Long-read sequencing improves the genetic diagnosis of retinitis pigmentosa by identifying an Alu retrotransposon insertion in the EYS gene.

BACKGROUND: Biallelic variants in EYS are the major cause of autosomal recessive retinitis pigmentosa (arRP) in certain populations, a clinically and genetically heterogeneous disease that may lead to legal blindness. EYS is one of the largest genes (~ 2 Mb) expressed in the retina, in which structural variants (SVs) represent a common cause of disease. However, their identification using short-read sequencing (SRS) is not always feasible. Here, we conducted targeted long-read sequencing (T-LRS) using adaptive sampling of EYS on the MinION sequencing platform (Oxford Nanopore Technologies) to definitively diagnose an arRP family, whose affected individuals (n = 3) carried the heterozygous pathogenic deletion of exons 32-33 in the EYS gene. As this was a recurrent variant identified in three additional families in our cohort, we also aimed to characterize the known deletion at the nucleotide level to assess a possible founder effect. RESULTS: T-LRS in family A unveiled a heterozygous AluYa5 insertion in the coding exon 43 of EYS (chr6(GRCh37):g.64430524_64430525ins352), which segregated with the disease in compound heterozygosity with the previously identified deletion. Visual inspection of previous SRS alignments using IGV revealed several reads containing soft-clipped bases, accompanied by a slight drop in coverage at the Alu insertion site. This prompted us to develop a simplified program using grep command to investigate the recurrence of this variant in our cohort from SRS data. Moreover, LRS also allowed the characterization of the CNV as a ~ 56.4kb deletion spanning exons 32-33 of EYS (chr6(GRCh37):g.64764235_64820592del). The results of further characterization by Sanger sequencing and linkage analysis in the four families were consistent with a founder variant. CONCLUSIONS: To our knowledge, this is the first report of a mobile element insertion into the coding sequence of EYS, as a likely cause of arRP in a family. Our study highlights the value of LRS technology in characterizing and identifying hidden pathogenic SVs, such as retrotransposon insertions, whose contribution to the etiopathogenesis of rare diseases may be underestimated.

Expanding the phenotype of THRB: a range of macular dystrophies as the major clinical manifestations in patients with a dominant splicing variant.

Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHß); however, none of the IRD patients exhibited RTHß. Genotype-phenotype correlations showed that RTHß can be caused by both truncating and missense variants, which are mainly located at the 3' (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRß1 and TRß2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5'-region of the gene that encodes the N-terminal domain of the TRß1 isoform protein, leaving the TRß2 isoform intact, which would explain the phenotypic variability observed between RTHß and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHß patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms.

Long-Term Effects of Inspiratory Muscle Training in Institutionalized Elderly Women: A Double-Blind Randomized Controlled Trial.

BACKGROUND: Inspiratory muscle training (IMT) has been applied to different populations, but evidence in elderly women and maintenance of improvements achieved during training is scarce. The objective of the study was to evaluate the effectiveness of IMT 6 months after finishing the training period in elderly women. METHODS: A controlled, randomized, double-blind trial was conducted, with allocation concealment performed on 26 institutionalized elderly women distributed into 2 groups: high loads and low loads. Over an 8-week period, an IMT protocol was followed; later, elderly women continued for 6 months with their daily normal activities. Inspiratory strength, expiratory strength, and functional capacity were measured pre-IMT, post-IMT, and 6 months after intervention. RESULTS: After an 8-week training, respiratory strength and functional capacity increased in both groups. Six months after finishing the intervention, these benefits were lost. Inspiratory strength decreased in the high-loads group but was 32% higher than initial values (p = 0.007); in the low-loads group, patients lost 41% (p = 0.015). Improvements were higher in the high-loads group after training (p = 0.000) and follow-up (p = 0.002). Functional capacity was similar to initial values in both groups. CONCLUSION: The improvements achieved with IMT in elderly women disappeared 6 months after the end of training. High loads were more effective to keep inspiratory strength than low loads.

A comprehensive WGS-based pipeline for the identification of new candidate genes in inherited retinal dystrophies.

To enhance the use of Whole Genome Sequencing (WGS) in clinical practice, it is still necessary to standardize data analysis pipelines. Herein, we aimed to define a WGS-based algorithm for the accurate interpretation of variants in inherited retinal dystrophies (IRD). This study comprised 429 phenotyped individuals divided into three cohorts. A comparison of 14 pathogenicity predictors, and the re-definition of its cutoffs, were performed using panel-sequencing curated data from 209 genetically diagnosed individuals with IRD (training cohort). The optimal tool combinations, previously validated in 50 additional IRD individuals, were also tested in patients with hereditary cancer (n = 109), and with neurological diseases (n = 47) to evaluate the translational value of this approach (validation cohort). Then, our workflow was applied for the WGS-data analysis of 14 individuals from genetically undiagnosed IRD families (discovery cohort). The statistical analysis showed that the optimal filtering combination included CADDv1.6, MAPP, Grantham, and SIFT tools. Our pipeline allowed the identification of one homozygous variant in the candidate gene CFAP20 (c.337 C > T; p.Arg113Trp), a conserved ciliary gene, which was abundantly expressed in human retina and was located in the photoreceptors layer. Although further studies are needed, we propose CFAP20 as a candidate gene for autosomal recessive retinitis pigmentosa. Moreover, we offer a translational strategy for accurate WGS-data prioritization, which is essential for the advancement of personalized medicine.

