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PURPOSE: We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory. METHODS: WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases. RESULTS: The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56 American College of Medical Genetics and Genomics-recommended genes, 6.2% (N = 129) had reportable pathogenic variants. In addition to cases with a definitive diagnosis, in 24.2% of cases a candidate gene was reported that may later be reclassified as being associated with a definitive diagnosis. CONCLUSION: Our experience with our first 3,040 WES cases suggests that analysis of trios significantly improves the diagnostic yield compared with proband-only testing for genetically heterogeneous disorders and facilitates identification of novel candidate genes.Genet Med 18 7, 696-704.
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Enfermedades Genéticas Congénitas/genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Exoma/genética , Enfermedades Genéticas Congénitas/clasificación , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/epidemiología , Humanos , Mutación , Análisis de Secuencia de ADN/métodosRESUMEN
PURPOSE: To describe the clinical, functional, and genetic findings in a young Caucasian girl and her father, in whom a mutation of the PAX6 gene was identified. METHODS: Detailed histories, eye examinations, and flash electroretinograms (ERGs) were acquired from both patients, and molecular genetic diagnostic testing was performed. Both patients were followed over a 2-year period. RESULTS: At presentation, the proband displayed congenital nystagmus, photophobia, posterior embryotoxon, foveal hypoplasia, and coarse peripheral retinal pigment epithelium mottling. Light-adapted cone-driven ERG responses were delayed and reduced. The father had similar findings, but additionally displayed corneal clouding and pannus, decreased best-corrected visual acuity, and his ERG demonstrated a larger reduction in ERG cone-driven responses. PAX6 testing of the proband revealed a heterozygous mutation in exon 13 resulting in a p.X423Lfs (p.Stop423Leufs) frameshift amino acid substitution, predicting aberrant protein elongation by either 14 or 36 amino acids (p.X423Lext14 or p.X423Lext36) and subsequent disruption of normal protein function. CONCLUSIONS: The p.X423Lfs mutation has previously been described in cases of atypical aniridia, but this is the first report demonstrating abnormal cone-driven ERG responses associated with this particular mutation of the PAX6 gene. ERG abnormalities have been documented in other mutations of the PAX6 gene, and we propose that the retinal pathology causing these ERG abnormalities may contribute to the photophobia experienced by patients with aniridia. Systematic ERG testing can aid in the diagnosis of PAX6-related disorders and may prove to be a useful tool to objectively assess responses to future treatments.
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Electrorretinografía , Proteínas del Ojo/genética , Mutación del Sistema de Lectura , Proteínas de Homeodominio/genética , Nistagmo Congénito/fisiopatología , Factores de Transcripción Paired Box/genética , Fotofobia/fisiopatología , Proteínas Represoras/genética , Células Fotorreceptoras Retinianas Conos/fisiología , Enfermedades de la Retina/fisiopatología , Femenino , Heterocigoto , Humanos , Lactante , Masculino , Nistagmo Congénito/genética , Factor de Transcripción PAX6 , Linaje , Estimulación Luminosa , Fotofobia/genética , Enfermedades de la Retina/genética , Adulto JovenRESUMEN
Recognition of the gene implicated in a Mendelian disorder subsequently leads to an expansion of potential phenotypes associated with mutations in that gene as patients with features beyond the core phenotype are identified by sequencing. Here, we present a young girl with developmental delay, short stature despite a markedly advanced bone age, hypertrichosis without elbow hair, renal anomalies, and dysmorphic facial features, found to have a heterozygous, de novo, intragenic deletion encompassing exons 2-10 of the KMT2A (MLL) gene detected by whole exome sequencing. Heterozygous mutations in this gene were recently demonstrated to cause Wiedemann-Steiner syndrome (OMIM 605130). Importantly, retrospective analysis of this patient's chromosomal microarray revealed decreased copy number of two probes corresponding to exons 2 and 9 of the KMT2A gene, though this result was not reported by the testing laboratory in keeping with standard protocols for reportable size cutoffs for array comparative genomic hybridization. This patient expands the clinical phenotype associated with mutations in KMT2A to include variable patterns of hypertrichosis and a significantly advanced bone age with premature eruption of the secondary dentition despite her growth retardation. This patient also represents the first report of Wiedemann-Steiner syndrome due to an exonic deletion, supporting haploinsufficiency as a causative mechanism. Our patient also illustrates the need for sensitive guidelines for the reporting of chromosomal microarray findings that are below traditional reporting size cutoffs, but that impact exons or other genomic regions of known function.
