ABSTRACT
Clinical research should conform to high standards of ethical and scientific integrity, given that human lives are at stake. However, economic incentives can generate conflicts of interest for investigators, who may be inclined to withhold unfavorable results or even tamper with data in order to achieve desired outcomes. To shed light on the integrity of clinical trial results, this paper systematically analyzes the distribution of P values of primary outcomes for phase II and phase III drug trials reported to the ClinicalTrials.gov registry. First, we detect no bunching of results just above the classical 5% threshold for statistical significance. Second, a density-discontinuity test reveals an upward jump at the 5% threshold for phase III results by small industry sponsors. Third, we document a larger fraction of significant results in phase III compared to phase II. Linking trials across phases, we find that early favorable results increase the likelihood of continuing into the next phase. Once we take into account this selective continuation, we can explain almost completely the excess of significant results in phase III for trials conducted by large industry sponsors. For small industry sponsors, instead, part of the excess remains unexplained.
Subject(s)
Clinical Trials as Topic/economics , Clinical Trials as Topic/standards , Research Report/standards , Biomedical Research/economics , Clinical Trials as Topic/statistics & numerical data , Drug Development/economics , Drug Development/organization & administration , Drug Industry/economics , Humans , Registries , Research Support as TopicABSTRACT
Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.
Subject(s)
Abnormalities, Multiple/genetics , Carrier Proteins/genetics , Cerebellum/abnormalities , Ciliopathies/genetics , Eye Abnormalities/genetics , Kidney Diseases, Cystic/genetics , Mutation/genetics , Nuclear Proteins/genetics , Retina/abnormalities , Abnormalities, Multiple/diagnostic imaging , Abnormalities, Multiple/pathology , Adolescent , Animals , Cerebellum/diagnostic imaging , Cerebellum/pathology , Child , Child, Preschool , Cilia/metabolism , Cilia/pathology , Ciliopathies/diagnostic imaging , Ciliopathies/pathology , Eye Abnormalities/diagnostic imaging , Eye Abnormalities/pathology , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Homozygote , Humans , Infant , Infant, Newborn , Kidney Diseases, Cystic/diagnostic imaging , Kidney Diseases, Cystic/pathology , Magnetic Resonance Imaging , Male , Pedigree , Phenotype , Retina/diagnostic imaging , Retina/pathology , Smoothened Receptor/metabolism , Young Adult , Zebrafish/geneticsABSTRACT
PurposeHearing loss is genetically extremely heterogeneous, making it suitable for next-generation sequencing (NGS). We identified a four-generation family with nonsyndromic mild to severe hearing loss of the mid- to high frequencies and onset from early childhood to second decade in seven members.MethodsNGS of 66 deafness genes, Sanger sequencing, genome-wide linkage analysis, whole-exome sequencing (WES), semiquantitative reverse-transcriptase polymerase chain reaction.ResultsWe identified a heterozygous nonsense mutation, c.6881G>A (p.Trp2294*), in the last coding exon of PTPRQ. PTPRQ has been linked with recessive (DFNB84A), but not dominant deafness. NGS and Sanger sequencing of all exons (including alternatively spliced 5' and N-scan-predicted exons of a putative "extended" transcript) did not identify a second mutation. The highest logarithm of the odds score was in the PTPRQ-containing region on chromosome 12, and p.Trp2294* cosegregated with hearing loss. WES did not identify other cosegregating candidate variants from the mapped region. PTPRQ expression in patient fibroblasts indicated that the mutant allele escapes nonsense-mediated decay (NMD).ConclusionKnown PTPRQ mutations are recessive and do not affect the C-terminal exon. In contrast to recessive loss-of-function mutations, c.6881G>A transcripts may escape NMD. PTPRQTrp2294* protein would lack only six terminal residues and could exert a dominant-negative effect, a possible explanation for allelic deafness, DFNA73, clinically and genetically distinct from DFNB84A.
