Utility of long-read sequencing for All of Us.

The All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compare the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis reveals substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also consider the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produce the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results lead to widespread improvements across AoU.

Design of large-insert jumping libraries for structural variant detection using Illumina sequencing.

Next-generation sequencing is an important and efficient tool for the identification of structural variation, particularly balanced chromosomal rearrangements, because such events are not routinely detected by microarray and localization of altered regions by karyotype is imprecise. Indeed, the degree of resolution that can be obtained through next-generation technologies enables elucidation of precise breakpoints and has facilitated the discovery of numerous pathogenic loci in human disease and congenital anomalies. The protocol described here explains one type of large-insert "jumping library" and the steps required to generate such a library for multiplexed sequencing using Illumina sequencing technology. This approach allows for cost-efficient multiplexing of samples and provides a very high yield of fragments with large inserts, or "jumping" fragments.

Disruption of MBD5 contributes to a spectrum of psychopathology and neurodevelopmental abnormalities.

Microdeletions of chromosomal region 2q23.1 that disrupt MBD5 (methyl-CpG-binding domain protein 5) contribute to a spectrum of neurodevelopmental phenotypes; however, the impact of this locus on human psychopathology has not been fully explored. To characterize the structural variation landscape of MBD5 disruptions and the associated human psychopathology, 22 individuals with genomic disruption of MBD5 (translocation, point mutation and deletion) were identified through whole-genome sequencing or cytogenomic microarray at 11 molecular diagnostic centers. The genomic impact ranged from a single base pair to 5.4 Mb. Parents were available for 11 cases, all of which confirmed that the rearrangement arose de novo. Phenotypes were largely indistinguishable between patients with full-segment 2q23.1 deletions and those with intragenic MBD5 rearrangements, including alterations confined entirely to the 5'-untranslated region, confirming the critical impact of non-coding sequence at this locus. We identified heterogeneous, multisystem pathogenic effects of MBD5 disruption and characterized the associated spectrum of psychopathology, including the novel finding of anxiety and bipolar disorder in multiple patients. Importantly, one of the unique features of the oldest known patient was behavioral regression. Analyses also revealed phenotypes that distinguish MBD5 disruptions from seven well-established syndromes with significant diagnostic overlap. This study demonstrates that haploinsufficiency of MBD5 causes diverse phenotypes, yields insight into the spectrum of resulting neurodevelopmental and behavioral psychopathology and provides clinical context for interpretation of MBD5 structural variations. Empirical evidence also indicates that disruption of non-coding MBD5 regulatory regions is sufficient for clinical manifestation, highlighting the limitations of exon-focused assessments. These results suggest an ongoing perturbation of neurological function throughout the lifespan, including risks for neurobehavioral regression.

Candidate genes and their interactions with other genetic/environmental risk factors in the etiology of schizophrenia.

Identification of causative factors for common, chronic disorders is a major focus of current human health science research. These disorders are likely to be caused by multiple etiological agents. Available evidence also suggests that interactions between the risk factors may explain some of their pathogenic effects. While progress in genomics and allied biological research has brought forth powerful analytic techniques, the predicted complexity poses daunting analytic challenges. The search for pathogenesis of schizophrenia shares most of these challenges. We have reviewed the analytic and logistic problems associated with the search for pathogenesis. Evidence for pathogenic interactions is presented for selected diseases and for schizophrenia. We end by suggesting 'recursive analyses' as a potential design to address these challenges. This scheme involves initial focused searches for interactions motivated by available evidence, typically involving identified individual risk factors, such as candidate gene variants. Putative interactions are tested rigorously for replication and for biological plausibility. Support for the interactions from statistical and functional analyses motivates a progressively larger array of interactants that are evaluated recursively. The risk explained by the interactions is assessed concurrently and further elaborate searches may be guided by the results of such analyses. By way of example, we summarize our ongoing analyses of dopaminergic polymorphisms, as well as infectious etiological factors in schizophrenia genesis.

Cognitive requirements for vestibular and ocular motor processing in healthy adults and patients with unilateral vestibular lesions.