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Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/genética , Exones , Proteína de la Leucemia Mieloide-Linfoide/genética , Eliminación de Secuencia , Preescolar , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Facies , Femenino , Mano/diagnóstico por imagen , Heterocigoto , Humanos , Fenotipo , Radiografía , SíndromeRESUMEN
X-linked Alport syndrome is a form of progressive renal failure caused by pathogenic variants in the COL4A5 gene. More than 700 variants have been described and a further 400 are estimated to be known to individual laboratories but are unpublished. The major genetic testing laboratories for X-linked Alport syndrome worldwide have established a Web-based database for published and unpublished COL4A5 variants ( https://grenada.lumc.nl/LOVD2/COL4A/home.php?select_db=COL4A5 ). This conforms with the recommendations of the Human Variome Project: it uses the Leiden Open Variation Database (LOVD) format, describes variants according to the human reference sequence with standardized nomenclature, indicates likely pathogenicity and associated clinical features, and credits the submitting laboratory. The database includes non-pathogenic and recurrent variants, and is linked to another COL4A5 mutation database and relevant bioinformatics sites. Access is free. Increasing the number of COL4A5 variants in the public domain helps patients, diagnostic laboratories, clinicians, and researchers. The database improves the accuracy and efficiency of genetic testing because its variants are already categorized for pathogenicity. The description of further COL4A5 variants and clinical associations will improve our ability to predict phenotype and our understanding of collagen IV biochemistry. The database for X-linked Alport syndrome represents a model for databases in other inherited renal diseases.
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Colágeno Tipo IV/genética , Bases de Datos de Ácidos Nucleicos , Nefritis Hereditaria/genética , Humanos , FenotipoRESUMEN
BACKGROUND AND AIMS: Anti-tumour necrosis factor [anti-TNF] therapy is widely used for the treatment of inflammatory bowel disease, yet many patients are primary non-responders, failing to respond to induction therapy. We aimed to identify blood gene expression differences between primary responders and primary non-responders to anti-TNF monoclonal antibodies [infliximab and adalimumab], and to predict response status from blood gene expression and clinical data. METHODS: The Personalised Anti-TNF Therapy in Crohn's Disease [PANTS] study is a UK-wide prospective observational cohort study of anti-TNF therapy outcome in anti-TNF-naive Crohn's disease patients [ClinicalTrials.gov identifier: NCT03088449]. Blood gene expression in 324 unique patients was measured by RNA-sequencing at baseline [week 0], and at weeks 14, 30, and 54 after treatment initiation [total sample sizeâ =â 814]. RESULTS: After adjusting for clinical covariates and estimated blood cell composition, baseline expression of major histocompatibility complex, antigen presentation, myeloid cell enriched receptor, and other innate immune gene modules was significantly higher in anti-TNF responders vs non-responders. Expression changes from baseline to week 14 were generally of consistent direction but greater magnitude [i.e. amplified] in responders, but interferon-related genes were upregulated uniquely in non-responders. Expression differences between responders and non-responders observed at week 14 were maintained at weeks 30 and 54. Prediction of response status from baseline clinical data, cell composition, and module expression was poor. CONCLUSIONS: Baseline gene module expression was associated with primary response to anti-TNF therapy in PANTS patients. However, these baseline expression differences did not predict response with sufficient sensitivity for clinical use.