Subject(s)
Deafness/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 3/genetics , Amino Acid Sequence , Codon, Nonsense/genetics , Exome/genetics , Exons/genetics , Family , Female , Genetic Linkage , Genome-Wide Association Study , Hearing Loss/genetics , Humans , Male , Mutation , Pedigree , Exome SequencingABSTRACT
Mutations affecting the integrity and function of cilia have been identified in various genes over the last decade accounting for a group of diseases called ciliopathies. Ciliopathies display a broad spectrum of phenotypes ranging from mild manifestations to lethal combinations of multiple severe symptoms and most of them share cystic kidneys as a common feature. Our starting point was a consanguineous pedigree with three affected fetuses showing an early embryonic phenotype with enlarged cystic kidneys, liver and pancreas and developmental heart disease. By genome-wide linkage analysis, we mapped the disease locus to chromosome 17q11 and identified a homozygous nonsense mutation in NEK8/NPHP9 that encodes a kinase involved in ciliary dynamics and cell cycle progression. Missense mutations in NEK8/NPHP9 have been identified in juvenile cystic kidney jck mice and in patients suffering from nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. This work confirmed a complete loss of NEK8 expression in the affected fetuses due to nonsense-mediated decay. In cultured fibroblasts derived from these fetuses, the expression of prominent polycystic kidney disease genes (PKD1 and PKD2) was decreased, whereas the oncogene c-MYC was upregulated, providing potential explanations for the observed renal phenotype. We furthermore linked NEK8 with NPHP3, another NPH protein known to cause a very similar phenotype in case of null mutations. Both proteins interact and activate the Hippo effector TAZ. Taken together, our study demonstrates that NEK8 is essential for organ development and that the complete loss of NEK8 perturbs multiple signalling pathways resulting in a severe early embryonic phenotype.
Subject(s)
Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Dandy-Walker Syndrome/genetics , Dandy-Walker Syndrome/metabolism , Gene Expression Regulation , Mutation , Pancreatic Cyst/genetics , Pancreatic Cyst/metabolism , Protein Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction , Abnormalities, Multiple/pathology , Cell Line , Consanguinity , Dandy-Walker Syndrome/pathology , Female , Fetus/abnormalities , Gene Frequency , Genome-Wide Association Study , Genotype , Hippo Signaling Pathway , Humans , Male , NIMA-Related Kinases , Pancreatic Cyst/pathology , Pedigree , Polymorphism, Single Nucleotide , Protein Binding , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolismABSTRACT
MYO1A is considered the gene underlying autosomal dominant nonsyndromic hearing loss DFNA48, based on six missense variants, one small in-frame insertion, and one nonsense mutation. Results from NGS targeting 66 deafness genes in 109 patients identified three families challenging this assumption: two novel nonsense (p.Tyr740* and p.Arg262*) and a known missense variant were identified heterozygously not only in index patients, but also in unaffected relatives. Deafness in these families clearly resulted from mutations in other genes (MYO7A, EYA1, and CIB2). Most of the altogether 10 MYO1A mutations are annotated in dbSNP, and population frequencies (dbSNP, 1000 Genomes, Exome Sequencing Project) above 0.1% contradict pathogenicity under a dominant model. One healthy individual was even homozygous for p.Arg262*, compatible with homozygous Myo1a knockout mice lacking any overt pathology. MYO1A seems dispensable for hearing and overall nonessential. MYO1A adds to the list of "erroneous disease genes", which will expand with increasing availability of large-scale sequencing data.
