Genome-wide identification of tandem repeats associated with splicing variation across 49 tissues in humans.

Tandem repeats (TRs) are one of the largest sources of polymorphism, and their length is associated with gene regulation. Although previous studies reported several tandem repeats regulating gene splicing in cis (spl-TRs), no large-scale study has been conducted. In this study, we established a genome-wide catalog of 9537 spl-TRs with a total of 58,290 significant TR-splicing associations across 49 tissues (false discovery rate 5%) by using Genotype-Tissue expression (GTex) Project data. Regression models explaining splicing variation by using spl-TRs and other flanking variants suggest that at least some of the spl-TRs directly modulate splicing. In our catalog, two spl-TRs are known loci for repeat expansion diseases, spinocerebellar ataxia 6 (SCA6) and 12 (SCA12). Splicing alterations by these spl-TRs were compatible with those observed in SCA6 and SCA12. Thus, our comprehensive spl-TR catalog may help elucidate the pathomechanism of genetic diseases.

Whole-exome sequencing reveals causative genetic variants for several overgrowth syndromes in molecularly negative Beckwith-Wiedemann spectrum.

Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by (epi)genetic alterations at 11p15. Because approximately 20% of patients test negative via molecular testing of peripheral blood leukocytes, the concept of Beckwith-Wiedemann spectrum (BWSp) was established to encompass a broader cohort with diverse and overlapping phenotypes. The prevalence of other overgrowth syndromes concealed within molecularly negative BWSp remains unexplored. Methods We conducted whole-exome sequencing (WES) on 69 singleton patients exhibiting molecularly negative BWSp. Variants were confirmed by Sanger sequencing or quantitative genomic PCR. We compared BWSp scores and clinical features between groups with classical BWS (cBWS), atypical BWS or isolated lateralised overgrowth (aBWS+ILO) and overgrowth syndromes identified via WES. Results Ten patients, one classified as aBWS and nine as cBWS, showed causative gene variants for Simpson-Golabi-Behmel syndrome (five patients), Sotos syndrome (two), Imagawa-Matsumoto syndrome (one), glycosylphosphatidylinositol biosynthesis defect 11 (one) or 8q duplication/9p deletion (one). BWSp scores did not distinguish between cBWS and other overgrowth syndromes. Birth weight and height in other overgrowth syndromes were significantly larger than in aBWS+ILO and cBWS, with varying intergroup frequencies of clinical features. Conclusion Molecularly negative BWSp encapsulates other syndromes, and considering both WES and clinical features may facilitate accurate diagnosis.

Detection of hidden intronic DDC variant in aromatic L-amino acid decarboxylase deficiency by adaptive sampling.

Aromatic l-amino acid decarboxylase (AADC) deficiency is an autosomal recessive neurotransmitter disorder caused by pathogenic DOPA decarboxylase (DDC) variants. We previously reported Japanese siblings with AADC deficiency, which was confirmed by the lack of enzyme activity; however, only a heterozygous missense variant was detected. We therefore performed targeted long-read sequencing by adaptive sampling to identify any missing variants. Haplotype phasing and variant calling identified a novel deep intronic variant (c.714+255 C > A), which was predicted to potentially activate the noncanonical splicing acceptor site. Minigene assay revealed that wild-type and c.714+255 C > A alleles had different impacts on splicing. Three transcripts, including the canonical transcript, were detected from the wild-type allele, but only the noncanonical cryptic exon was produced from the variant allele, indicating that c.714+255 C > A was pathogenic. Target long-read sequencing may be used to detect hidden pathogenic variants in unresolved autosomal recessive cases with only one disclosed hit variant.

A heterozygous germline deletion within USP8 causes severe neurodevelopmental delay with multiorgan abnormalities.

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in deubiquitinating the enhanced epidermal growth factor receptor for escape from degradation. Somatic variants at a hotspot in USP8 are a cause of Cushing's disease, and a de novo germline USP8 variant at this hotspot has been described only once previously, in a girl with Cushing's disease and developmental delay. In this study, we investigated an exome-negative patient with severe developmental delay, dysmorphic features, and multiorgan dysfunction by long-read sequencing, and identified a 22-kb de novo germline deletion within USP8 (chr15:50469966-50491995 [GRCh38]). The deletion involved the variant hotspot, one rhodanese domain, and two SH3 binding motifs, and was presumed to be generated through nonallelic homologous recombination through Alu elements. Thus, the patient may have perturbation of the endosomal sorting system and mitochondrial autophagy through the USP8 defect. This is the second reported case of a germline variant in USP8.

