases, one of the leading causes of blindness. Introducing a comprehensive genetic testing strategy by combining single gene Sanger sequencing, next-generation sequencing (NGS) including whole exome sequencing (WES), and a specific hereditary eye disease enrichment panel (HEDEP) sequencing, to identify the disease-causing variants of 800 Chinese probands affected with non-syndromic IRDs. Retrospective analysis. Eight hundred Chinese non-syndromic IRDs probands and their families. A total of 149 patients were subjected to Sanger sequencing. Of the 651 patients subjected to NGS, 86 patients underwent WES and 565 underwent HEDEP. Patients that likely carried copy number variations (CNVs) detected by HEDEP were further validated by multiplex ligation-dependent probe amplification (MLPA) or quantitative fluorescence PCR (QF-PCR). The diagnostic rate. (Likely) pathogenic variants were determined in 481 cases (60.13% detection rate). The detection rates of single gene Sanger sequencing, WES and HEDEP were 86.58%, 31.40% and 56.99%, respectively. Approximately 11.64% of 481 cases carried autosomal dominant variants, 72.97% carried AR variants and 15.39% were found to be X-linked. CNVs were confirmed by MLPA or QF-PCR in 17 families. Fourteen genes that each caused disease in 1% or more of the cohort were detected, and these genes were collectively responsible for disease in almost one half (46.38%) of the families. Sanger sequencing is ideal to detect pathogenic variants of clinical homogeneous diseases, whereas NGS is more appropriate for patients without an explicit clinical diagnosis.

Characteristics of genotype and phenotype in Chinese patients with Bardet-Biedl syndrome.

To investigate complex and different phenotypes in seven Chinese patients diagnosed with Bardet-Biedl syndrome (BBS) and carrying pathogenic mutations. Seven unrelated BBS patients were enrolled. Their medical and ophthalmic histories were reviewed, and comprehensive clinical examinations, such as fundus photography, optical coherence tomography, and medical imaging, were performed. A specific hereditary eye disease enrichment panel based on exome-capture technology was used to collect and amplify the protein-coding regions of 441 targeted hereditary eye disease genes, followed by high-throughput sequencing using the Illumina HiSeq platform. All patients exhibited the primary clinical phenotype of BBS. Seven BBS mutations were found in five patients (BBS7 in two patients, BBS10 in two patients, BBS12 in one patient), for a detection rate of 71% (5/7). The ratio of novel to known BBS mutations was 5:2. This study showed the phenotypic and genotypic spectrum of BBS patients from China, and the findings underscore the importance of obtaining comprehensive clinical observations and molecular analyses for ciliopathies.

Novel mutations of RPGRIP1 gene in a family with Leber congenital amaurosis

Objective To identify the pathogenic gene mutations in a family with Leber congenital amaurosis (LCA). Methods In October 2018, 1 patient and 3 normal family members from a LCA family was enrolled in this retrospective study. Detailed medical history of proband was obtained and fixation test, cycloplegic refraction, slit-lamp, fundus color photography and full-field ERG were performed. And other family members underwent BCVA, refraction slit-lamp, fundus biomicroscopy with the slit lamp, fundus color photography and full-field ERG. The family was investigated with a specific hereditary eye disease enrichment panel which contained 441 known pathogenic genes and based on targeted exome capture technology first to indentify the potential pathogenic genes and mutations. Then the potential pathogenic mutations were conformed by Sanger sequencing. Finally, the results were analyzed via bioinformatics analysis. Results The proband showed no trace object from childhood, but had obvious photophobia and nystagmus. No positive changes were found in the anterior segment, vitreous and retina in both eyes. Both cone and rod system function decreased significantly in full-field ERG in both eyes. Gene tests showed the proband carried both RPGRIP1 c.1635dupA and c.3565C> T, which composited a heterozygous mutation. Bioinformatics analysis showed RPGRIP1 c.1635dupA was a pathogenic mutation, and RPGRIP1 c.3565C> T which was a novel potential pathogenic mutation in LCA. Conclusion The compound heterozygous mutation, c.1635dupA and c.3565C> T in RPGRIP1 may be responsible for the pathogenesis in this Chinese Han LCA pedigree. Key words: Leber congenital amaurosis/etiology; Genes; Mutation; RPGRIP1 gene

Simultaneous expression of two pathogenic genes in four Chinese patients affected with inherited retinal dystrophy.