This study investigated the role of cognition in the vestibulo-ocular reflex (VOR) and ocular pursuit using a dual-task paradigm in patients with unilateral peripheral vestibular loss and healthy adults. We hypothesized that cognitive resources are involved in successful processing and integration of vestibular and ocular motor sensory information, and this requirement would be greater in patients with vestibular dysfunction. Sixteen well-compensated patients with surgically confirmed absent unilateral peripheral vestibular function and 16 healthy age- and sex-matched controls underwent seven combinations of vestibular-only, visual-only, and visual-vestibular stimuli while performing three different information processing tasks. Visual-vestibular stimuli included a semicircular canal and an otolith stimulus provided through seated chair rotations; fixation on a laser target and sinusoidal smooth pursuit while still; and fixation on a head-fixed laser target during chair rotations. The information processing tasks were three different auditory reaction time (RT) tasks: (1) simple RT, (2) disjunctive RT, and (3) choice RT. Our results showed increases in RTs in both patients and controls under all vestibular-only stimulation conditions and during ocular pursuit. Patients showed greater increases in RTs during vestibular stimulation and the more complex disjunctive and choice RT tasks. No differences between the groups were found during the visual-only or visual-vestibular interaction conditions. These results reveal interference between vestibulo-ocular processing and a concurrent RT task, suggesting that the VOR and the ocular motor system are dependent upon cognitive resources to some extent, and thus, are not fully automatic systems. We speculate that this interference with cognition occurs as a result of the sensory integration required for resolving inputs from multiple sensory streams. The particularly large decrement in information processing task performance of the patients compared with controls during vestibular stimulation suggests that compensation for unilateral vestibular loss requires continued cognitive resources.

Association and linkage analysis of RGS4 polymorphisms with schizophrenia and bipolar disorder in Brazil.

Linkage and association studies in five independently ascertained samples have suggested that polymorphisms of the regulator of G-protein signaling 4 (RGS4) may confer risk for schizophrenia (SCZ). Suggestive evidence for association with bipolar disorder (BD) has also been presented. However, the associated alleles and haplotypes have differed among the samples. Data from other independent samples may clarify the putative associations. Hence, we investigated an independent, ethnically diverse Brazilian population comprising patients with SCZ (n=271) or BD1 (n=306), who were contrasted with 576 community-based controls. Parents of 49 SCZ cases and 44 BD cases were available for transmission disequilibrium tests (TDTs). Four RGS4 single-nucleotide polymorphisms (SNPs) 1, 4, 7 and 18 putatively associated with SCZ were investigated. In the SCZ samples, significant case-control differences were not observed for individual SNPs or haplotypes, though the TDT suggested transmission distortion similar to that observed in the initial report. For the BD sample, case-control comparisons revealed no significant differences for individual SNPs, but an omnibus test suggested differences in the overall distribution of haplotypes bearing all four SNPs (SNP-EM Omnibus likelihood ratio test; P=0.003). The TDT revealed over-transmission of allele A at SNP7 (P=0.016), as well as haplotypes incorporating this allele. However, global tests incorporating all haplotypes yielded only suggestive trends for association (P=0.19). In conclusion, association with SCZ was not detected in the present analyses. The failure to detect an association may be related to inadequate power or to confounds related to ethnic admixture. Suggestive associations with BD detected here require further investigation in a larger sample.

Genetic polymorphisms of the RGS4 and dorsolateral prefrontal cortex morphometry among first episode schizophrenia patients.

Polymorphisms of the gene encoding the regulator of G-protein signaling subtype 4 (RGS4) may confer risk for schizophrenia.(1) DNA microarray studies of postmortem brain samples have shown RGS4 underexpression in the dorsolateral prefrontal cortex (DLPFC, area 9), motor and visual cortices in schizophrenia patients relative to control subjects.(2) Underexpression of RGS4 in DLPFC is pathophysiologically significant because DLPFC pathology in schizophrenia has been supported by neurocognitive,(3,4) structural(5) and functional(6,7) imaging, postmortem,(8) cellular(9,10) and molecular(11) pathological studies. For these reasons, we examined the association of DLPFC gray matter volume with RGS4 polymorphisms in a series of antipsychotic-naive first-episode schizophrenia patients and control subjects. We hypothesized that volumetric alterations of the DLPFC would be associated with RGS4 polymorphisms and that these differences would be more pronounced in patients than in controls. We observed robust volumetric differences across the genotypes in the pooled sample of patients and control subjects; when separately analyzed, we observed differences within the patient group (n = 30) but not in control subject (n = 27) group. The findings suggest that RGS4 polymorphisms may contribute to structural alterations in the DLPFC.