Novel missense variants cause intermediate phenotypes in the phenotypic spectrum of SLC5A6-related disorders.

SLC5A6 encodes the sodium-dependent multivitamin transporter, a transmembrane protein that uptakes biotin, pantothenic acid, and lipoic acid. Biallelic SLC5A6 variants cause sodium-dependent multivitamin transporter deficiency (SMVTD) and childhood-onset biotin-responsive peripheral motor neuropathy (COMNB), which both respond well to replacement therapy with the above three nutrients. SMVTD usually presents with various symptoms in multiple organs, such as gastrointestinal hemorrhage, brain atrophy, and global developmental delay, at birth or in infancy. Without nutrient replacement therapy, SMVTD can be lethal in early childhood. COMNB is clinically milder and has a later onset than SMVTD, at approximately 10 years of age. COMNB symptoms are mostly limited to peripheral motor neuropathy. Here we report three patients from one Japanese family harboring novel compound heterozygous missense variants in SLC5A6, namely NM_021095.4:c.[221C>T];[642G>C] p.[(Ser74Phe)];[(Gln214His)]. Both variants were predicted to be deleterious through multiple lines of evidence, including amino acid conservation, in silico predictions of pathogenicity, and protein structure considerations. Drosophila analysis also showed c.221C>T to be pathogenic. All three patients had congenital brain cysts on neonatal cranial imaging, but no other morphological abnormalities. They also had a mild motor developmental delay that almost completely resolved despite no treatment. In terms of severity, their phenotypes were intermediate between SMVTD and COMNB. From these findings we propose a new SLC5A6-related disorder, spontaneously remitting developmental delay with brain cysts (SRDDBC) whose phenotypic severity is between that of SMVTD and COMNB. Further clinical and genetic evidence is needed to support our suggestion.

Novel compound heterozygous ABCA2 variants cause IDPOGSA, a variable phenotypic syndrome with intellectual disability.

The gene for ATP binding cassette subfamily A member 2 (ABCA2) is located at chromosome 9q34.3. Biallelic ABCA2 variants lead to intellectual developmental disorder with poor growth and with or without seizures or ataxia (IDPOGSA). In this study, we identified novel compound heterozygous ABCA2 variants (NM_001606.5:c.[5300-17C>A];[6379C>T]) by whole exome sequencing in a 28-year-old Korean female patient with intellectual disability. These variants included intronic and nonsense variants of paternal and maternal origin, respectively, and are absent from gnomAD. SpliceAI predicted that the intron variant creates a cryptic acceptor site. Reverse transcription-PCR using RNA extracted from a lymphoblastoid cell line of the patient confirmed two aberrant transcripts. Her clinical features are compatible with those of IDPOGSA.

Complete nanopore repeat sequencing of SCA27B (GAA-FGF14 ataxia) in Japanese.

BACKGROUND: Although pure GAA expansion is considered pathogenic in SCA27B, non-GAA repeat motif is mostly mixed into longer repeat sequences. This study aimed to unravel the complete sequencing of FGF14 repeat expansion to elucidate its repeat motifs and pathogenicity. METHODS: We screened FGF14 repeat expansion in a Japanese cohort of 460 molecularly undiagnosed adult-onset cerebellar ataxia patients and 1022 controls, together with 92 non-Japanese controls, and performed nanopore sequencing of FGF14 repeat expansion. RESULTS: In the Japanese population, the GCA motif was predominantly observed as the non-GAA motif, whereas the GGA motif was frequently detected in non-Japanese controls. The 5'-common flanking variant was observed in all Japanese GAA repeat alleles within normal length, demonstrating its meiotic stability against repeat expansion. In both patients and controls, pure GAA repeat was up to 400 units in length, whereas non-pathogenic GAA-GCA repeat was larger, up to 900 units, but they evolved from different haplotypes, as rs534066520, located just upstream of the repeat sequence, completely discriminated them. Both (GAA)≥250 and (GAA)≥200 were enriched in patients, whereas (GAA-GCA)≥200 was similarly observed in patients and controls, suggesting the pathogenic threshold of (GAA)≥200 for cerebellar ataxia. We identified 14 patients with SCA27B (3.0%), but their single-nucleotide polymorphism genotype indicated different founder alleles between Japanese and Caucasians. The low prevalence of SCA27B in Japanese may be due to the lower allele frequency of (GAA)≥250 in the Japanese population than in Caucasians (0.15% vs 0.32%-1.26%). CONCLUSIONS: FGF14 repeat expansion has unique features of pathogenicity and allelic origin, as revealed by a single ethnic study.