Novel BEST1 mutations and clinical characteristics of autosomal recessive bestrophinopathy in a Spanish patient.

PURPOSE: To describe the clinical and genetic characteristics (novel mutation in BEST1 gene) of a Spanish patient with autosomal recessive bestrophinopathy (ARB). METHODS: The detailed ophthalmological examination included best corrected visual acuity (BCVA), color and autofluorescence photography, fluorescein angiography, optical coherence tomography, and electrophysiology tests. A next-generation sequencing (NGS) strategy was applied to the index patient, and then sequenced in an Illumina NextSeq500 system. RESULTS: A 55-year-old male presented with a BCVA of 20/25 in the right eye and 20/20 in the left eye. Fundoscopy revealed perifoveal yellow flecked-like lesions. Fluorescein angiography and fundus autofluorescence results were consistent with pattern dystrophy. A homozygous frameshift mutation in BEST1 (c.341_342del; p.(Leu114Glnfs*57)) was identified as the cause of the disease. CONCLUSION: ARB is a genetic disease that leads to irreversible visual loss. In this report we found a novel mutation responsible for this disease.

A Multi-Strategy Sequencing Workflow in Inherited Retinal Dystrophies: Routine Diagnosis, Addressing Unsolved Cases and Candidate Genes Identification.

The management of unsolved inherited retinal dystrophies (IRD) cases is challenging since no standard pipelines have been established. This study aimed to define a diagnostic algorithm useful for the diagnostic routine and to address unsolved cases. Here, we applied a Next-Generation Sequencing-based workflow, including a first step of panel sequencing (PS) followed by clinical-exome sequencing (CES) and whole-exome sequencing (WES), in 46 IRD patients belonging to 42 families. Twenty-six likely causal variants in retinal genes were found by PS and CES. CES and WES allowed proposing two novel candidate loci (WDFY3 and a X-linked region including CITED1), both abundantly expressed in human retina according to RT-PCR and immunohistochemistry. After comparison studies, PS showed the best quality and cost values, CES and WES involved similar analytical efforts and WES presented the highest diagnostic yield. These results reinforce the relevance of panels as a first step in the diagnostic routine and suggest WES as the next strategy for unsolved cases, reserving CES for the simultaneous study of multiple conditions. Standardizing this algorithm would enhance the efficiency and equity of clinical genetics practice. Furthermore, the identified candidate genes could contribute to increase the diagnostic yield and expand the mutational spectrum in these disorders.

Unmasking Retinitis Pigmentosa complex cases by a whole genome sequencing algorithm based on open-access tools: hidden recessive inheritance and potential oligogenic variants.

BACKGROUND: Retinitis Pigmentosa (RP) is a clinically and genetically heterogeneous disorder that results in inherited blindness. Despite the large number of genes identified, only ~ 60% of cases receive a genetic diagnosis using targeted-sequencing. The aim of this study was to design a whole genome sequencing (WGS) based approach to increase the diagnostic yield of complex Retinitis Pigmentosa cases. METHODS: WGS was conducted in three family members, belonging to one large apparent autosomal dominant RP family that remained unsolved by previous studies, using Illumina TruSeq library preparation kit and Illumina HiSeq X platform. Variant annotation, filtering and prioritization were performed using a number of open-access tools and public databases. Sanger sequencing of candidate variants was conducted in the extended family members. RESULTS: We have developed and optimized an algorithm, based on the combination of different open-access tools, for variant prioritization of WGS data which allowed us to reduce significantly the number of likely causative variants pending to be manually assessed and segregated. Following this algorithm, four heterozygous variants in one autosomal recessive gene (USH2A) were identified, segregating in pairs in the affected members. Additionally, two pathogenic alleles in ADGRV1 and PDZD7 could be contributing to the phenotype in one patient. CONCLUSIONS: The optimization of a diagnostic algorithm for WGS data analysis, accompanied by a hypothesis-free approach, have allowed us to unmask the genetic cause of the disease in one large RP family, as well as to reassign its inheritance pattern which implies differences in the clinical management of these cases. These results contribute to increasing the number of cases with apparently dominant inheritance that carry causal mutations in recessive genes, as well as the possible involvement of various genes in the pathogenesis of RP in one patient. Moreover, our WGS-analysis approach, based on open-access tools, can easily be implemented by other researchers and clinicians to improve the diagnostic yield of additional patients with inherited retinal dystrophies.

Searching the second hit in patients with inherited retinal dystrophies and monoallelic variants in ABCA4, USH2A and CEP290 by whole-gene targeted sequencing.