Subject(s)
Genetic Predisposition to Disease , Hearing Loss, Sensorineural/genetics , Mutation, Missense , Myosin Heavy Chains/genetics , Myosin Type I/genetics , Adolescent , Adult , Animals , Child , Child, Preschool , Databases, Genetic , Female , Hearing Loss, Sensorineural/etiology , Hearing Loss, Sensorineural/pathology , Humans , Infant , Mice , Mice, Knockout , Middle Aged , Pedigree , Polymorphism, Single NucleotideABSTRACT
BACKGROUND: An emerging number of clinically and genetically heterogeneous diseases now collectively termed ciliopathies have been connected to the dysfunction of primary cilia. We describe an 8-year-old girl with a complex phenotype that did not clearly match any familiar syndrome. CASE-DIAGNOSIS/TREATMENT: Hypotonia, facial dysmorphism and retardation were noted shortly after birth. Other features included short stature, mild skeletal anomalies, strabism, deafness, subdural hygroma, hepatosplenomegaly and end-stage renal failure. Renal biopsy revealed tubular atrophy, interstitial fibrosis and segmental glomerulosclerosis. After exclusion of a chromosomal abnormality by array-comparative genomic hybridization (CGH), we performed next-generation sequencing (NGS) using a customized panel that targeted 131 genes known or hypothesized to cause ciliopathies. We identified the novel homozygous WDR19 mutation c.1483G > C (p.Gly495Arg) that affects an evolutionarily highly conserved residue in the intraflagellar transport protein IFT144, is absent from databases and is predicted to be pathogenic by all bioinformatic sources used. CONCLUSION: Mutations in WDR19 encoding the intraflagellar transport component IFT144 have recently been described in single families with the clinically overlapping skeletal ciliopathies Jeune and Sensenbrenner syndromes, combined or isolated nephronophthisis (NPHP) and retinitis pigmentosa (RP) (Senior-Loken syndrome). Our patient emphasizes the usefulness and efficiency of a comprehensive NGS panel approach in patients with unclassified ciliopathies. It further suggests that WDR19 mutations can cause a broad spectrum of ciliopathies that extends to Jeune and Sensenbrenner syndromes, RP and renal NPHP-like phenotypes.
Subject(s)
Cilia/pathology , Kidney Diseases/genetics , Proteins/genetics , Child , Cytoskeletal Proteins , Exons/genetics , Female , Growth/physiology , Homozygote , Humans , Intracellular Signaling Peptides and Proteins , Kidney Diseases/pathology , Mutation/genetics , Sequence Analysis, DNAABSTRACT
Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.
Subject(s)
Biological Transport/genetics , Cilia/metabolism , Kidney Diseases/genetics , Mutation , Animals , Cerebellar Ataxia/genetics , Child , Cohort Studies , Disease Progression , Exome , Humans , Kidney Diseases/pathology , Male , Mice , Retinitis Pigmentosa/geneticsABSTRACT
OBJECTIVE AND AIM: Severe cases of bone atrophy in the maxilla or mandible are often reconstructed using bone from extraoral donor sides. Most commonly, grafts from the iliac crest are used for augmentation, however, frequently associated with bone resorption as possible late complication. Calvarial bone grafts, often reported to show less resorption, are an alternative. The aim of this study was to compare the bone stability of vertical bone grafts from the iliac crest and the calvarium. PATIENTS AND METHODS: Twenty-three patients receiving vertical onlay bone grafts were included in this retrospective cohort study. In nine patients alveolar ridge defects were treated with bone from the iliac crest. Fourteen patients were reconstructed using calvarial bone grafts. To quantify bone resorption, the data of digital panographs were evaluated. Radiographs were taken prior to bone grafting, after augmentation surgery, 6 months after bone healing, prior to implant surgery, after implant surgery and at yearly intervals thereafter. RESULTS: Postoperative complications at the recipient site occurred equally in both groups. The complication rate was 35.7% for the calvarial group and 33.3% in the iliac crest group. No donor-site complications were reported in either group. After bone augmentation procedure, a mean vertical bone gain of 8.55 mm (SD 5.96) was measured. Bone grafts from the iliac crest showed a significantly higher bone loss of 24.16% (SD 8.47) than grafts from the calvarium (8.44%, SD 3.64) at the time of implant placement (P = 0.0003). Implant survival was similar in both groups. DISCUSSION: Both bone-grafting approaches are successful and reliable techniques, enabling implant placement in even highly atrophied alveolar ridges and with identical implant survival rates, although bone resorption differs. Within the limitations of this study bone from the calvarium shows higher bone stability in the early healing phase.