Long-term clinical observation of patients with heterozygous KIF1A variants.

KIF1A-related disorders (KRDs) encompass recessive and dominant variants with wide clinical variability. Recent genetic investigations have expanded the clinical phenotypes of heterozygous KIF1A variants. However, there have been a few long-term observational studies of patients with heterozygous KIF1A variants. A retrospective chart review of consecutive patients diagnosed with spastic paraplegia at Miyagi Children's Hospital from 2016 to 2020 identified six patients with heterozygous KIF1A variants. To understand the long-term changes in clinical symptoms, we examined these patients in terms of their characteristics, clinical symptoms, results of electrophysiological and neuroimaging studies, and genetic testing. The median follow-up period was 30 years (4-44 years). This long-term observational study showed that early developmental delay and equinus gait, or unsteady gait, are the first signs of disease onset, appearing with the commencement of independent walking. In addition, later age-related progression was observed in spastic paraplegia, and the appearance of axonal neuropathy and reduced visual acuity were characteristic features of the late disease phenotype. Brain imaging showed age-related progression of cerebellar atrophy and the appearance of hyperintensity of optic radiation on T2WI and FLAIR imaging. Long-term follow-up revealed a pattern of steady progression and a variety of clinical symptoms, including spastic paraplegia, peripheral neuropathy, reduced visual acuity, and some degree of cerebellar ataxia. Clinical variability between patients was observed to some extent, and therefore, further studies are required to determine the phenotype-genotype correlation.

Taz1-Shelterin Promotes Facultative Heterochromatin Assembly at Chromosome-Internal Sites Containing Late Replication Origins.

Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 or other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes derepression of associated genes. Moreover, the late-origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We explore the connection between facultative heterochromatin and replication control and show that heterochromatin machinery affects replication timing. These analyses reveal the role of Shelterin in facultative heterochromatin assembly at late origins, which has important implications for genome stability and gene regulation.

NOTCH2NLC GGC Repeat Expansion in Patients With Vascular Leukoencephalopathy.

BACKGROUND: Neuronal intranuclear inclusion disease (NIID), caused by GGC (guanine-guanine-cytosine) repeat expansion in NOTCH2NLC, has several clinical and radiological features akin to cerebral small vessel disease (cSVD). The present study tested the hypothesis that NOTCH2NLC GGC expansion may contribute to cSVD. METHODS: One hundred and ninety-seven unrelated patients with genetically unsolved vascular leukoencephalopathy without NOTCH3, HTRA1, and mitochondrial m.3243A>G mutations and 730 healthy individuals were screened for NOTCH2NLC GGC repeat expansion using repeat-primed polymerase chain reaction, fragment analysis, Southern blot analysis, or nanopore sequencing with Cas9 (CRISPR associated protein 9)-mediated enrichment. The clinical and neuroimaging features of the patients were compared between individuals with and without NOTCH2NLC GGC repeat expansion. RESULTS: Six of the 197 (3.0%) patients with unsolved vascular leukoencephalopathy and none of the controls carried the GGC repeat expansion (P=0.00009). Skin biopsy of 1 patient revealed eosinophilic, ubiquitin-positive, and p62-positive intranuclear inclusions in the cells of sweat gland and capillary, providing pathologic evidence for the involvement of small vessels in NIID. For the 6 patients, gait disturbance and cognitive decline were common manifestations with a median onset age of 65 (59-69) years. They all had multiple neuroimaging features suggestive of cSVD, including diffuse white matter hyperintensities, lacunes, and enlarged perivascular space in all 6 patients, cerebral microbleeds in 5, and old intracerebral hemorrhage in 4. Four patients had linear hyperintensity in the corticomedullary junction on diffusion-weighted imaging-the characteristic neuroimaging feature of NIID. There was no difference in the severity of cSVD imaging features between the patients with and without the GGC expansion but more pronounced brain atrophy in the patients with the GGC expansion. CONCLUSIONS: NOTCH2NLC GGC repeat expansion accounted for 3% of genetically unsolved Taiwanese vascular leukoencephalopathy cases after excluding participants with cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). NIID should be considered in patients manifesting cSVD, especially in those with characteristic neuroimaging feature of NIID.