Inherited Retinal Dystrophies are clinically and genetically heterogeneous disorders affecting the photoreceptors. Although NGS has shown to be helpful for the molecular diagnosis of these conditions, some cases remain unsolved. Among these, several individuals harboured monoallelic variants in a recessive gene, suggesting that a comprehensive screening could improve the overall diagnosis. In order to assess the contribution of non-coding variations in a cohort of 29 patients, 25 of them with monoallelic mutations, we performed targeted NGS. The design comprised the entire genomic sequence of three genes (USH2A, ABCA4 and CEP290), the coding exons of 76 genes and two disease-associated intronic regions in OFD1 and PRPF31. As a result, likely causative mutations (8 novel) were identified in 17 probands (diagnostic rate: 58.62%), including two copy-number variations in USH2A (one deletion of exons 22-55 and one duplication of exons 46-47). Possibly damaging deep-intronic mutations were identified in one family, and another with a monoallelic variant harboured causal mutations in a different locus. In conclusion, due to the high prevalence of carriers of IRD mutations and the results obtained here, sequencing entire genes do not seem to be the approach of choice for detecting the second hit in IRD patients with monoallelic variants.

Unravelling the genetic basis of simplex Retinitis Pigmentosa cases.

Retinitis Pigmentosa (RP) is the most common form of inherited retinal dystrophy (IRD) characterized ultimately by photoreceptors degeneration. Exhibiting great clinical and genetic heterogeneity, RP can be inherited as an autosomal dominant (ad), autosomal recessive (ar) and X-linked (xl) disorder. Although the relative prevalence of each form varies somewhat between populations, a major proportion (41% in Spain) of patients represent simplex cases (sRP) in which the mode of inheritance is unknown. Molecular genetic diagnostic is crucial, but also challenging, for sRP patients because any of the 81 RP genes identified to date may be causative. Herein, we report the use of a customized targeted gene panel consisting of 68 IRD genes for the molecular characterization of 106 sRP cases. The diagnostic rate was 62.26% (66 of 106) with a proportion of clinical refinements of 30.3%, demonstrating the high efficiency of this genomic approach even for clinically ambiguous cases. The high number of patients diagnosed here has allowed us to study in detail the genetic basis of the sRP. The solved sRP cohort is composed of 62.1% of arRP cases, 24.2% of adRP and 13.6% of xlRP, which implies consequences for counselling of patients and families.

Identification of epistatic interactions through genome-wide association studies in sporadic medullary and juvenile papillary thyroid carcinomas.

BACKGROUND: The molecular mechanisms leading to sporadic medullary thyroid carcinoma (sMTC) and juvenile papillary thyroid carcinoma (PTC), two rare tumours of the thyroid gland, remain poorly understood. Genetic studies on thyroid carcinomas have been conducted, although just a few loci have been systematically associated. Given the difficulties to obtain single-loci associations, this work expands its scope to the study of epistatic interactions that could help to understand the genetic architecture of complex diseases and explain new heritable components of genetic risk. METHODS: We carried out the first screening for epistasis by Multifactor-Dimensionality Reduction (MDR) in genome-wide association study (GWAS) on sMTC and juvenile PTC, to identify the potential simultaneous involvement of pairs of variants in the disease. RESULTS: We have identified two significant epistatic gene interactions in sMTC (CHFR-AC016582.2 and C8orf37-RNU1-55P) and three in juvenile PTC (RP11-648k4.2-DIO1, RP11-648k4.2-DMGDH and RP11-648k4.2-LOXL1). Interestingly, each interacting gene pair included a non-coding RNA, providing thus support to the relevance that these elements are increasingly gaining to explain carcinoma development and progression. CONCLUSIONS: Overall, this study contributes to the understanding of the genetic basis of thyroid carcinoma susceptibility in two different case scenarios such as sMTC and juvenile PTC.

Next-generation-based targeted sequencing as an efficient tool for the study of the genetic background in Hirschsprung patients.

BACKGROUND: The development of next-generation sequencing (NGS) technologies has a great impact in the human variation detection given their high-throughput. These techniques are particularly helpful for the evaluation of the genetic background in disorders of complex genetic etiology such as Hirschsprung disease (HSCR). The purpose of this study was the design of a panel of HSCR associated genes as a rapid and efficient tool to perform genetic screening in a series of patients. METHODS: We have performed NGS-based targeted sequencing (454-GS Junior) using a panel containing 26 associated or candidate genes for HSCR in a group of 11 selected HSCR patients. RESULTS: The average percentage of covered bases was of 97%, the 91.4% of the targeted bases were covered with depth above 20X and the mean coverage was 422X. In addition, we have found a total of 13 new coding variants and 11 new variants within regulatory regions among our patients. These outcomes allowed us to re-evaluate the genetic component associated to HSCR in these patients. CONCLUSIONS: Our validated NGS panel constitutes an optimum method for the identification of new variants in our patients. This approach could be used for a fast, reliable and more thorough genetic screening in future series of patients.