Ryan K. C. Yuen

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect. Ninety percent of isolated cases remain genotype-elusive despite short-read genome sequencing (GS). Our goal was to assess genomic yield with long-read GS compared with short-read GS in CHD. METHODS: We performed Illumina short-read GS on 1101 CHD probands. In a subset of 46 genotype-elusive probands, we performed additional PacBio long-read GS as well as parental sequencing of 7 complete trios. We compared variant calls genome-wide, including across dark and CHD genes in samples with paired short-read and long-read GS. RESULTS: Paired analysis of the 46 probands revealed that genome-wide, long-read GS had 1.01- to 7.93-fold higher call rates of single nucleotide variants, deletions, duplications, and insertions but fewer indels and inversions compared with short-read GS. With a genome-wide sequencing depth of 18.3×, long-read GS had higher coverage for 10 Tier 1 CHD genes and dark genes, but nonuniform and low sequencing depth (ZEB2 in a CHD proband who had an extracardiac phenotype overlapping with Mowat-Wilson syndrome. CONCLUSIONS: Long-read GS demonstrated higher genome-wide variant call yield compared with short-read GS, better coverage of several cardiac-relevant genes, and better resolution of complex structural variants, including tandem repeat expansions. Long-read GS may provide an option for a subset of patients with CHD who remain genotype-elusive on short-read GS.

Congenital heart disease (CHD) affects approximately 1% of live births, yet a substantial proportion of cases remain genetically unexplained. Tetralogy of Fallot (TOF) is among the most common cyanotic CHDs. Tandem repeat expansions (TREs) can alter gene regulation and have been implicated in complex cardiac disease, but their contribution to structural CHD remains poorly understood. We performed genome-wide analysis of rare TREs using short-read genome sequencing in 835 individuals with TOF and 386 controls, complemented by PacBio HiFi long-read sequencing, DNA methylation profiling, myocardial RNA sequencing, and fetal human heart single-cell RNA-seq. We further compared TRE-associated genes and biological processes between TOF and a previously characterized cardiomyopathy cohort. We identified 1,043 rare TREs in individuals with TOF, corresponding to 1.25 rare TREs per individual, compared with 0.85 per individual in controls. TOF-associated rare TREs were enriched for GC-rich motifs, more often found in the 5′ untranslated regions (UTR; OR = 7.0, p = 2.1 x 10^-2) and splicing regions (OR = 3.0, p = 9 x 10^-4), and located closer to transcription start sites and splice junctions, consistent with a regulatory role. We identified 91 recurrent genic TRE loci, including recurrent 5′ UTR TREs absent from controls and TREs in the CHD-associated genes PACS1 and TRIP4. Long-read analysis identified 36 loci at which repeat size was significantly associated with DNA methylation, revealing both expansion-associated hypermethylation and hypomethylation. TRE-associated genes were preferentially expressed in smooth muscle cells, pericytes, and cardiomyocytes in the developing human heart. TRE-associated biological pathways were largely distinct between TOF and cardiomyopathy. Notably, rare TRE burden was higher among individuals without positive clinical genetic testing results. We estimated an excess burden of rare genic TREs of approximately 4.4% in the TOF cohort. Rare TREs represent an underrecognized class of genetic variation contributing to TOF. Their enrichment in regulatory regions, association with locus-specific DNA methylation and transcriptional alterations, and increased burden among genetically unexplained cases support a role for TRE-mediated regulatory dysfunction in CHD pathogenesis and suggest that assessment of tandem repeat variation may improve genetic characterization of TOF.