To describe the complex, overlapping phenotype of four Chinese patients with inherited retinal dystrophies (IRDs) who harbored two pathogenic genes simultaneously. This retrospective study included 4 patients affected with IRDs. Medical and ophthalmic histories were obtained, and clinical examinations were performed. A specific Hereditary Eye Disease Enrichment Panel (HEDEP) based on exome capture technology was used for genetic screening. Four patients were identified to harbor disease-causing variants in two different genes. Patient retinitis pigmentosa (RP) 01-II:1 exhibited both classical ABCA4-induced Stargardt disease (STGD) 1 and USH2A-associated RP, patient RP02-III:2 exhibited both classical ABCA4-induced STGD1 and CDH23-associated RP, patient RP03-II:1 exhibited both USH2A-induced autosomal recessive retinitis pigmentosa (arRP) syndrome and SNRNP200-induced autosomal dominant retinitis pigmentosa (adRP), and patient RP04-II:2 exhibited USH2A-induced arRP syndrome and EYS-induced arRP at the same time. Our study demonstrates that genotype-phenotype correlations and comprehensive genetic screening is crucial for diagnosing IRDs and helping family planning for patients suffering from the disease.

Application of Whole Exome and Targeted Panel Sequencing in the Clinical Molecular Diagnosis of 319 Chinese Families with Inherited Retinal Dystrophy and Comparison Study.

Inherited retinal dystrophies (IRDs) are a group of clinically and genetically heterogeneous diseases involving more than 280 genes and no less than 20 different clinical phenotypes. In this study, our aims were to identify the disease-causing gene variants of 319 Chinese patients with IRD, and compare the pros and cons of targeted panel sequencing and whole exome sequencing (WES). Patients were assigned for analysis with a hereditary eye disease enrichment panel (HEDEP) or WES examination based on time of recruitment. This HEDEP was able to capture 441 hereditary eye disease genes, which included 291 genes related to IRD. As RPGR ORF15 was difficult to capture, all samples were subjected to Sanger sequencing for this region. Among the 163 disease-causing variants identified in this study, 73 had been previously reported, and the other 90 were novel. Genes most commonly implicated in different inheritances of IRDs in this cohort were presented. HEDEP and WES achieved diagnostic yield with 41.2% and 33.0%, respectively. In addition, nine patients were found to carry pathogenic mutations in the RPGR ORF15 region with Sanger sequencing. Our study demonstrates that HEDEP can be used as a first-tier test for patients with IRDs.

Dependable and Efficient Clinical Molecular Diagnosis of Chinese RP Patient with Targeted Exon Sequencing.

Retinitis pigmentosa (RP) is the most common inherited retinal disease. It is a clinically and genetically heterogeneous disorder, which is why it is particularly challenging to diagnose. The aim of this study was to establish a targeted next-generation sequencing (NGS) approach for the comprehensive, rapid, and cost-effective clinical molecular diagnosis of RP. A specific hereditary eye disease enrichment panel (HEDEP) based on exome capture technology was used to collect the protein coding regions of 371 targeted hereditary eye disease genes, followed by high-throughput sequencing on the Illumina HiSeq2000 platform. From a cohort of 34 Chinese RP families, 13 families were successfully diagnosed; thus, the method achieves a diagnostic rate of approximately 40%. Of 16 pathogenic mutations identified, 11 were novel. Our study demonstrates that targeted capture sequencing offers a rapid and effective method for the molecular diagnosis of RP, which helps to provide a more accurate clinical diagnosis and paves the way for genetic counseling, family planning, and future gene-targeted treatment.

Identification of a Novel NHS Mutation in a Chinese Family with Nance-Horan Syndrome

Purpose: To identiy the disease causing mutation in a Chinese family presenting with early-onset cataract and dental anomalies.Materials and Methods: A specific Hereditary Eye Disease Enrichment Panel (HEDEP) (personalized customization by MyGenostics, Baltimore, MD) based on targeted exome capture technology was used to collect the protein coding regions of 30 early-onset cataract associated genes, and high throughput sequencing was done with Illumina HiSeq 2000 platform. The identified variant was confirmed with Sanger sequencing.Results: A novel deletion in exon 4 (c.852delG) of NHS gene was identified; the identified 1 bp deletion altered the reading frame and was predicted to result in a premature stop codon after the addition of twelve novel amino acid (p.S285PfsX13). This mutation co-segregated in affected males and obligate female carriers, but was absent in 100 matched controls.Conclusions: Our findings broaden the spectrum of NHS mutations causing Nance-Horan syndrome and phenotypic spectrum of the disease in Chinese patients.

Targeted exome capture and sequencing identifies novel PRPF31 mutations in autosomal dominant retinitis pigmentosa in Chinese families

ObjectivesTo identify disease-causing mutations in two Chinese families with autosomal dominant retinitis pigmentosa (adRP).DesignProspective analysis.PatientsTwo Chinese adRP families underwent genetic diagnosis. A specific hereditary eye disease enrichment panel (HEDEP) based...