Subject(s)
Alveolar Bone Loss/etiology , Alveolar Ridge Augmentation/methods , Autografts/transplantation , Bone Transplantation/classification , Postoperative Complications , Transplant Donor Site/surgery , Adult , Aged , Cohort Studies , Dental Implantation, Endosseous/instrumentation , Dental Implants , Female , Follow-Up Studies , Humans , Ilium/surgery , Image Processing, Computer-Assisted/methods , Male , Mandibular Diseases/surgery , Maxillary Diseases/surgery , Middle Aged , Radiography, Panoramic/methods , Plastic Surgery Procedures/methods , Retrospective Studies , Skull/surgery , Survival Analysis , Tomography, X-Ray Computed/methodsABSTRACT
The Bitcoin network has scalability problems. To increase its transaction rate and speed, micropayment channel networks have been proposed; however, these require to lock funds into specific channels. Moreover, the available space in the blockchain does not allow scaling to a worldwide payment system. We propose a new layer that sits in between the blockchain and the payment channels. The new layer addresses the scalability problem by enabling trustless off-blockchain channel funding. It consists of shared accounts of groups of nodes that flexibly create one-to-one channels for the payment network. The new system allows rapid changes of the allocation of funds to channels and reduces the cost of opening new channels. Instead of one blockchain transaction per channel, each user only needs one transaction to enter a group of nodes-within the group the user can create arbitrarily many channels. For a group of 20 users with 100 intra-group channels, the cost of the blockchain transactions is reduced by 90% compared to 100 regular micropayment channels opened on the blockchain. This can be increased further to 96% if Bitcoin introduces Schnorr signatures with signature aggregation.
ABSTRACT
BACKGROUND: Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). METHODS: Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. RESULTS: A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3, genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. CONCLUSION: Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.
ABSTRACT
The authors describe a 58-year-old man with sudden onset of a unilateral tremor caused by a midbrain lesion that affected the substantia nigra and the cerebellothalamic pathway. There were also clinical and neuroimaging signs of a communicating chronic hydrocephalus. The patient was severely handicapped by this tremor, which was a typical Holmes tremor with rest, posture, and intention components. Parkinson disease or multiple-system atrophy as causes for the tremor could be ruled out by DaTSCAN and 123I iodobenzamide and single-photon emission computerized tomography (SPECT), respectively. The tremor was completely supressed by temporary and permanent cerebrospinal fluid release after ventriculoperitoneal shunt placement, without any additional medication, for a period of 6 months. Afterward, the tremor returned, and the patient had to be treated by a stereotactic electrode implantation in the contralateral ventralis intermedius nucleus, which led to complete tremor suppression during the 1.5-year follow-up period. In this case report, the authors present the clinical description and the electrophysiological, SPECT, and magnetic resonance imaging data of a rare combination of symptoms and their surgical treatment.
Subject(s)
Brain Ischemia/complications , Cerebral Ventricles/pathology , Hydrocephalus/complications , Hydrocephalus/etiology , Tremor/etiology , Electrophysiology , Humans , Hydrocephalus/therapy , Magnetic Resonance Imaging , Male , Middle Aged , Recurrence , Tomography, Emission-Computed, Single-Photon , Treatment Outcome , Ventriculoperitoneal ShuntABSTRACT
Renal cysts are clinically and genetically heterogeneous conditions. Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent life-threatening genetic disease and mainly caused by mutations in PKD1. The presence of six PKD1 pseudogenes and tremendous allelic heterogeneity make molecular genetic testing challenging requiring laborious locus-specific amplification. Increasing evidence suggests a major role for PKD1 in early and severe cases of ADPKD and some patients with a recessive form. Furthermore it is becoming obvious that clinical manifestations can be mimicked by mutations in a number of other genes with the necessity for broader genetic testing. We established and validated a sequence capture based NGS testing approach for all genes known for cystic and polycystic kidney disease including PKD1. Thereby, we demonstrate that the applied standard mapping algorithm specifically aligns reads to the PKD1 locus and overcomes the complication of unspecific capture of pseudogenes. Employing careful and experienced assessment of NGS data, the method is shown to be very specific and equally sensitive as established methods. An additional advantage over conventional Sanger sequencing is the detection of copy number variations (CNVs). Sophisticated bioinformatic read simulation increased the high analytical depth of the validation study and further demonstrated the strength of the approach. We further raise some awareness of limitations and pitfalls of common NGS workflows when applied in complex regions like PKD1 demonstrating that quality of NGS needs more than high coverage of the target region. By this, we propose a time- and cost-efficient diagnostic strategy for comprehensive molecular genetic testing of polycystic kidney disease which is highly automatable and will be of particular value when therapeutic options for PKD emerge and genetic testing is needed for larger numbers of patients.