Autism spectrum disorder (ASD) is a highly heritable and heterogeneous neurodevelopmental condition for which currently identified genetic variants explain only a subset of cases. Tandem repeat expansions (TREs) have recently emerged as an underappreciated source of genetic risk for ASD and other complex disorders. Advances in genome sequencing technologies, from short-read to long-read platforms, have substantially expanded the discovery of TREs and their contribution to ASD-risk, implicating their involvement in ASD etiology. In this review, we summarize recent advances in the genetics of ASD and discuss how the molecular mechanisms of established TREs provide mechanistic insights into ASD biology. We highlight the FMR1 CGG and DMPK CTG repeat expansions, which, through distinct pathogenic mechanisms, contribute to the high prevalence of ASD in fragile X syndrome and congenital myotonic dystrophy type 1, respectively. Emerging evidence suggests that disruption of post-transcriptional gene regulation, particularly developmental RNA splicing, may represent a convergent pathway across syndromic and idiopathic ASD. Finally, we discuss the emerging therapeutic strategies and consider how advances in TRE disease biology may facilitate further development of potential therapies for ASD.

Schizophrenia develops in one in every four individuals with a pathogenic 22q11.2 deletion, yet the genetic modifiers influencing the manifestation of schizophrenia in this high-risk group remain incompletely understood. Here, we identify rare tandem repeat expansions (TREs) as significant contributors to schizophrenia risk in this population. Genome sequencing of 438 unrelated individuals with 22q11.2 deletions revealed a marked enrichment of rare genic TREs among those with schizophrenia, with effect sizes comparable to common polygenic risk. These TREs are disproportionately located in intronic and splice-adjacent regions relative to other genomic regions, with evidence suggesting that they disrupt gene regulation through mechanisms including altered methylation and splicing. Cell-type-specific analyses indicate that TREs are primarily associated with differentially expressed genes in excitatory and inhibitory neurons in the prefrontal cortex. Affected genes, including DLGAP2 and DMPK, are involved in neurodevelopment and synaptic organization. These findings extend the role of TREs as genetic modifiers, providing new insights into the molecular mechanisms underlying schizophrenia in this ultra-high-risk population and into the broader biology of idiopathic schizophrenia.

Short tandem repeat (STR) length is a known determinant of pathogenicity in a variety of human disorders. The repeat sequence itself can modulate disease severity and penetrance; however, the broader impact of STR sequence variation on gene expression in the general population remains poorly understood. Here, we analyze the sequence composition of STRs across two general population cohorts of unrelated individuals (n = 3,150) and report that ~ 7% of STRs exhibit sequence variability, with distinct patterns observed among different ethnic groups. These variable repeats are more prone to expansion and are frequently found in proximity to Alu elements. Notably, STRs with variable motifs are often found near splice junctions of genes involved in brain and neuronal functions. This is supported by the differential expression of genes associated with neuron and cellular projection functions, driven by the presence of distinct STR sequences. Our findings underscore the previously unrecognized role of STR sequence variability in modulating gene expression and contributing to human phenotypic diversity.

Psychiatric conditions share common genes, but mechanisms that differentiate diagnoses remain unclear. We present a multidimensional framework for functional analysis of rare copy number variants (CNVs) across 6 diagnostic categories, including schizophrenia (SCZ), autism (ASD), bipolar disorder (BD), depression (MDD), PTSD, and ADHD (N = 574,965). Using gene-set burden analysis (GSBA), we tested duplication (DUP) and deletion (DEL) burden across 2,645 functional gene sets defined by the intersections of pathways, cell types, and cortical regions. While diagnoses converge on shared pathways, mixed-effects modeling revealed divergence of pathway effects by cell type, brain region, and gene dosage. Factor analysis identified latent dimensions aligned with clinical axes. A primary factor (F1) captured reciprocal dose-dependent effects of DUP and DEL in SCZ reflecting positive and negative effects in excitatory versus inhibitory neurons and association versus sensory cortex. SCZ and ASD were both strongly aligned with F1 but with opposing directionalities. Orthogonal factors highlighted neuronal versus non-neuronal effects in mood disorders (F2) and differential spatial distributions of DEL effects in ADHD and MDD (F3). High-impact CNVs at 16p11.2 and 22q11.2 were enriched for combinations of cell-type-specific genes involved in pathways consistent with our broader findings. These results reveal molecular and cellular mechanisms that are broadly shared across psychiatric traits but differ between diagnostic categories in context and directionality.