44Forty-four genomic sites that exhibited differential variation between recurrent and primary samples

Figure S1. Targeted gene information and clinical breast cancer samples used for deep sequencing.

We report a case of adrenomyeloneuropathy caused by a novel ABCD1 variant complicated by recurrent herpes simplex virus type 2 meningitis, with no evidence of either any clinical or radiological progression. Given previous reports describing temporal associations between infections and the recognition of cerebral adrenoleukodystrophy (ALD), careful continuous observation of the present case with attention to the development of cerebral ALD is required. The early recognition of cerebral ALD is mandatory to achieve better outcomes with hematopoietic stem cell transplantation.

Patients’ characteristics and clinical information.

Figure S5. Comparison of non-coding variant sites and their counts between primary and recurrent samples.

Figure S6. Oncoprint analysis of the 26 targeted loci in clinical breast cancer samples from the TCGA study.

Detection of recurrence and metastasis in patient samples using targeted cfDNA nucleosome profiling. A, Schematic illustration of nucleosome occupancy based on a per-base WPS, calculated by subtracting the number of fragment endpoints within a 120-bp window (indicated by B) from the number of fragments completely spanning the window (indicated by A). B, Hierarchical clustering of cfDNA WPS value at 44 genomic sites that were differentially altered (Wilcoxon rank-sum test, Padj < 0.5), based on the log2 FC between primary (gray) and recurrent (pink) samples. C, Volcano plots showing differential nucleosome occupancy based on cfDNA WPS values, plotted as log2FC vs. −log10 (Padj). Solid and dashed horizontal lines indicate Padj thresholds of 0.3 and 0.05, respectively. Representative genomic loci, including the top four sites with open or closed nucleosomes in the ranked list (Supplementary Table S4), are indicated. D, Mean adjusted WPS values for primary (N = 105; gray) and recurrent (N = 45; pink) samples at the top four sites indicated in C. E, Comparison of nucleosome score calculated as z-scores of WPS across 19 genomic sites (Padj < 0.3) between primary (N = 105) and recurrent (N = 45) samples. Asterisks denote statistical significance with Mann-Whitney U test (****, P < 0.0001). F, Relationship between nucleosome scores and ctDNA aneuploidy fraction assessed by Spearman’s correlation in primary (N = 105; pink) and recurrent samples (N = 45; blue). G, Comparison of nucleosome scores calculated as z-scores of WPS at the top two genomic loci (Padj < 0.05), including the SYNPO2 locus and two 10-nt windows upstream and downstream, between primary (N = 105) and recurrent (N = 45) samples. Asterisks denote statistical significance with Mann-Whitney U test (****, P < 0.0001). H, ROC curves of nucleosome scores and histologic grade in primary (N = 105) and recurrent (N = 45) samples. The 95% confidence intervals for the AUC are shown.

WPS classification by log2 fold change between recurrent and primary samples

PURPOSE:Despite advances in diagnostics, many inherited peripheral neuropathies remain genetically unexplained. We investigated whether biallelic variants in FAT3 (FAT Atypical Cadherin 3) are implicated in inherited axonal neuropathies. METHODS:We identified biallelic FAT3 variants in three unrelated individuals among 3315 Japanese patients with inherited peripheral neuropathies. Variants were evaluated by segregation analysis, in silico modeling, and functional studies in Drosophila and mouse models. RESULTS:All patients exhibited progressive distal muscle weakness and cranial nerve involvement, including tongue atrophy, dysarthria, and facial weakness. Two required ventilatory support because of respiratory muscle paralysis. One patient additionally showed central hypomyelination, autonomic dysfunction, and developmental anomalies, such as congenital scoliosis and intestinal pseudo-obstruction. Identified variants were ultrarare, affected conserved residues, segregated with disease, and were predicted to impair domain stability. FAT3 knockdown in Drosophila resulted in rough eye phenotype, shortened lifespan, impaired motor function, and defective motor neuron branching. Fat3 knockout and knockin mice displayed perinatal lethality, sciatic nerve axonal degeneration, and central nervous system abnormalities despite preserved motor performance. CONCLUSION:Our findings establish FAT3 as a novel gene for autosomal-recessive axonal neuropathies and support the concept of a FAT3-related multisystem neurodevelopmental disorder characterized by motor neuron degeneration and systemic abnormalities.