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Enfermedad de Crohn , Humanos , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/genética , Redes Reguladoras de Genes , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Estudios Prospectivos , Inmunoterapia , Factor de Necrosis Tumoral alfaRESUMEN
Importance: The involvement of chronic inflammation in the pathogenesis of age-related macular degeneration (AMD) opens therapeutic possibilities to AMD management. Objective: To determine whether Janus kinase inhibitors (JAKis) are associated with a reduced risk of AMD development in patients with autoimmune diseases. Design, Setting, and Participants: This retrospective observational cohort study used administrative claims data from Merative MarketScan research databases (Commercial and Medicare Supplemental) and Optum Clinformatics Data Mart databases between January 1, 2010, and January 31, 2022. Patients with autoimmune diseases satisfying study eligibility criteria and who received JAKi treatment (9126 in MarketScan and 5667 in Optum) were propensity score matched (1:1) to identical numbers of study-eligible patients who received non-JAKi-based immunotherapy. Exposure: Treatment duration of 6 months or longer. Main Outcomes and Measures: Incidence rates of AMD (exudative and nonexudative) over the first 6 to 18 months of treatment were determined, and bayesian Poisson regression models were used to estimate incidence rate ratios, 95% CIs, and posterior probabilities of AMD. Results: After matching, female sex represented the majority of the patient population in both MarketScan and Optum (14â¯019/18â¯252 [76.6%] and 8563/3364 [75.2%], respectively in the JAKi patient population). More than 60% of the patient population was older than 55 years of age in both cohorts. Over the specified treatment period, a 49% relative reduction in incidence of AMD was observed among patients who received JAKi therapy (10/9126 events; adjusted incidence rate ratio [AIRR], 0.51; 95% CI, 0.19-0.90) vs those who received non-JAKi therapy (43/9126 events; AIRR, 1 [reference]) in MarketScan, and a 73% relative reduction in incidence of AMD was observed among patients who received JAKi therapy (3/5667 events; AIRR, 0.27; 95% CI, 0.03-0.74) vs those who received non-JAKi therapy (21/5667 events; AIRR, 1 [reference]) in Optum. The absolute percentage reductions were 0.36% (MarketScan) and 0.32% (Optum), favoring patients who received JAKi therapy. Posterior probabilities of the adjusted risk being less than unity were 97.6% (MarketScan) and 98.9% (Optum) for those who received JAKi therapy vs those who received non-JAKi therapy in MarketScan and Optum, respectively. Conclusions and Relevance: JAKi use may be associated with a reduced risk of incident AMD in US adults with major autoimmune diseases. The absolute percentage reduction is consistent with a potential role for JAKi in this population. Future studies with long-term follow-up are recommended to investigate the association between JAKi use and incident AMD in other disease indications. Investigation into the role of systemic inflammation and JAK-signal transducers and activators of transcription signaling in AMD may improve understanding of the pathophysiology of AMD and lead to new treatment options.
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PURPOSE: X-linked juvenile retinoschisis (XLRS) is a vitreoretinal dystrophy characterized by schisis (splitting) of the inner layers of the neuroretina. Mutations within the retinoschisis (RS1) gene are responsible for this disease. The mutation spectrum consists of amino acid substitutions, splice site variations, small indels, and larger genomic deletions. Clinically, genomic deletions are rarely reported. Here, we characterize two novel full exonic deletions: one encompassing exon 1 and the other spanning exons 4-5 of the RS1 gene. We also report the clinical findings in these patients with XLRS with two different exonic deletions. METHODS: Unrelated XLRS men and boys and their mothers (if available) were enrolled for molecular genetics evaluation. The patients also underwent ophthalmologic examination and in some cases electroretinogram (ERG) recording. All the exons and the flanking intronic regions of the RS1 gene were analyzed with direct sequencing. Two patients with exonic deletions were further evaluated with array comparative genomic hybridization to define the scope of the genomic aberrations. After the deleted genomic region was identified, primer walking followed by direct sequencing was used to determine the exact breakpoints. RESULTS: Two novel exonic deletions of the RS1 gene were identified: one including exon 1 and the other spanning exons 4 and 5. The exon 1 deletion extends from the 5' region of the RS1 gene (including the promoter) through intron 1 (c.(-35)-1723_c.51+2664del4472). The exon 4-5 deletion spans introns 3 to intron 5 (c.185-1020_c.522+1844del5764). CONCLUSIONS: Here we report two novel exonic deletions within the RS1 gene locus. We have also described the clinical presentations and hypothesized the genomic mechanisms underlying these schisis phenotypes.
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Exones/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Retinosquisis/genética , Eliminación de Secuencia/genética , Niño , Rotura Cromosómica , Proteínas del Ojo/genética , Femenino , Fondo de Ojo , Pruebas Genéticas , Humanos , Masculino , Tomografía de Coherencia ÓpticaRESUMEN
BACKGROUND AND AIMS: This study assessed whether baseline triggering receptor expressed on myeloid cells [TREM-1] whole blood gene expression predicts response to anti-TNF therapy in patients with UC or CD. METHODS: TREM-1 whole blood gene expression was analysed by RNA sequencing [RNA-seq] in patients with moderately to severely active UC or CD treated with adalimumab in the Phase 3 SERENE-UC and SERENE-CD clinical trials. The predictive value of baseline TREM-1 expression was evaluated and compared according to endoscopic and clinical response vs non-response, and remission vs non-remission, at Weeks 8 and 52 [SERENE-UC], and Weeks 12 and 56 [SERENE-CD]. RESULTS: TREM-1 expression was analysed in 95 and 106 patients with UC and CD, respectively, receiving standard-dose adalimumab induction treatment. In SERENE-UC, baseline TREM-1 expression was not predictive of endoscopic response [p=0.48], endoscopic remission [p=0.53], clinical response [p=0.58] or clinical remission [p=0.79] at Week 8, or clinical response [p=0.60] at Week 52. However, an association was observed with endoscopic response [p=0.01], endoscopic remission [p=0.048], and clinical remission [p=0.04997] at Week 52. For SERENE-CD, baseline TREM-1 expression was not predictive of endoscopic response [p=0.56], endoscopic remission [p=0.33], clinical response [p=0.07], clinical remission [p=0.65] at Week 12, or endoscopic response [p=0.61], endoscopic remission [p=0.51], clinical response [p=0.62] or clinical remission [p=0.97] at Week 56. CONCLUSIONS: Baseline TREM-1 gene expression did not uniformly predict adalimumab response in SERENE clinical trials. Further research is needed to identify potential blood-based biomarkers predictive of response to anti-TNF therapy in patients with IBD.
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BACKGROUND AND AIMS: Anti-TNF treatment failure in patients with inflammatory bowel disease (IBD) is common and frequently related to low drug concentrations. In order to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy, we sought to define epigenetic biomarkers in whole blood at baseline associated with anti-TNF drug concentrations at week 14. METHODS: DNA methylation from 1,104 whole blood samples from 385 patients in the Personalised Anti-TNF Therapy in Crohn's disease (PANTS) study were assessed using the Illumina EPIC Beadchip (v1.0) at baseline, weeks 14, 30 and 54. We compared DNA methylation profiles in anti-TNF-treated patients who experienced primary non-response at week 14 and if they were assessed at subsequent time points, were not in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 94), with patients who responded at week 14 and when assessed at subsequent time points, were in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 93). RESULTS: Overall, between baseline and week 14, we observed 4,999 differentially methylated probes (DMPs) annotated to 2376 genes following anti-TNF treatment. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses.Epigenome-wide association (EWAS) analysis identified 323 DMPs annotated to 210 genes at baseline associated with higher anti-TNF drug concentrations at week 14. Of these, 125 DMPs demonstrated shared associations with other common traits (proportion of shared CpGs compared to DMPs) including body mass index (23.2%), followed by CRP (11.5%), smoking (7.4%), alcohol consumption per day (7.1%) and IBD type (6.8%). EWAS of primary non-response to anti-TNF identified 20 DMPs that were associated with both anti-TNF drug concentration and primary non-response to anti-TNF with a strong correlation of the coefficients (Spearman's rho = -0.94, p < 0.001). CONCLUSION: Baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14 to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy.
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PURPOSE: Mendelian disorders are most commonly caused by mutations identifiable by DNA sequencing. Exonic deletions and duplications can go undetected by sequencing, and their frequency in most Mendelian disorders is unknown. METHODS: We designed an array comparative genomic hybridization (CGH) test with probes in exonic regions of 589 genes. Targeted testing was performed for 219 genes in 3,018 patients. We demonstrate for the first time the utility of exon-level array CGH in a large clinical cohort by testing for 136 autosomal dominant, 53 autosomal recessive, and 30 X-linked disorders. RESULTS: Overall, 98 deletions and two duplications were identified in 53 genes, corresponding to a detection rate of 3.3%. Approximately 40% of positive findings were deletions of only one or two exons. A high frequency of deletions was observed for several autosomal dominant disorders, with a detection rate of 2.9%. For autosomal recessive disorders, array CGH was usually performed after a single mutation was identified by sequencing. Among 138 individuals tested for recessive disorders, 10.1% had intragenic deletions. For X-linked disorders, 3.5% of 313 patients carried a deletion or duplication. CONCLUSION: Our results demonstrate that exon-level array CGH provides a robust option for intragenic copy number analysis and should routinely supplement sequence analysis for Mendelian disorders.
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Hibridación Genómica Comparativa/métodos , Exones/genética , Enfermedades Genéticas Congénitas/diagnóstico , Mutación/genética , Estudios de Cohortes , Femenino , Eliminación de Gen , Dosificación de Gen , Duplicación de Gen , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Humanos , Masculino , Análisis de la Aleatorización Mendeliana , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
OBJECTIVES: To identify the frequency and distribution of familial Mediterranean fever (FMF) gene (MEFV) mutations in Tunisian patients. PATIENTS AND METHODS: This study was performed in the Genetic Department of Tunis University Hospital. A clinical diagnosis of FMF was made according to published criteria. Mutation screening of the MEFV gene was performed in the Human Genetic Laboratory of the "Faculté de Medecine de Tunis" for 8 mutations including the 5 most common known mutations M694V, V726A, M694l, M680l, and E148Q. The tests performed were polymerase chain reaction (PCR) restriction-digestion for M694V, V726A, M680l, R761H, E148Q; amplification refractory mutation system for A744S, M694l; and PCR-electrophoresis assay for l692del. RESULTS: Of the 139 unrelated patients investigated, 61 (44%) had 1 or 2 mutations. In 78 (56%) probands no mutation was identified: 28 patients were homozygous; 16 were compound-heterozygous; 2 had complex alleles; and 17 had only 1 identifiable mutation. Of the mutations, M680l, M694V, M694l, V726A, A744S, R761H, l692DEL, and E148Q accounted for 32, 27, 13, 5, 3, 1, 1, and 18%, respectively. CONCLUSION: The profile of the MEFV gene mutations in the Tunisian population is concordant with other Arab populations but with some differences. M680l is the most common mutation, while V726A, the commonest mutation among Arabs, is rare in our population.
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Proteínas del Citoesqueleto/genética , Fiebre Mediterránea Familiar/genética , Predisposición Genética a la Enfermedad , Mutación , Adolescente , Adulto , Niño , Preescolar , Análisis Mutacional de ADN , Fiebre Mediterránea Familiar/etnología , Fiebre Mediterránea Familiar/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pirina , Túnez/epidemiologíaRESUMEN
PURPOSE: To study Bardet-Biedl syndrome (BBS) in the Tunisian population and determine the presence of triallelism in the eight identified BBS genes. METHODS: DNA samples were collected from 19 consanguineous Tunisian families with BBS. Genome-wide scans were performed with microsatellite markers in 12 families, and two-point linkage analyses were performed. Direct sequencing was used to screen patients with BBS for mutations in all eight identified BBS genes. RESULTS: Mutations in the BBS genes were identified in nine families. In addition, a large consanguineous family (57004) showed linkage to the BBS7 locus, although no mutation was identified. Five novel mutations were present in the nine families: one in BBS2 (c.565C>T, p.ArgR189Stop), one in BBS5 (c.123delA, p.Gly42GlufsX11), one in BBS7 (g.47247455_47267458del20004insATA, p.Met284LysfsX7), and two in BBS8 (c.459+1G>A, p.Pro101LeufsX12 and c.355_356insGGTGGAAGGCCAGGCA, p.Thr124ArgfsX43). CONCLUSIONS: All families in which mutations were identified show changes in both copies of the mutant gene, and inheritance patterns in all families are consistent with autosomal recessive inheritance excluding any evidence of triallelism in the BBS genes in Tunisia.
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Alelos , Síndrome de Bardet-Biedl/genética , Consanguinidad , Femenino , Genes Recesivos , Prueba de Complementación Genética , Ligamiento Genético , Pruebas Genéticas , Genotipo , Humanos , Masculino , Repeticiones de Microsatélite , Mutación , Linaje , Proteínas/genética , TúnezAsunto(s)
Anomalías Múltiples/patología , Duplicación Cromosómica/genética , Cromosomas Humanos Par 11/genética , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Fenotipo , Proteínas Represoras/genética , Anomalías Múltiples/genética , Niño , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Humanos , Microcefalia/genética , Microcefalia/patología , Hipotonía Muscular/genética , Hipotonía Muscular/patología , Factor de Transcripción PAX6 , Convulsiones/genética , Convulsiones/patologíaRESUMEN
Cleft palate with ankyloglossia (CPX; OMIM 303400) is inherited as a Mendelian semidominant X-Linked disorder. Linkage studies resulted in mapping CPX to Xq13-q 21-31 region. TBX22 was identified as causing CPX. We report a new mutation in a Tunisian family and the first Arab family with X-Linked cleft palate and ankyloglossia. The family includes 6 affected members, 4 males and 2 females. Linkage study was performed using 9 microsatellite markers surrounding the CPX locus with a maximum lod score 1.81 at theta=0 with several markers. Sequence analysis of TBX22 gene revealed a novel change c.358C>T in exon 3 (R120W) located in the T-BOX domain; this change was present in all affected members and none of the 100 controls. A second modification in exon 4 (c.559G>A) predicted to result in a nonconservative substitution (E187 K) was present in the affected members but also in 2 controls, suggesting a polymorphism which functional role cannot be excluded without further study.
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Fisura del Paladar/genética , Familia , Mutación , Proteínas de Dominio T Box/genética , Femenino , Humanos , Masculino , Linaje , TúnezRESUMEN
OBJECTIVE: To determine the cause and course of a novel syndrome with progressive encephalopathy and brain atrophy in children. METHODS: Clinical whole-exome sequencing was performed for global developmental delay and intellectual disability; some patients also had spastic paraparesis and evidence of clinical regression. Six patients were identified with de novo missense mutations in the kinesin gene KIF1A. The predicted functional disruption of these mutations was assessed in silico to compare the calculated conformational flexibility and estimated efficiency of ATP binding to kinesin motor domains of wild-type (WT) versus mutant alleles. Additionally, an in vitro microtubule gliding assay was performed to assess the effects of de novo dominant, inherited recessive, and polymorphic variants on KIF1A motor function. RESULTS: All six subjects had severe developmental delay, hypotonia, and varying degrees of hyperreflexia and spastic paraparesis. Microcephaly, cortical visual impairment, optic neuropathy, peripheral neuropathy, ataxia, epilepsy, and movement disorders were also observed. All six patients had a degenerative neurologic course with progressive cerebral and cerebellar atrophy seen on sequential magnetic resonance imaging scans. Computational modeling of mutant protein structures when compared to WT kinesin showed substantial differences in conformational flexibility and ATP-binding efficiency. The de novo KIF1A mutants were nonmotile in the microtubule gliding assay. INTERPRETATION: De novo mutations in KIF1A cause a degenerative neurologic syndrome with brain atrophy. Computational and in vitro assays differentiate the severity of dominant de novo heterozygous versus inherited recessive KIF1A mutations. The profound effect de novo mutations have on axonal transport is likely related to the cause of progressive neurologic impairment in these patients.
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PURPOSE: To map the locus and identify the gene causing autosomal recessive congenital cataracts in a large consanguineous Tunisian family. METHODS: DNA was extracted from blood samples from a large Tunisian family with an autosomal recessive, congenital, total white cataract. A genome-wide scan was performed with microsatellite markers. All exons and the splice sites of the HSF4 gene were sequenced in all members of the Tunisian family and in control individuals. RT-PCR was used to detect different transcripts of the HSF4 gene in the human lens. The transcripts were cloned in a TA cloning vector and sequenced. RESULTS: Two-point linkage analyses showed linkage to markers on 16q22 with a maximum lod score of 17.78 at theta = 0.01 with D16S3043. Haplotype analysis refined the critical region to a 1.8-cM (4.8-Mb) interval, flanked by D16S3031 and D16S3095. This region contains HSF4, some mutations of which cause the autosomal dominant Marner cataract. Sequencing of HSF4 showed a homozygous mutation in the 5' splice site of intron 12 (c.1327+4A-->G), which causes the skipping of exon 12. A more detailed study of the transcripts resulting from alternative splicing of the HSF4 gene in the lens is also reported, showing the major transcript HSF4b. CONCLUSIONS: This is the first report describing association of an autosomal recessive cataract with the HSF4 locus on 16q21-q22.1 and the first description of HSF4 splice variants in the lens showing that HSF4b is the major transcript.
