RESUMO
North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.
Assuntos
Distrofias Hereditárias da Córnea , Tomografia de Coerência Óptica , Adulto , Animais , Humanos , Linhagem , Retina/metabolismo , Xenopus laevis/genéticaRESUMO
Purpose: To clinically and molecularly investigate a new family with North Carolina macular dystrophy (NCMD) from Turkey, a previously unreported geographic origin for this phenotype. Methods: Clinical ophthalmic examinations, including fundus imaging and spectral domain-optical coherence tomography (SD-OCT), were performed on eight members of a two-generation non-consanguineous family from southern Turkey. Whole genome sequencing (WGS) was performed on two affected subjects, followed by variant filtering and copy number variant (CNV) analysis. Junction PCR and Sanger sequencing were used to confirm and characterize the duplication involving PRDM13 at the nucleotide level. The underlying mechanism was assessed with in silico analyses. Results: The proband presented with lifelong bilateral vision impairment and displayed large grade 3 coloboma-like central macular lesions. Five of her six children showed similar macular malformations, consistent with autosomal dominant NCMD. The severity grades in the six affected individuals from two generations are not evenly distributed. CNV analysis of WGS data of the two affected family members, followed by junction PCR and Sanger sequencing, revealed a novel 56.2 kb tandem duplication involving PRDM13 (chr6:99560265-99616492dup, hg38) at the MCDR1 locus. This duplication cosegregates with the NCMD phenotype in the five affected children. No other (likely) pathogenic variants in known inherited retinal disease genes were found in the WGS data. Bioinformatics analyses of the breakpoints suggest a replicative-based repair mechanism underlying the duplication. Conclusions: We report a novel tandem duplication involving the PRDM13 gene in a family with NCMD from a previously unreported geographic region. The duplication size is the smallest that has been reported thus far and may correlate with the particular phenotype.
Assuntos
Povo Asiático/genética , Distrofias Hereditárias da Córnea/genética , Duplicação Gênica , Histona-Lisina N-Metiltransferase/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Pré-Escolar , Cromossomos Humanos Par 6/genética , Distrofias Hereditárias da Córnea/diagnóstico por imagem , Feminino , Ligação Genética , Humanos , Masculino , Linhagem , Reação em Cadeia da Polimerase , Tomografia de Coerência Óptica , Turquia/epidemiologia , Sequenciamento Completo do GenomaRESUMO
Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities.
Assuntos
Alelos , Proteínas de Ciclo Celular/genética , Distrofias de Cones e Bastonetes/genética , Efeito Fundador , Perda Auditiva/genética , Infertilidade Masculina/genética , Mutação de Sentido Incorreto , Adolescente , Proteínas de Ciclo Celular/química , Cílios/metabolismo , Cílios/ultraestrutura , Distrofias de Cones e Bastonetes/diagnóstico , Análise Mutacional de DNA , Feminino , Fibroblastos/metabolismo , Genótipo , Perda Auditiva/diagnóstico , Humanos , Infertilidade Masculina/diagnóstico , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Fenótipo , Conformação Proteica , Relação Estrutura-Atividade , Síndrome , Sequenciamento do ExomaRESUMO
PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Genes Recessivos/genética , Oligonucleotídeos Antissenso/genética , Distrofias Retinianas/genética , Adulto , Alelos , Estudos de Coortes , Éxons/genética , Feminino , Frequência do Gene , Células HEK293 , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Oligonucleotídeos Antissenso/farmacologia , Linhagem , Fenótipo , Distrofias Retinianas/patologiaRESUMO
PURPOSE: RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease. METHODS: Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members. RESULTS: Biallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry. CONCLUSION: This study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.
Assuntos
Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Retinose Pigmentar/genética , Fatores de Transcrição/genética , Adulto , Análise Mutacional de DNA/métodos , Proteínas do Olho/metabolismo , Proteínas do Olho/fisiologia , Feminino , Genes Recessivos/genética , Estudos de Associação Genética/métodos , Genótipo , Haplótipos/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Retina/metabolismo , Retina/patologia , Doenças Retinianas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , População Branca/genéticaRESUMO
The original version of this Article contained an incorrect version of Fig. 3, which included two variants initially shown in black text in Fig. 3a that the authors removed from the final manuscript. The correct version of Fig. 3 without the two variants now appears in the PDF and HTML versions of the Article.
RESUMO
For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.
Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Neoplasias Ovarianas/genética , Sítios de Splice de RNA , Simulação por Computador , DNA Complementar , Éxons/genética , Feminino , Variação Genética , Humanos , Mutação , RNA Mensageiro/genéticaRESUMO
PURPOSE: We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD). METHODS: Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of mutation were investigated using luciferase assays, localization studies, and RNA-seq. RESULTS: We identified a novel heterozygous NR5A1 mutation, c.274C>T p.(Arg92Trp), in three unrelated patients. The Arg92 residue is highly conserved and located in the Ftz-F1 region, probably involved in DNA-binding specificity and stability. There were no consistent changes in transcriptional activation or subcellular localization. Transcriptomics in patient-derived lymphocytes showed upregulation of MAMLD1, a direct NR5A1 target previously associated with 46,XY DSD. In gonads of affected individuals, ovarian FOXL2 and testicular SRY-independent SOX9 expression observed. CONCLUSIONS: We propose NR5A1, previously associated with 46,XY DSD and 46,XX primary ovarian insufficiency, as a novel gene for 46,XX (ovo)testicular DSD. We hypothesize that p.(Arg92Trp) results in decreased inhibition of the male developmental pathway through downregulation of female antitestis genes, thereby tipping the balance toward testicular differentiation in 46,XX individuals. In conclusion, our study supports a role for NR5A1 in testis differentiation in the XX gonad.Genet Med 19 4, 367-376.
Assuntos
Proteínas de Ligação a DNA/genética , Sequenciamento do Exoma/métodos , Perfilação da Expressão Gênica/métodos , Proteínas Nucleares/genética , Transtornos Ovotesticulares do Desenvolvimento Sexual/genética , Análise de Sequência de RNA/métodos , Fator Esteroidogênico 1/genética , Fatores de Transcrição/genética , Feminino , Predisposição Genética para Doença , Haplótipos , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Ovário/metabolismo , Transtornos Ovotesticulares do Desenvolvimento Sexual/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/metabolismo , Testículo/metabolismo , Regulação para Cima , Adulto JovemRESUMO
Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:- and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.
Assuntos
Elementos de DNA Transponíveis , Flagelina/genética , Mutagênese Insercional , Regiões Promotoras Genéticas , Salmonella typhimurium/genética , Animais , Bélgica , DNA Bacteriano/química , DNA Bacteriano/genética , Variação Genética , Carne/microbiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Infecções por Salmonella/microbiologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/classificação , Salmonella typhimurium/isolamento & purificação , Análise de Sequência de DNA , SuínosRESUMO
SNARE proteins comprise a conserved protein family responsible for catalyzing membrane fusion during vesicle traffic. Syntaxin18 (STX18) is a poorly characterized endoplasmic reticulum (ER)-resident t-SNARE. Recently, together with TANGO1 and SLY1, its involvement was shown in ER to Golgi transport of collagen II during chondrogenesis. We report a fetus with a severe osteochondrodysplasia in whom we identified a homozygous substitution of the highly conserved p.Arg10 to Pro of STX18. CRISPR/Cas9-mediated Stx18 deficiency in zebrafish reveals a crucial role for Stx18 in cartilage and bone development. Furthermore, increased expression of multiple components of the Stx18 SNARE complex and of COPI and COPII proteins suggests that Stx18 deficiency impairs antero- and retrograde vesicular transport in the crispant stx18 zebrafish. Taken together, our studies highlight a new candidate gene for a recessive form of osteochondrodysplasia, thereby possibly broadening the SNAREopathy phenotypic spectrum and opening new doors toward future research avenues. © 2023 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Osteocondrodisplasias , Peixe-Zebra , Animais , Humanos , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Osteocondrodisplasias/metabolismo , Complexo de Golgi/metabolismo , Cartilagem/metabolismo , Desenvolvimento Ósseo , Transporte ProteicoRESUMO
Phospholipase A/acyltransferase 3 (PLAAT3) is a phospholipid-modifying enzyme predominantly expressed in neural and white adipose tissue (WAT). It is a potential drug target for metabolic syndrome, as Plaat3 deficiency in mice protects against diet-induced obesity. We identified seven patients from four unrelated consanguineous families, with homozygous loss-of-function variants in PLAAT3, who presented with a lipodystrophy syndrome with loss of fat varying from partial to generalized and associated with metabolic complications, as well as variable neurological features including demyelinating neuropathy and intellectual disability. Multi-omics analysis of mouse Plaat3-/- and patient-derived WAT showed enrichment of arachidonic acid-containing membrane phospholipids and a strong decrease in the signaling of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipocyte differentiation. Accordingly, CRISPR-Cas9-mediated PLAAT3 inactivation in human adipose stem cells induced insulin resistance, altered adipocyte differentiation with decreased lipid droplet formation and reduced the expression of adipogenic and mature adipocyte markers, including PPARγ. These findings establish PLAAT3 deficiency as a hereditary lipodystrophy syndrome with neurological manifestations, caused by a PPARγ-dependent defect in WAT differentiation and function.