Hypomyelinating leukodystrophy 15 (HLD15) results from biallelic pathogenic variants in EPRS1, but exonic deletions have not been reported. We describe a 40-year-old woman with mild intellectual disability, ataxia, dystonia, and MRI showing hypomyelination. Whole-exome sequencing identified a heterozygous missense variant in the prolyl-tRNA synthetase domain of EPRS1 (c.3430 C > G; p.Leu1144Val, NM_004446.3), without second variant. Whole-genome sequencing revealed a heterozygous 220-bp deletion spanning exon 15 (c.1743-30_1932del), and segregation analysis confirmed compound heterozygosity. RT-PCR from lymphoblastoid cells demonstrated exon-15 skipping leading to a frameshift (p.Asn582Serfs*10) and nonsense-mediated decay, leaving predominant expression of the paternally inherited missense allele. These findings support loss-of-function for the deletion and classify c.3430 C > G as likely pathogenic under ACMG/AMP criteria (PM1, PM2, PM3, PP3). This case represents the first exonic deletion reported in EPRS1. The relatively mild, adult-onset phenotype broadens both mutational and clinical spectra of HLD15 and highlights the importance of structural-variant analysis when only a single candidate variant is detected in recessive leukodystrophy.

Targeted cfDNA sequencing to assess nucleosome architecture, genomic variants, and CNAs. A, Schematic overview of study design and analytic workflow. InDel, insertion and deletion; SNV, single-nucleotide variation. B, Per-sample overview of nucleosome features, genomic variants, CNAs, tumor fraction, and clinical information. Each vertical line indicates an individual patient sample, and each horizontal line indicates the analyzed data, as shown by the corresponding color scheme on the right. ctDNA aneuploidy fraction and VC, SD of CNV, and nucleosome architecture are shown in C and D, Figs. 2 and 3, respectively. TNBC, triple-negative breast cancer. C, The ctDNA aneuploidy fraction in cfDNA estimated using ichorCNA in primary (N = 105; gray) and recurrent (N = 45; pink) samples. D, Comparison of VC coding and noncoding regions and SVs detected in primary samples (N = 105) vs. recurrent samples (N = 45).

Figure S7. Quantification of cfDNA coverage across 26 genomic loci and nucleosome scores in the HER2-positive subset.

Distribution of fragment lengths and cfDNA coverage across 26 genomic loci. A, Fragment length frequency distribution of genomic (top) and mitochondrial (bottom) fraction from primary (gray) and recurrent (pink) samples. B, Comparison of fragment ratios of targeted genes in relation to CNA, calculated as the ratio of fragments shorter than 140 bp to peak fragment counts, in primary (N = 105; gray) and recurrent (N = 45; pink) samples. C, Quantification of cfDNA coverage across the 26 genomic loci. D, Comparison of cfDNA coverage of targeted genes between primary and recurrent/metastatic samples. Asterisks denote statistical significance with Welch’s t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001 for the indicated comparison. E, Comparison of the SD of CNAs between primary (N = 105; gray) and recurrent/metastatic (N = 45; pink) samples. F, Relationship between CNAs and ctDNA aneuploidy fraction assessed by Spearman’s correlation in primary (N = 105; pink) and recurrent (N = 45; blue) samples.

