RESUMO
Autosomal dominant Kabuki syndrome (KS) is a rare multiple congenital anomalies/neurodevelopmental disorder caused by heterozygous inactivating variants or structural rearrangements of the lysine-specific methyltransferase 2D (KMT2D) gene. While it is often recognizable due to a distinctive gestalt, the disorder is clinically variable, and a phenotypic scoring system has been introduced to help clinicians to reach a clinical diagnosis. The phenotype, however, can be less pronounced in some patients, including those carrying postzygotic mutations. The full spectrum of pathogenic variation in KMT2D has not fully been characterized, which may hamper the clinical classification of a portion of these variants. DNA methylation (DNAm) profiling has successfully been used as a tool to classify variants in genes associated with several neurodevelopmental disorders, including KS. In this work, we applied a KS-specific DNAm signature in a cohort of 13 individuals with KMT2D VUS and clinical features suggestive or overlapping with KS. We succeeded in correctly classifying all the tested individuals, confirming diagnosis for three subjects and rejecting the pathogenic role of 10 VUS in the context of KS. In the latter group, exome sequencing allowed to identify the genetic cause underlying the disorder in three subjects. By testing five individuals with postzygotic pathogenic KMT2D variants, we also provide evidence that DNAm profiling has power to recognize pathogenic variants at different levels of mosaicism, identifying 15% as the minimum threshold for which DNAm profiling can be applied as an informative diagnostic tool in KS mosaics.
Assuntos
Anormalidades Múltiplas , Metilação de DNA , Proteínas de Ligação a DNA , Face , Doenças Hematológicas , Mosaicismo , Proteínas de Neoplasias , Doenças Vestibulares , Humanos , Doenças Vestibulares/genética , Doenças Vestibulares/diagnóstico , Face/anormalidades , Doenças Hematológicas/genética , Doenças Hematológicas/diagnóstico , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/diagnóstico , Proteínas de Ligação a DNA/genética , Masculino , Feminino , Proteínas de Neoplasias/genética , Criança , Pré-Escolar , Adolescente , Mutação em Linhagem Germinativa , Lactente , Fenótipo , AdultoRESUMO
BACKGROUND: Studies suggest that Wilms tumours (WT) are caused by underlying genetic (5%-10%) and epigenetic (2%-29%) mechanisms, yet studies covering both aspects are sparse. METHODS: We performed prospective whole-genome sequencing of germline DNA in Danish children diagnosed with WT from 2016 to 2021, and linked genotypes to deep phenotypes. RESULTS: Of 24 patients (58% female), 3 (13%, all female) harboured pathogenic germline variants in WT risk genes (FBXW7, WT1 and REST). Only one patient had a family history of WT (3 cases), segregating with the REST variant. Epigenetic testing revealed one (4%) additional patient (female) with uniparental disomy of chromosome 11 and Beckwith-Wiedemann syndrome (BWS). We observed a tendency of higher methylation of the BWS-related imprinting centre 1 in patients with WT than in healthy controls. Three patients (13%, all female) with bilateral tumours and/or features of BWS had higher birth weights (4780 g vs 3575 g; p=0.002). We observed more patients with macrosomia (>4250 g, n=5, all female) than expected (OR 9.98 (95% CI 2.56 to 34.66)). Genes involved in early kidney development were enriched in our constrained gene analysis, including both known (WT1, FBXW7) and candidate (CTNND1, FRMD4A) WT predisposition genes. WT predisposing variants, BWS and/or macrosomia (n=8, all female) were more common in female patients than male patients (p=0.01). CONCLUSION: We find that most females (57%) and 33% of all patients with WT had either a genetic or another indicator of WT predisposition. This emphasises the need for scrutiny when diagnosing patients with WT, as early detection of underlying predisposition may impact treatment, follow-up and genetic counselling.
Assuntos
Síndrome de Beckwith-Wiedemann , Neoplasias Renais , Tumor de Wilms , Masculino , Feminino , Humanos , Proteína 7 com Repetições F-Box-WD/genética , Macrossomia Fetal/genética , Impressão Genômica , Tumor de Wilms/genética , Genótipo , Síndrome de Beckwith-Wiedemann/patologia , Metilação de DNA/genética , Suscetibilidade a Doenças , Neoplasias Renais/genética , Células Germinativas/patologiaRESUMO
FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.