Subject(s)
Molecular Diagnostic Techniques/methods , Polycystic Kidney Diseases/diagnosis , Polycystic Kidney Diseases/genetics , DNA Copy Number Variations , False Negative Reactions , Gene Duplication , Genetic Loci/genetics , High-Throughput Nucleotide Sequencing , Humans , Kidney/metabolism , Protein Serine-Threonine Kinases/genetics , Pseudogenes/genetics , Pyruvate Dehydrogenase Acetyl-Transferring KinaseABSTRACT
OBJECTIVES: Patients with continuous bone defects of the mandible after ablative tumor surgery need bony reconstruction for proper function and aesthetics. Free microvascular reanastomized bone grafts provide a clinically proven option for such patients, yet the optimal source of donor tissue has not yet been established. The aim of this study was to evaluate and compare the bone volume stability of vascularized bone grafts, particularly in the early highly resorptive phase, from the iliac crest (DCIA) and the fibula and to assess the implantologic rehabilitations. MATERIALS AND METHODS: Thirty-six patients with mandibular continuity defects due to tumor resection were reconstructed by the use of vascularized bone grafts; 21 patients received DCIA flaps and 15 patients received a composite free fibular flap, depending on the size and location of the defect. Bone resorption was assessed using digital panographs. Radiographs were taken immediately after bone reconstruction, 6 months postoperatively, prior to implant surgery, and at prosthetic loading. RESULTS: After a mean observation period of 6 months, vertical bone resorption was 6.79% for the patients of the iliac crest group (DCIA), 10.20% after 11 months, and 12.58% after 17 months. Fibular grafts showed a bone resorption of 5.30% after a mean observation time of 6 months, 8.26% after 11 months, and 16.95% after 17 months. Eighteen patients received 71 implants for implant-retained dental reconstructions. CONCLUSIONS: Microvascular reanastomized bone grafts represent a reliable treatment option for reconstruction in cases of large defects of the mandible, with low graft resorption in the early healing phase. Additionally, the compared grafts provide sufficient bone volume to permit implant rehabilitation.
Subject(s)
Bone Resorption/etiology , Bone Transplantation/methods , Fibula/physiology , Graft Survival , Ilium/physiology , Mandibular Reconstruction/methods , Transplant Donor Site/physiology , Anastomosis, Surgical/methods , Autografts/transplantation , Cohort Studies , Dental Implantation, Endosseous/methods , Female , Fibula/surgery , Follow-Up Studies , Free Tissue Flaps/transplantation , Gingiva/transplantation , Humans , Ilium/surgery , Male , Mandibular Neoplasms/surgery , Radiography, Panoramic/methods , Retrospective Studies , Scapula/physiology , Scapula/surgery , Skin Transplantation/methods , Transplant Donor Site/surgery , Treatment Outcome , Vestibuloplasty/methodsABSTRACT
OBJECTIVE: A great benefit of FRC technology is that, in case of minor failure events, restorations can be repaired or reinserted. However, various FRC materials are available, that differ in matrix composition and fiber pre-treatment. The aim of this investigation was, therefore, to evaluate original and repair bond strength of FRC materials. METHODS: Five fully pre-impregnated, unidirectional FRCs were selected, one semi-interpenetrating polymer network FRC and four cross-linked-polymer FRCs. The primary endpoint was the evaluation of shear bond strength (SBS) between FRC and composite resin, which was performed by a universal testing machine. For each FRC specimens were divided into control (original SBS, resin to fresh FRC with oxygen inhibition layer (OIL), n=30) and test groups (repair SBS, resin to FRC after removal of OIL and adhesive infiltration, n=30). RESULTS: The cross-linked-polymer FRC GrandTec(®) (12.4±5.4 MPa) yielded the highest control SBS, followed by the semi-interpenetrating polymer network FRC (everStick(®), 9.2±3.5 MPa). With everStick(®), repair led to a significant increase in the test SBS (14.6±5.8 MPa, p=0.01). SIGNIFICANCE: Control SBS was best with GrandTec(®) indicating that the material is superior in direct clinical application. Test SBS was significantly increased with everStick(®) which points at potential reparability and advantages in semi-direct or indirect fabrication of fiber-reinforced fixed partial dentures.