BackgroundAnti-amyloid-β (anti-Aβ) therapy slows cognitive decline but paradoxically accelerates whole-brain gray matter atrophy, complicating outcome prediction.ObjectiveTo identify early imaging markers that predict long-term prognoses.MethodsThis longitudinal cohort study prospectively enrolled participants with early Alzheimer's disease, initiating anti-Aβ therapy. We performed automated T1-weighted MRI volumetric analysis of the whole-brain gray matter, hippocampus, and white matter hyperintensities (WMH). Temporal dynamics were characterized using piecewise linear regression, and predictive utility was assessed using multivariate linear regression analyses.ResultsTwenty-two participants (74 ± 11 years; 14 women) were followed for 376 ± 146 days. Significant volume changes occurred in all regions (p < 0.01). No overt amyloid-related edema was detected, and hemorrhagic events were mild to moderate. Piecewise regression revealed an initial WMH surge that decelerated after 60 days (β=-0.19 [95%CI: -0.33, -0.042], p = 0.01), whereas gray matter regions showed linear volume decline. Early subthreshold WMH expansion predicted long-term atrophy independent of baseline covariates (age, sex, amyloid burden, mini-mental state examination score, drug, and hemorrhagic events) in the hippocampus (β=-0.058 [95%CI: -0.092, -0.025], p = 0.003), and whole-brain gray matter (β=-0.033 [95%CI: -0.065, -0.002], p = 0.04). Early whole-brain gray matter atrophy correlated with the WMH surge (β=-0.53 [95%CI: -0.69, -0.38], p < 0.001) and predicted long-term hippocampal atrophy (β=0.098 [95%CI: 0.038, 0.16], p = 0.004).ConclusionsA transient WMH surge within the first 60 days of therapy mirrors the known time course of amyloid-related imaging abnormalities. Along with concurrent early whole-brain volume loss, this surge independently predicts accelerated long-term hippocampal and whole-brain atrophy, highlighting its potential as an early prognostic imaging marker.

Germline variants in leukemia predisposition genes are increasingly detected in pediatric patients. Nevertheless, the rare variants identified in diagnostic samples may be misinterpreted without variant-level functional evaluation and cautious clinical interpretation. Whole-exome sequencing was performed on 28 individuals from five kindreds in which at least two children developed acute leukemia. Results identified a rare ETV6 variant, c.604C > G (p.Arg202Gly), in monozygotic twins with ETV6::RUNX1-positive B-cell precursor acute lymphoblastic leukemia. To support variant interpretation, HeLa-cell populations stably expressing FLAG-tagged wild-type ETV6 and p.Arg202Gly and the known loss-of-function control p.Pro214Leu were established. Subcellular localization was assessed via immunofluorescence microscopy with quantitative scoring. Transcriptional repression was evaluated using luciferase reporter assays driven by ETV6 target promoters (MMP3 and PF4). p.Arg202Gly predominantly showed nuclear localization comparable to WT, whereas p.Pro214Leu was enriched in the cytoplasm. In the reporter assays, similar to WT, p.Arg202Gly retained repression activity. Meanwhile, p.Pro214Leu failed to repress both reporters. The in-silico prediction of nuclear export sequences suggested an additional export signal in p.Pro214Leu, but not in p.Arg202Gly. Collectively, these findings indicate that p.Arg202Gly behaves as WT-like in the assays performed and do not support a loss-of-function effect. Our study emphasizes the importance of variant-level functional assessment for rare ETV6 variants to inform clinical interpretation and avoid overestimation of pathogenicity.

Somatic variants analyzed using the maftools

Figure S2. Comparison of variant counts in coding and non-coding regions.

BackgroundDistal hereditary motor neuropathy (dHMN) is characterized by slowly progressive distal muscle weakness and amyotrophy, and it exhibits clinical overlap with Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis (ALS). Biallelic variants in SIGMAR1, encoding sigma nonopioid intracellular receptor 1, have been linked to autosomal recessive dHMN with pyramidal features. This study investigated the clinical and genetic features of patients with dHMN associated with SIGMAR1 variants in Japan.MethodsWe conducted genetic screening of Japanese patients with clinically suspected inherited peripheral neuropathies using targeted gene panels and whole-exome sequencing. SIGMAR1 variants were evaluated via segregation analysis using Sanger sequencing. Detailed clinical and electrophysiological data were systematically reviewed.ResultsBiallelic SIGMAR1 variants, including three novel variants and one previously reported variant, were identified in six patients from five unrelated families. The genotypes comprised compound heterozygous variants in four patients and homozygous variants in two patients. All patients presented with early-onset distal muscle weakness and atrophy. Enhanced tendon reflexes and pyramidal tract signs were frequently observed, whereas bulbar or respiratory involvement was absent. Nerve conduction studies consistently revealed motor-predominant axonal neuropathy with minimal sensory involvement. Disease progression was slow, and all patients remained ambulatory for years to decades after onset.ConclusionOur findings expand the clinical and genetic spectrum of SIGMAR1-associated disease and support its classification as dHMN rather than ALS. SIGMAR1 variants should be considered in the genetic evaluation of early-onset motor neuropathies, particularly in patients with dHMN accompanied by pyramidal features.