Assuntos
Lipodistrofia , PPAR gama , Humanos , Animais , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Adipócitos , Adipogenia/genética , Lipodistrofia/genética , Lipodistrofia/metabolismo , FosfolipasesRESUMO
Nine influenza viruses isolated from gulls and shorebirds in Belgium (2008-2010), including H3N8, H5N2, H6N1, H11N9, H13N6, H13N8, and H16N3 subtypes, were targeted using random amplification and next-generation sequencing. The gene segments of these viruses segregated into three phylogeographic lineage types: (1) segments circulating in waterfowl in Eurasia with sporadic introduction in other species and in the Americas ("Eurasian avian"), (2) segments circulating in American waterfowl with sporadic introduction to other species and regions ("American avian"), and (3) segments circulating exclusively in gulls and shorebirds and having increased connectivity between the two hemispheres ("Charadriiformes specific"). Notably, an H6N1 and an H5N2 isolated from L. argentatus had mainly Eurasian avian genes but shared a matrix segment of American avian origin (first documentation in European gulls of transhemispheric reassortment). These data support the growing evidence of an important role of Charadriiformes birds in the dynamic nature of avian influenza ecology.
Assuntos
Charadriiformes/virologia , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Filogenia , Animais , Bélgica/epidemiologia , Variação Genética , Influenza Aviária/diagnóstico , Influenza Aviária/epidemiologia , Técnicas de Amplificação de Ácido Nucleico , Filogeografia , Análise de Sequência de RNARESUMO
BACKGROUND: During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (Anas platyrhynchos) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests. METHODS: To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes. RESULTS: Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool. CONCLUSIONS: These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.
Assuntos
Infecções por Avulavirus/veterinária , Avulavirus/genética , Avulavirus/isolamento & purificação , Patos/virologia , Variação Genética , Genoma Viral , RNA Viral/genética , Animais , Avulavirus/classificação , Infecções por Avulavirus/virologia , Bélgica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , SorotipagemRESUMO
Presence of pseudogenes is a dreadful issue in next generation sequencing (NGS), because their contamination can interfere with the detection of variants in the genuine gene and generate false positive and false negative variants.In this chapter we focus on issues related to the application of NGS strategies for analysis of genes with pseudogenes in a clinical setting. The degree to which a pseudogene impacts the ability to accurately detect and map variants in its parent gene depends on the degree of similarity (homology) with the parent gene itself. Hereby, target enrichment and mapping strategies are crucial factors to avoid "contaminating" pseudogene sequences. For target enrichment, we describe advantages and disadvantages of PCR- and capture-based strategies. For mapping strategies, we discuss crucial parameters that need to be considered to accurately distinguish sequences of functional genes from pseudogenic sequences. Finally, we discuss some examples of genes associated with Mendelian disorders, for which interesting NGS approaches are described to avoid interference with pseudogene sequences.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular , Pseudogenes , Sequência de Bases , Análise de Dados , Eletroforese Capilar/métodos , Genes BRCA1 , Genes da Neurofibromatose 1 , Humanos , Mutação INDEL , Peptídeos e Proteínas de Sinalização Intercelular/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sondas de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Canais de Cátion TRPP/genéticaRESUMO
We describe both phenotype and pathogenesis in two male siblings with typical retinitis pigmentosa (RP) and the potentially X-linked RP (XLRP) carrier phenotype in their mother. Two affected sons, two unaffected daughters, and their mother underwent detailed ophthalmological assessments including Goldmann perimetry, color vision testing, multimodal imaging and ISCEV-standard electroretinography. Genetic testing consisted of targeted next-generation sequencing (NGS) of known XLRP genes and whole exome sequencing (WES) of known inherited retinal disease genes (RetNet-WES). Variant validation and segregation analysis were performed by Sanger sequencing. The mutational load of the RHO variant in the mother was assessed in DNA from leucocytes, buccal cells and hair follicles using targeted NGS. Both affected sons showed signs of classical RP, while the mother displayed patches of hyperautofluorescence on blue light autofluorescence imaging and regional, intraretinal, spicular pigmentation, reminiscent of a carrier phenotype of XLRP. XLRP testing was negative. RetNet-WES testing revealed RHO variant c.404G > C p.(Arg135Pro) in a mosaic state (21% of the reads) in the mother and in a heterozygous state in both sons. Targeted NGQSS of the RHO variant in different maternal tissues showed a mutation load between 25.06% and 41.72%. We report for the first time that somatic mosaicism of RHO variant c.404G > C p.(Arg135Pro) mimics the phenotype of a female carrier of XLRP, in combination with heterozygosity for the variant in the two affected sons.