Assuntos
Síndrome de Rett , Humanos , Encéfalo/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Fatores de Transcrição Forkhead/genética , Proteínas do Tecido NervosoRESUMO
Disease-specific DNA methylation patterns (DNAm signatures) have been established for an increasing number of genetic disorders and represent a valuable tool for classification of genetic variants of uncertain significance (VUS). Sample size and batch effects are critical issues for establishing DNAm signatures, but their impact on the sensitivity and specificity of an already established DNAm signature has not previously been tested. Here, we assessed whether publicly available DNAm data can be employed to generate a binary machine learning classifier for VUS classification, and used variants in KMT2D, the gene associated with Kabuki syndrome, together with an existing DNAm signature as proof-of-concept. Using publicly available methylation data for training, a classifier for KMT2D variants was generated, and individuals with molecularly confirmed Kabuki syndrome and unaffected individuals could be correctly classified. The present study documents the clinical utility of a robust DNAm signature even for few affected individuals, and most importantly, underlines the importance of data sharing for improved diagnosis of rare genetic disorders.
Assuntos
Anormalidades Múltiplas , Doenças Hematológicas , Doenças Vestibulares , Humanos , Metilação de DNA , Anormalidades Múltiplas/genética , Doenças Hematológicas/genética , Doenças Vestibulares/genéticaRESUMO
Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder caused by the expansion of a CTG repeat in the 3'-UTR of DMPK, which is transcribed to a toxic gain-of-function RNA that affects splicing of a range of genes. The expanded repeat is unstable in both germline and somatic cells. The variable age at disease onset and severity of symptoms have been linked to the inherited CTG repeat length, non-CTG interruptions, and methylation levels flanking the repeat. In general, the genetic biomarkers are investigated separately with specific methods, making it tedious to obtain an overall characterisation of the repeat for a given individual. In the present study, we employed Oxford nanopore sequencing in a pilot study to simultaneously determine the repeat lengths, investigate the presence and nature of repeat interruptions, and quantify methylation levels in the regions flanking the CTG-repeats in four patients with DM1. We determined the repeat lengths, and in three patients, we observed interruptions which were not detected using repeat-primed PCR. Interruptions may thus be more common than previously anticipated and should be investigated in larger cohorts. Allele-specific analyses enabled characterisation of aberrant methylation levels specific to the expanded allele, which greatly increased the sensitivity and resolved cases where the methylation levels were ambiguous.
Assuntos
Distrofia Miotônica , Miotonina Proteína Quinase , Metilação de DNA , Humanos , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Projetos Piloto , Splicing de RNA , Expansão das Repetições de TrinucleotídeosRESUMO
Tic spectrum disorder (TSD) is an umbrella term which includes Gilles de la Tourette syndrome (GTS) and chronic tic disorder (CTD). They are considered highly heritable, yet the genetic components remain largely unknown. In this study we aimed to investigate disease-associated DNA methylation differences to identify genes and pathways which may be implicated in TSD aetiology. For this purpose, we performed an exploratory analysis of the genome-wide DNA methylation patterns in whole blood samples of 16 monozygotic twin pairs, of which eight were discordant and six concordant for TSD, while two pairs were asymptomatic. Although no sites reached genome-wide significance, we identified several sites and regions with a suggestive significance, which were located within or in the vicinity of genes with biological functions associated with neuropsychiatric disorders. The two top genes identified (TSC1 and CRYZ/TYW3) and the enriched pathways and components (phosphoinosides and PTEN pathways, and insulin receptor substrate binding) are related to, or have been associated with, the PI3K/AKT/mTOR pathway. Genes in this pathway have previously been associated with GTS, and mTOR signalling has been implicated in a range of neuropsychiatric disorders. It is thus possible that altered mTOR signalling plays a role in the complex pathogenesis of TSD.
Assuntos
Metilação de DNA , Epigênese Genética , Serina-Treonina Quinases TOR/genética , Síndrome de Tourette/genética , Gêmeos Monozigóticos/genética , Feminino , Humanos , Masculino , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Síndrome de Tourette/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Gilles de la Tourette syndrome (GTS) is a complex neurodevelopmental disorder characterized by motor and vocal tics. Most of the GTS individuals have comorbid diagnoses, of which obsessive-compulsive disorder (OCD) and attention deficit-hyperactivity disorder (ADHD) are the most common. Several neurotransmitter systems have been implicated in disease pathogenesis, and amongst these, the dopaminergic and the serotonergic pathways are the most widely studied. In this study, we aimed to investigate whether the serotonin transporter (SERT) gene (SLC6A4) was differentially expressed among GTS individuals compared to healthy controls, and whether DNA variants (the SERT-linked polymorphic region 5-HTTLPR, together with the associated rs25531 and rs25532 variants, and the rare Ile425Val variant) or promoter methylation of SLC6A4 were associated with gene expression levels or with the presence of OCD as comorbidity. We observed that SLC6A4 expression is upregulated in GTS individuals compared to controls. Although no specific genotype, allele or haplotype was overrepresented in GTS individuals compared to controls, we observed that the LAC/LAC genotype of the 5-HTTLPR/rs25531/rs25532 three-locus haplotype was associated with higher SLC6A4 mRNA expression levels in GTS individuals, but not in the control group.
Assuntos
Regulação da Expressão Gênica , Mutação de Sentido Incorreto , Polimorfismo Genético , Proteínas da Membrana Plasmática de Transporte de Serotonina , Síndrome de Tourette , Substituição de Aminoácidos , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Humanos , Masculino , Transtorno Obsessivo-Compulsivo/genética , Transtorno Obsessivo-Compulsivo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Síndrome de Tourette/genética , Síndrome de Tourette/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystem disorder mainly characterized by gradual muscle loss, weakness, and delayed relaxation after muscle contraction. It is caused by an expanded CTG repeat in the 3' UTR of DMPK, which is transcribed into a toxic gain-of-function mRNA that affects the splicing of a range of other genes. The repeat is unstable, with a bias towards expansions both in somatic cells and in the germline, which results in a tendency for earlier onset with each generation, as longer repeat lengths generally correlate with earlier onset. Previous studies have found hypermethylation in the regions flanking the repeat in congenital onset DM1 and in some patients with non-congenital DM1. We used pyrosequencing to investigate blood methylation levels in 68 patients with non-congenital DM1, compare the methylation levels between the blood and muscle, and assess whether methylation levels change over time in the blood. We found higher methylation levels in the blood of DM1 patients than in healthy controls and especially in the patients who had inherited the disease allele maternally. The methylation levels remained relatively stable over time and are a strong biomarker of the disease, as well as of the maternal inheritance of the disease.
Assuntos
Ilhas de CpG , Metilação de DNA , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Expansão das Repetições de Trinucleotídeos , Adolescente , Adulto , Alelos , Sequência de Bases , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Padrões de Herança , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Miotônica/diagnóstico , Fenótipo , Curva ROC , Adulto JovemRESUMO
Immediate freezing is perhaps the most preferred method used for preserving gut microbial samples, but research on sample preservation has been principally based around samples from mammalian species, and little is known about the advantages or disadvantages relating to different storage methods for fish guts. Fish gut samples may pose additional challenges due to the different chemical and enzymatic profile, as well as the higher water content, which might affect the yield and purity of DNA recovered. To explore this, we took gut content and mucosal scrape samples from 10 rainbow trout (Oncorhynchus mykiss), and tested whether different preservation methods have any effect on the ability to construct high quality genomic libraries for shotgun and 16S rRNA gene sequencing. Four different storage methods were compared for the gut content samples (immediate freezing on dry ice, 96% ethanol, RNAlater and DNA/RNA shield), while two different methods were compared for mucosal scrape samples (96% ethanol and RNAlater). The samples were thereafter stored at -80⯰C. Our findings concluded that 96% ethanol outperforms the other storage methods when considering DNA quantity, quality, cost and labor. Ethanol works consistently well for both gut content and mucosal scrape samples, and enables construction of DNA sequencing libraries of sufficient quantity and with a fragment length distribution suitable for shotgun sequencing. Two main conclusions from our study are i) sample storage optimisation is an important part of establishing a microbiome research program in a new species or sample type system, and ii) 96% ethanol is the preferred method for storing rainbow trout gut content and mucosal scrape samples.