Subject(s)
Bisphenol A-Glycidyl Methacrylate/chemistry , Composite Resins/chemistry , Methacrylates/chemistry , Dental Materials/chemistry , Dental Stress Analysis , In Vitro Techniques , Light-Curing of Dental Adhesives , Shear StrengthABSTRACT
Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.
Subject(s)
DNA Copy Number Variations , Exons/genetics , High-Throughput Nucleotide Sequencing , Retinal Dystrophies/genetics , Sequence Analysis, DNA , Adolescent , Adult , Child , Child, Preschool , Female , Heterozygote , Humans , Male , Middle Aged , Mutation , Pedigree , Retinal Dystrophies/diagnosis , Young AdultABSTRACT
BACKGROUND: Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family. METHODS: Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. RESULTS: Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype. CONCLUSION: Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis.
Subject(s)
Codon, Nonsense , Homozygote , Monoacylglycerol Lipases/genetics , Sequence Analysis, DNA/methods , Usher Syndromes/genetics , Female , Genetic Linkage , Humans , Male , Pedigree , Polymorphism, Single NucleotideABSTRACT
OBJECTIVE: Deep brain stimulation (DBS) is widely accepted in the treatment of advanced Parkinson's disease (PD) and other movement disorders. The standard implantation procedure is performed under local anesthesia (LA). Certain groups of patients may not be eligible for surgery under LA because of clinical reasons, such as massive fear, reduced cooperativity, or coughing attacks. Microrecording (MER) has been shown to be helpful in DBS surgery. The purpose of this study was to evaluate the feasibility of MER for DBS surgery under general anesthesia (GA) and to compare the data of intraoperative MER as well as the clinical data with that of the current literature of patients undergoing operation under LA. CLINICAL PRESENTATION: The data of nine patients with advanced PD (mean Hoehn and Yahr status, 4.2) who were operated with subthalamic nucleus (STN) DBS under GA, owing to certain clinical circumstances ruling out DBS under LA, were retrospectively analyzed. All operations were performed under analgosedation with propofol or remifentanil and intraoperative MER. For MER, remifentanil was ceased completely and propofol was lowered as far as possible. INTERVENTION: The STN could be identified intraoperatively in all patients with MER. The typical bursting pattern was identified, whereas a widening of the baseline noise could not be as adequately detected as in patients under LA. The daily off phases of the patients were reduced from 50 to 17%, whereas the Unified Parkinson's Disease Rating Scale III score was reduced from 43 (preoperative, medication off) to 19 (stimulation on, medication off) and 12 (stimulation on, medication on). Two patients showed a transient neuropsychological deterioration after surgery, but both also had preexisting episodes of disorientation. One implantable pulse generator infection was noticed. No further significant clinical complications were observed. CONCLUSION: STN surgery for advanced PD with MER guidance is possible with good clinical results under GA. Intraoperative MER of the STN region can be performed under GA with a special anesthesiological protocol. In this setting, the typical STN bursting pattern can be identified, whereas the typical widening of the background noise baseline while entering the STN region is obviously absent. This technique may enlarge the group of patients eligible for STN surgery. Although the clinical improvements and parameter settings in this study were within the range of the current literature, further randomized controlled studies are necessary to compare the results of STN DBS under GA and LA, respectively.