Assuntos
Mosaicismo , Retinose Pigmentar/genética , Rodopsina/genética , Adulto , Sequência de Bases , Feminino , Dosagem de Genes , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Mutação Puntual , Retinose Pigmentar/congênito , Retinose Pigmentar/metabolismo , Rodopsina/metabolismo , Adulto JovemRESUMO
Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient's materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.
RESUMO
(1) Background: The proportion and spectrum of germline pathogenic variants (PV) associated with an increased risk for pancreatic ductal adenocarcinoma (PDAC) varies among populations. (2) Methods: We analyzed 72 Belgian and 226 Czech PDAC patients by multigene panel testing. The prevalence of pathogenic variants (PV) in relation to personal/family cancer history were evaluated. PDAC risks were calculated using both gnomAD-NFE and population-matched controls. (3) Results: In 35/298 (11.7%) patients a PV in an established PDAC-predisposition gene was found. BRCA1/2 PV conferred a high risk in both populations, ATM and Lynch genes only in the Belgian subgroup. PV in other known PDAC-predisposition genes were rarer. Interestingly, a high frequency of CHEK2 PV was observed in both patient populations. PV in PDAC-predisposition genes were more frequent in patients with (i) multiple primary cancers (12/38; 32%), (ii) relatives with PDAC (15/56; 27%), (iii) relatives with breast/ovarian/colorectal cancer or melanoma (15/86; 17%) but more rare in sporadic PDAC (5/149; 3.4%). PV in homologous recombination genes were associated with improved overall survival (HR = 0.51; 95% CI 0.34-0.77). (4) Conclusions: Our analysis emphasizes the value of multigene panel testing in PDAC patients, especially in individuals with a positive family cancer history, and underlines the importance of population-matched controls for risk assessment.
RESUMO
BACKGROUND: This study characterizes the second hit spectrum in BRCA1 and BRCA2-associated breast and ovarian cancers at both gene loci to investigate if second hit mechanisms are mutually exclusive or able to coincide within the same tumor. METHODS: Loss of heterozygosity, somatic point mutations and copy number alterations along with promoter methylation were studied in 56 breast and 15 ovarian cancers from BRCA1 and BRCA2 germline mutation carriers. A mathematical methodology was introduced to quantify the tumor cell population carrying a second hit. RESULTS: Copy neutral LOH was the most prevalent LOH mechanism in this cohort (BC 69%, OC 67%). However, only 36% of BC and 47% of OC showed LOH in all cancerous cells. Somatic intragenic deletions and methylated subclones were also found in combination with (partial) loss of heterozygosity. Unequivocal deleterious somatic point mutations were not identified in this cohort. CONCLUSION: Different mechanisms inactivating the wild type allele are present within the same tumor sample at various extents. Results indicate that BRCA1/2-linked breast and ovarian cancer cells are predominantly characterized by LOH, but harbor a complex combination of second hits at various frequencies.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Análise de Sequência de DNA/métodos , Estudos de Coortes , Variações do Número de Cópias de DNA , Metilação de DNA , Epigênese Genética , Feminino , Mutação em Linhagem Germinativa , Humanos , Perda de Heterozigosidade , Modelos Teóricos , Mutação Puntual , Regiões Promotoras GenéticasRESUMO
The complete and fully annotated genome sequence of a bovine polyomavirus type 1 (BPyV/BEL/1/2014) from aborted cattle was assembled from a metagenomics data set. The 4,697-bp circular dsDNA genome contains 6 protein-coding genes. Bovine polyomavirus is unlikely to be causally related to the abortion cases.
RESUMO
The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals.