Father-to-offspring transmission of extremely long NOTCH2NLC repeat expansions with contractions: genetic and epigenetic profiling with long-read sequencing

Abstract

Background GGC repeat expansions in NOTCH2NLC are associated with neuronal intranuclear inclusion disease. Very recently, asymptomatic carriers with NOTCH2NLC repeat expansions were reported. In these asymptomatic individuals, the CpG island in NOTCH2NLC is hypermethylated, suggesting that two factors repeat length and DNA methylation status should be considered to evaluate pathogenicity. Long-read sequencing can be used to simultaneously profile genomic and epigenomic alterations. We analyzed four sporadic cases with NOTCH2NLC repeat expansion and their phenotypically normal parents. The native genomic DNA that retains base modification was sequenced on a per-trio basis using both PacBio and Oxford Nanopore long-read sequencing technologies. A custom workflow was developed to evaluate DNA modifications. With these two technologies combined, long-range DNA methylation information was integrated with complete repeat DNA sequences to investigate the genetic origins of expanded GGC repeats in these sporadic cases. Results In all four families, asymptomatic fathers had longer expansions (median: 522, 390, 528 and 650 repeats) compared with their affected offspring (median: 93, 117, 162 and 140 repeats, respectively). These expansions are much longer than the disease-causing range previously reported (in general, 41–300 repeats). Repeat lengths were extremely variable in the father, suggesting somatic mosaicism. Instability is more frequent in alleles with uninterrupted pure GGCs. Single molecule epigenetic analysis revealed complex DNA methylation patterns and epigenetic heterogeneity. We identified an aberrant gain-of-methylation region (2.2 kb in size beyond the CpG island and GGC repeats) in asymptomatic fathers. This methylated region was unmethylated in the normal allele with bilateral transitional zones with both methylated and unmethylated CpG dinucleotides, which may be protected from methylation to ensure NOTCH2NLC expression. Conclusions We clearly demonstrate that the four sporadic NOTCH2NLC-related cases are derived from the paternal GGC repeat contraction associated with demethylation. The entire genetic and epigenetic landscape of the NOTCH2NLC region was uncovered using the custom workflow of long-read sequence data, demonstrating the utility of this method for revealing epigenetic/mutational changes in repetitive elements, which are difficult to characterize by conventional short-read/bisulfite sequencing methods. Our approach should be useful for biomedical research, aiding the discovery of DNA methylation abnormalities through the entire genome.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

“…Another advantage of the approach demonstrated hereby is that PCR-free analysis potentially allows the direct assessment of DNA methylation, as already reported for other repeat-linked diseases (Fukuda et al 2021;Giesselmann et al 2019). This can provide additional information concerning the impact of expansions on the functionality of the CNBP gene.…”

Trinucleotide CGG Repeat Diseases: An Expanding Field of Polyglycine Proteins?

“…), there are only few examples of anticipation reported in NIID and OPDM families, and a limited correlation between the size of CGG expansions and the age of onset in OPDM1 and OPDM2, with a r 2 of 0.188 and 0.158, respectively, ( Kumutpongpanich et al, 2021 ; Xi et al, 2021 ). On the contrary, there are increasing reports of individuals carrying large CGG expansions (>200–300 repeats) in the NOTCH2NLC , GIPC1, and LRP12 genes and who are free of overt clinical manifestations ( Deng et al, 2020 ; Ogasawara et al, 2020 ; Deng et al, 2021 ; Fukuda et al, 2021 ; Kumutpongpanich et al, 2021 ; Yu et al, 2021 ). Molecular analyses indicate that these individuals present DNA hypermethylation associated to promoter silencing and loss of expression of the allele carrying these large CGG expansions.…”

“…Second, these data also provide some molecular bases to the complex pedigree observed in these diseases, with increasing reports of asymptomatic ascendants who nevertheless carry the causative mutation, but of longer size (over 200–300 CGG repeats) compared to their affected descendants who typically carry an allele with 60 to 200–300 CGG repeats. Interestingly, of the ∼10 currently reported examples of asymptomatic individuals with large expansions and who have transmitted shorter repeats to their NIID or OPDM-affected descendants, all were father to infant transmission cases, suggesting a bias of expansions contraction in male germinal cells ( Deng et al, 2020 ; Deng et al, 2021 ; Fukuda et al, 2021 ; Kumutpongpanich et al, 2021 ; Yu et al, 2021 ). Of interest, a similar bias of transmission was observed in carriers of the FMR1 CGG repeat premutation where contraction occurred more often in paternal than in maternal transmission ( Nolin et al, 2019 ).…”

Les myopathies oculo-pharyngo-distales : des nouvelles maladies à expansions de répétitions CGG

“…Toutefois, dans les maladies causées par des expansions de répétitions CGG situées dans des régions 5'UTR, l'existence d'un mécanisme d'anticipation et/ou une corrélation exacte entre la taille de l'expansion et la sévérité des symptômes ne font pas actuellement consensus [7]. En effet, certains individus étaient porteurs de longues expansions de répétitions CGG dans les gènes LRP12, GIPC1 ou NOTCH2NLC mais ne développaient pas les symptômes de l'OPDM [2,38,39]. Par exemple, dans l'OPDM2, un individu asymptomatique était porteur d'une expansion de plus de 500 répétitions CGG dans le gène GIPC1 alors que ses deux enfants sont porteurs d'expansions de répétitions CGG plus courtes (117 et 113 répétitions) et ont tous deux développé des formes classiques d'OPDM [2].…”

“…De manière similaire, des patients possédant un nombre de répétitions CGG supérieur à 300 ne développent pas de symptômes particuliers contrairement à leurs enfants porteurs d'expansions plus courtes et qui développent une NIID et/ou une OPDM3. Il a été montré récemment que ces longues expansions (200-300 répétitions CGG) conduisent à des modifications épigénétiques du promoteur et donc à une perte d'expression du gène et de la protéine NOTCH2NLC, suggérant ainsi qu'un mécanisme de perte de fonction est peu probable dans la NIID et l'OPDM3 [38]. Ces données sont à mettre en perspective avec la physiopathologie du FXS et du FXTAS.…”