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Catarata/genética , Cromosomas Humanos Par 16/genética , Proteínas de Unión al ADN/genética , Mutación , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Factores de Transcripción/genética , Catarata/congénito , Mapeo Cromosómico , Consanguinidad , Análisis Mutacional de ADN , Femenino , Genes Recesivos , Haplotipos , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Intrones/genética , Escala de Lod , Masculino , Repeticiones de Microsatélite , Linaje , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TúnezRESUMEN
PURPOSE: To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the retinoschisin protein by gene delivery can reverse the abnormal "electronegative" electroretinogram (ERG) retinal response. METHODS: An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functional analysis was performed. RESULTS: No RS protein was detected by Western blot analysis or immunohistochemistry in the Rs1h-KO mouse. Dark-adapted ERG responses showed an electronegative configuration, with b-wave reduction in both Rs1h(-/Y) and Rs1h-/- mice, typical of XLRS in humans. Histologic examination of Rs1h-KO mice showed disorganization of multiple retinal layers, including duplication and mislocalization of ganglion cells, laminar dissection through the inner plexiform layer, disorganization of the outer plexiform layer, loss of regularity of the outer nuclear layer, and shortening of the inner/outer segments with mislocalization of photoreceptor nuclei into this layer. After intraocular administration of AAV(2/2)-CMV-Rs1h, immunohistochemistry showed retinoschisin expression in all retinal layers of Rs1h(-/Y) mice, and ERG recordings showed reversal of the electronegative waveform and restoration of the normal positive b-wave. CONCLUSIONS: The RS-KO mouse mimics structural features of human X-linked juvenile retinoschisis with dissection through, and disorganization of, multiple retinal layers. The Rs1h-KO functional deficit results in an electronegative ERG waveform that is characteristic of human retinoschisis disease and that implicates a synaptic transmission deficit in the absence of retinoschisin protein. Replacement therapy by supplementing normal Rs1h protein in the adult Rs1h-KO mouse restored the normal ERG configuration. This indicates that gene therapy is a viable strategy of therapeutic intervention even in the postdevelopmental adult stage of XLRS disease.
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Proteínas del Ojo/genética , Técnicas de Transferencia de Gen , Retina/fisiopatología , Retinosquisis/fisiopatología , Animales , Moléculas de Adhesión Celular , Niño , Adaptación a la Oscuridad , Electrorretinografía , Proteínas del Ojo/metabolismo , Vectores Genéticos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Retina/metabolismo , Retina/patología , Retinosquisis/genética , Retinosquisis/patologíaRESUMEN
Advances in genetic technology and analytical algorithms have greatly accelerated elucidation of the genetic contribution to cataractogenesis. Currently, 27 isolated or primary cataract loci have been identified by linkage analysis or mutational screening, and 20 are associated with specific genes. These are beginning to provide a framework for thinking of congenital cataracts. In addition to clues provided by the study of congenital and childhood cataracts, new experimental approaches to age-related cataracts are beginning to provide insights into its genetic origins.
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Catarata/genética , Catarata/congénito , Humanos , Biología MolecularRESUMEN
Whole exome sequencing made it possible to identify novel de novo mutations in genes that might be linked to human syndromes (genotype first analysis). We describe a female patient with a novel de novo SPOCK1 variant, which has not been previously been associated with a human phenotype. Her features include intellectual disability with dyspraxia, dysarthria, partial agenesis of corpus callosum, prenatal-onset microcephaly and atrial septal defect with aberrant subclavian artery. Previous genetic, cytogenomic and metabolic studies were unrevealing. At age 13 years, exome sequencing on the patient and her parents revealed a de novo novel missense mutation in SPOCK1 (coding for Testican-1) on chromosome 5q31: c.239A>T (p.D80V). This mutation affects a highly evolutionarily conserved area of the gene, replacing a polar aspartic acid with hydrophobic nonpolar valine, and changing the chemical properties of the protein product, likely representing a pathogenic variant. Previous microdeletions of 5q31 including SPOCK1 have suggested genes on 5q31 as candidates for intellectual disability. No mutations or variants in other genes potentially linked to her phenotype were identified. Testicans are proteoglycans belonging to the BM-40/SPARC/osteonectin family of extracellular calcium-binding proteins. Testican-1 is encoded by the SPOCK1 gene, and mouse models have been shown it to be strongly expressed in the brain and to be involved in neurogenesis. We hypothesize that because this gene function is critical for neurogenesis, mutations could potentially lead to a phenotype with developmental delay and microcephaly.