De novo ARF3 variants cause neurodevelopmental disorder with brain abnormality.

An optimal Golgi transport system is important for mammalian cells. The adenosine diphosphate (ADP) ribosylation factors (ARF) are key proteins for regulating cargo sorting at the Golgi network. In this family, ARF3 mainly works at the trans-Golgi network (TGN), and no ARF3-related phenotypes have yet been described in humans. We here report the clinical and genetic evaluations of two unrelated children with de novo pathogenic variants in the ARF3 gene: c.200A > T (p.Asp67Val) and c.296G > T (p.Arg99Leu). Although the affected individuals presented commonly with developmental delay, epilepsy and brain abnormalities, there were differences in severity, clinical course and brain lesions. In vitro subcellular localization assays revealed that the p.Arg99Leu mutant localized to Golgi apparatus, similar to the wild-type, whereas the p.Asp67Val mutant tended to show a disperse cytosolic pattern together with abnormally dispersed Golgi localization, similar to that observed in a known dominant negative variant (p.Thr31Asn). Pull-down assays revealed that the p.Asp67Val had a loss-of-function effect and the p.Arg99Leu variant had increased binding of the adaptor protein, Golgi-localized, γ-adaptin ear-containing, ARF-binding protein 1 (GGA1), supporting the gain of function. Furthermore, in vivo studies revealed that p.Asp67Val transfection led to lethality in flies. In contrast, flies expressing p.Arg99Leu had abnormal rough eye, as observed in the gain-of-function variant p.Gln71Leu. These data indicate that two ARF3 variants, the possibly loss-of-function p.Asp67Val and the gain-of-function p.Arg99Leu, both impair the Golgi transport system. Therefore, it may not be unreasonable that they showed different clinical features like diffuse brain atrophy (p.Asp67Val) and cerebellar hypoplasia (p.Arg99Leu).

De Novo Truncating Variants in the Last Exon of SEMA6B Cause Progressive Myoclonic Epilepsy.

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10-8; exome-wide threshold: 2.5 × 10-6). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 × 10-13) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

Complete SAMD12 repeat expansion sequencing in a four-generation BAFME1 family with anticipation.

Benign adult familial myoclonic epilepsy type 1 (BAFME1) is an autosomal dominant, adult-onset neurological disease caused by SAMD12 repeat expansion. In BAFME1, anticipation, such as the earlier onset of tremor and/or seizures in the next generation, was reported. This could be explained by intergenerational repeat instability, leading to larger expansions in successive generations. We report a four-generation BAFME1-affected family with anticipation. Using Nanopore long-read sequencing, detailed information regarding the sizes, configurations, and compositions of the expanded SAMD12 repeats across generations was obtained. Unexpectedly, a grandmother-mother-daughter triad showed similar repeat structures but with slight repeat expansions, despite quite variable age of onset of seizures (range: 52-14 years old), implying a complex relationship between the SAMD12 repeat expansion sequence and anticipation. This study suggests that different factor(s) from repeat expansion could modify the anticipation in BAFME1.

Distal arthrogryposis in a girl arising from a novel TNNI2 variant inherited from paternal somatic mosaicism.

TNNI2 at 11p15.5 encodes troponin I2, fast skeletal type, which is a member of the troponin I gene family and a component of the troponin complex. Distal arthrogryposis (DA) is characterized by congenital limb contractures without primary neurological or muscular effects. DA is inherited in an autosomal dominant fashion and is clinically and genetically heterogeneous. Exome sequencing identified a causative variant in TNNI2 [NM_003282.4:c.532T>C p.(Phe178Leu)] in a Japanese girl with typical DA2b. Interestingly, the familial study using Sanger sequencing suggested a mosaic variant in her healthy father. Subsequent targeted amplicon-based deep sequencing detected the TNNI2 variant with variant allele frequencies of 9.4-17.7% in genomic DNA derived from peripheral blood leukocytes, saliva, hair, and nails in the father. We confirmed a disease-causing variant in TNNI2 in the proband inherited from her asymptomatic father with its somatic variant. Our case demonstrates that careful clinical and genetic evaluation is required in DA.

A novel homozygous CHMP1A variant arising from segmental uniparental disomy causes pontocerebellar hypoplasia type 8.

Pontocerebellar hypoplasia (PCH) is currently classified into 16 subgroups. Using mostly next-generation sequencing, pathogenic variants have been identified in as many as 24 PCH-associated genes. PCH type 8 (PCH8) is a rare heterogeneous disorder. Its clinical presentation includes severe development delay, increased muscle tone, microcephaly, and magnetic resonance imaging (MRI) abnormalities such as reduced cerebral white matter, a thin corpus callosum, and brainstem and cerebellar hypoplasia. To date, only two variants in the CHMP1A gene (MIM: 164010), NM_002768.5: c.88 C > T (p.Glu30*) and c.28-13 G > A, have been identified homozygously in seven patients with PCH8 from four families (MIM: 614961). CHMP1A is a subunit of the endosomal sorting complex required for transport III (ESCRT-III), which regulates the formation and release of extracellular vesicles. Biallelic CHMP1A loss of function impairs the ESCRT-III-mediated release of extracellular vesicles, which causes impaired progenitor proliferation in the developing brain. Herein, we report a patient with PCH8 who had a homozygous CHMP1A variant, c.122delA (p.Asn41Metfs*2), which arose from segmental uniparental disomy. Although our patient had similar MRI findings to those of previously reported patients, with no progression, we report some novel neurological and developmental findings that expand our knowledge of the clinical consequences associated with CHMP1A variants.

Long-read sequencing revealing intragenic deletions in exome-negative spastic paraplegias.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and weakness in the lower extremities. To date, a total of 88 types of SPG are known. To diagnose HSP, multiple technologies, including microarray, direct sequencing, multiplex ligation-dependent probe amplification, and short-read next-generation sequencing, are often chosen based on the frequency of HSP subtypes. Exome sequencing (ES) is commonly used. We used ES to analyze ten cases of HSP from eight families. We identified pathogenic variants in three cases (from three different families); however, we were unable to determine the cause of the other seven cases using ES. We therefore applied long-read sequencing to the seven undetermined HSP cases (from five families). We detected intragenic deletions within the SPAST gene in four families, and a deletion within PSEN1 in the remaining family. The size of the deletion ranged from 4.7 to 12.5 kb and involved 1-7 exons. All deletions were entirely included in one long read. We retrospectively performed an ES-based copy number variation analysis focusing on pathogenic deletions, but were not able to accurately detect these deletions. This study demonstrated the efficiency of long-read sequencing in detecting intragenic pathogenic deletions in ES-negative HSP patients.

Three KINSSHIP syndrome patients with mosaic and germline AFF3 variants.

AFF3 at 2q11.2 encodes the nuclear transcriptional activator AF4/FMR2 Family Member 3. AFF3 constitutes super elongation complex like 3, which plays a role in promoting the expression of genes involved in neurogenesis and development. The degron motif in AFF3 with nine highly conserved amino acids is recognized by E3 ubiquitin ligase to induce protein degradation. Recently, AFF3 missense variants in this region and variants featuring deletion including this region were identified and shown to cause KINSSHIP syndrome. In this study, we identified two novel and one previously reported missense variants in the degron of AFF3 in three unrelated Japanese patients. Notably, two of these three variants exhibited mosaicism in the examined tissues. This study suggests that mosaic variants also cause KINSSHIP syndrome, showing various phenotypes.

Neuronal intranuclear inclusion disease in patients with adult-onset non-vascular leukoencephalopathy.

Neuronal intranuclear inclusion disease (NIID), caused by an expansion of GGC repeats in the 5'-untranslated region of NOTCH2NLC, is an important but underdiagnosed cause of adult-onset leukoencephalopathies. The present study aimed to investigate the prevalence, clinical spectrum and brain MRI characteristics of NIID in adult-onset nonvascular leukoencephalopathies and assess the diagnostic performance of neuroimaging features. One hundred and sixty-one unrelated Taiwanese patients with genetically undetermined nonvascular leukoencephalopathies were screened for the NOTCH2NLC GGC repeat expansions using fragment analysis, repeat-primed PCR, Southern blot analysis and/or nanopore sequencing with Cas9-mediated enrichment. Among them, 32 (19.9%) patients had an expanded NOTCH2NLC allele and were diagnosed with NIID. We enrolled another two affected family members from one patient for further analysis. The size of the expanded NOTCH2NLC GGC repeats in the 34 patients ranged from 73 to 323 repeats. Skin biopsies from five patients all showed eosinophilic, p62-positive intranuclear inclusions in the sweat gland cells and dermal adipocytes. Among the 34 NIID patients presenting with nonvascular leukoencephalopathies, the median age at symptom onset was 61 years (range, 41-78 years) and the initial presentations included cognitive decline (44.1%; 15/34), acute encephalitis-like episodes (32.4%; 11/34), limb weakness (11.8%; 4/34) and parkinsonism (11.8%; 4/34). Cognitive decline (64.7%; 22/34) and acute encephalitis-like episodes (55.9%; 19/34) were also the most common overall manifestations. Two-thirds of the patients had either bladder dysfunction or visual disturbance. Comparing the brain MRI features between the NIID patients and individuals with other undetermined leukoencephalopathies, corticomedullary junction curvilinear lesions on diffusion weighted images were the best biomarkers for diagnosing NIID with high specificity (98.4%) and sensitivity (88.2%). However, this diffusion weighted imaging abnormality was absent in 11.8% of the NIID patients. When only fluid-attenuated inversion recovery images were available, the presence of white matter hyperintensity lesions either in the paravermis or middle cerebellar peduncles also favoured the diagnosis of NIID with a specificity of 85.3% and sensitivity of 76.5%. Among the MRI scans of 10 patients, performed within 5 days of the onset of acute encephalitis-like episodes, five showed cortical hyperintense lesions on diffusion weighted images and two revealed focal brain oedema. In conclusion, NIID accounts for 19.9% (32/161) of patients with adult-onset genetically undiagnosed nonvascular leukoencephalopathies in Taiwan. Half of the NIID patients developed encephalitis-like episodes with restricted diffusion in the cortical regions on diffusion weighted images at the acute stage. Corticomedullary junction hyperintense lesions, white matter hyperintensities in the paravermis or middle cerebellar peduncles, bladder dysfunction and visual disturbance are useful hints to diagnosing NIID.

Repeat conformation heterogeneity in cerebellar ataxia, neuropathy, vestibular areflexia syndrome.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, slow-progressing multisystem neurodegenerative disorder. Biallelic AAGGG repeat expansion in RFC1 has been identified as causative of this disease, and repeat conformation heterogeneity (ACAGG repeat) was also recently implied. To molecularly characterize this disease in Japanese patients with adult-onset ataxia, we accumulated and screened 212 candidate families by an integrated approach consisting of flanking PCR, repeat-primed PCR, Southern blotting and long-read sequencing using Sequel II, GridION or PromethION. We identified 16 patients from 11 families, of whom seven had ACAGG expansions [(ACAGG)exp/(ACAGG)exp] (ACAGG homozygotes), two had ACAGG and AAGGG expansions [(ACAGG)exp/(AAGGG)exp] (ACAGG/AAGGG compound heterozygotes) and seven had AAGGG expansions [(AAGGG)exp/(AAGGG)exp] (AAGGG homozygotes). The overall detection rate was 5.2% (11/212 families including one family having two expansion genotypes). Long-read sequencers revealed the entire sequence of both AAGGG and ACAGG repeat expansions at the nucleotide level of resolution. Clinical assessment and neuropathology results suggested that patients with ACAGG expansions have similar clinical features to previously reported patients with homozygous AAGGG expansions, although motor neuron involvement was more notable in patients with ACAGG expansions (even if one allele was involved). Furthermore, a later age of onset and slower clinical progression were implied in patients with ACAGG/AAGGG compound heterozygous expansions compared with either ACAGG or AAGGG homozygotes in our very limited cohort. Our study clearly shows the occurrence of repeat conformation heterogeneity, with possible different impacts on the affected nervous systems. The difference in disease onset and progression between compound heterozygotes and homozygotes might also be suspected but with very limited certainty due to the small sample number of cases in our study. Studies of additional patients are needed to confirm this.