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Genome analysis of individuals affected by retinitis pigmentosa (RP) identified two rare nucleotide substitutions at the same genomic location on chromosome 11 (g.61392563 [GRCh38]), 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (c.-69G>A, c.-69G>T [GenBank: NM_001173991.3]), in individuals of South Asian and African ancestry, respectively. Genotypes included 71 homozygotes and 3 mixed heterozygotes in trans with a predicted loss-of-function allele. Haplotype analysis showed single-nucleotide variants (SNVs) common across families, suggesting ancestral alleles within the two distinct ethnic populations. Clinical phenotype analysis of 62 available individuals from 49 families indicated a similar clinical presentation with night blindness in the first decade and progressive peripheral field loss thereafter. No evident systemic ciliopathy features were noted. Functional characterization of these variants by luciferase reporter gene assay showed reduced promotor activity. Nanopore sequencing confirmed the lower transcription of the TMEM216 c.-69G>T allele in blood-derived RNA from a heterozygous carrier, and reduced expression was further recapitulated by qPCR, using both leukocytes-derived RNA of c.-69G>T homozygotes and total RNA from genome-edited hTERT-RPE1 cells carrying homozygous TMEM216 c.-69G>A. In conclusion, these variants explain a significant proportion of unsolved cases, specifically in individuals of African ancestry, suggesting that reduced TMEM216 expression might lead to abnormal ciliogenesis and photoreceptor degeneration.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

There have been major advances in the armamentarium for hepatocellular carcinoma (HCC) since the last official update of the Barcelona Clinic Liver Cancer prognosis and treatment strategy published in 2018. Whilst there have been advances in all areas, we will focus on those that have led to a change in strategy and we will discuss why, despite being encouraging, data for select interventions are still too immature for them to be incorporated into an evidence-based model for clinicians and researchers. Finally, we describe the critical insight and expert knowledge that are required to make clinical decisions for individual patients, considering all of the parameters that must be considered to deliver personalised clinical management.

Pathogenic variants in the ATP-binding cassette transporter A4 (ABCA4) gene cause a continuum of retinal disease phenotypes, including Stargardt disease. Noncanonical splice site (NCSS) and deep-intronic variants constitute a large fraction of disease-causing alleles, defining the functional consequences of which remains a challenge. We aimed to determine the effect on splicing of nine previously reported or unpublished NCSS variants, one near exon splice variant and nine deep-intronic variants in ABCA4, using in vitro splice assays in human embryonic kidney 293T cells. Reverse transcription-polymerase chain reaction and Sanger sequence analysis revealed splicing defects for 12 out of 19 variants. Four deep-intronic variants create pseudoexons or elongate the upstream exon. Furthermore, eight NCSS variants cause a partial deletion or skipping of one or more exons in messenger RNAs. Among the 12 variants, nine lead to premature stop codons and predicted truncated ABCA4 proteins. At least two deep-intronic variants affect splice enhancer and silencer motifs and, therefore, these conserved sequences should be carefully evaluated when predicting the outcome of NCSS and deep-intronic variants.

The aim of the study was to analyze the association between the liver uptake of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA) in the hepatobiliary phase (HBP) in cirrhotic patients and the presence of clinically significant portal hypertension (CSPH), and how these features impact on hepatocellular carcinoma (HCC) detection in the HBP.

Introduction: Long-read sequencing (LRS) enables accurate structural variant detection and variant phasing. When a molecular diagnosis is suspected, target enrichment can reduce the cost and duration of sequencing. Methods: LRS was conducted in five inherited retinal dystrophy (IRD) patients harboring a monoallelic variant in RPE65 that remained uncharacterized after clinical exome sequencing (CES). CRISPR-Cas9 guide RNA probes were designed to target a 31 kb region, including the entire RPE65 locus. The DNA was sequenced on a MinION platform. Short-read ×30 whole-genome sequencing (WGS) was performed for five patients to validate nanopore results. Results: The nanopore sequencing process yielded a median of 271 reads within the targeted region, with a mean depth of 109 and a median read size of 8 kb. All variants identified by CES have been detected using this approach, and no additional RPE65 gene causative variants were found. Nanopore variant detection demonstrated performance akin to short-read WGS at similar coverage levels, although exhibiting increased false positive calls at lower coverage. Discussion: In this study, we explore the advantages of using a targeted approach together with long-read sequencing to identify variants associated with IRD. The results underscore the utility of targeted long reads for characterizing patients affected by rare diseases when first-tier diagnostic tests are non-conclusive. [ABSTRACT FROM AUTHOR]

Duodeno-duodenostomy (DD) has been proposed as a more physiological alternative to conventional duodeno-jejunostomy (DJ) for pancreas transplantation. Accessibility of percutaneous biopsies in these grafts has not yet been assessed. We conducted a retrospective study including all pancreatic percutaneous graft biopsies requested between November 2009 and July 2021. Whenever possible, biopsies were performed under ultrasound (US) guidance or computed tomography (CT) guidance when the US approach failed. Patients were classified into two groups according to surgical technique (DJ and DD). Accessibility, success for histological diagnosis and complications were compared. Biopsy was performed in 93/136 (68.4%) patients in the DJ group and 116/132 (87.9%) of the DD group (p = 0.0001). The graft was not accessible for biopsy mainly due to intestinal loop interposition (n = 29 DJ, n = 10 DD). Adequate sample for histological diagnosis was obtained in 86/93 (92.5%) of the DJ group and 102/116 (87.9%) of the DD group (p = 0.2777). One minor complication was noted in the DD group. The retrocolic position of the DD pancreatic graft does not limit access to percutaneous biopsy. This is a safe technique with a high histological diagnostic success rate. [ABSTRACT FROM AUTHOR]

Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action. [ABSTRACT FROM AUTHOR]

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses. The entire 140 kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation revealing, on average, 200 intronic variants per sample. Filtering of these data resulted in 141 candidates for new mutations. Two variants were detected in four samples, two in three samples, and 20 variants in two samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 12 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. No copy number variation (large deletions and insertions) was detected in any patient suggesting that it is a very rare event in the ABCA4 locus. Many variants were excluded since they were not conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants. The sequence variability in the ABCA4 locus is extensive and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Defining disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and extensive analyses by a combination of various approaches.

Introduction: The incidence of hepatocellular carcinoma (HCC) in Budd-Chiari syndrome (BCS) is unknown and there is no validated diagnostic work-up to define the liver nodules with arterial phase hyperenhancement (APHE), suggesting malignancy. This prospective study evaluates HCC incidence in a Western cohort of patients with BCS and assesses the performance of MRI with hepatobiliary contrast (HB-MRI) for nodule characterization. Methods: Patients with BCS followed in our hospital were prospectively evaluated by MRI with extracellular contrast (EC-MRI). Nodules with APHE categorized as non-conclusively benign by 2 radiologists were studied by HB-MRI and reviewed by 2 radiologists blinded to the EC-MRI results. A new EC-MRI 1 year later and clinical, analytical, and sonographic follow-up every 6 months for a median of 10 years was performed. Results: A total of 55 non-conclusively benign nodules with APHE were detected at EC-MRI in 41 patients. While 32 of them were suggestive of HCC by EC-MRI, all the 55 nodules showed increased uptake of hepatobiliary contrast. An unequivocal central scar was seen in 12/55 nodules at HB-MRI regardless of it was not detected on the EC-MRI. None of the nodules was hypointense in the hepatobiliary phase (HBP). HCC was not detected during a median of 10 years of follow-up. Conclusions: Detection of nodules with APHE is frequent in patients with BCS, but HCC is rare in Western patients with BCS. While EC-MRI may detect nodules suggesting malignancy, the identification of contrast uptake in the HBP at HB-MRI may help categorize them as benign. [ABSTRACT FROM AUTHOR]

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants., Introduction SARS-CoV-2 continues its diversification worldwide, and a new variant termed Omicron (B.l.1.529), carrying a large number of mutations, was recently described in South Africa and classified as a potential [...]