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PURPOSE: Mendelian disease genomic research has undergone a massive transformation over the past decade. With increasing availability of exome and genome sequencing, the role of Mendelian research has expanded beyond data collection, sequencing, and analysis to worldwide data sharing and collaboration. METHODS: Over the past 10 years, the National Institutes of Health–supported Centers for Mendelian Genomics (CMGs) have played a major role in this research and clinical evolution. RESULTS: We highlight the cumulative gene discoveries facilitated by the program, biomedical research leveraged by the approach, and the larger impact on the research community. Beyond generating a list of gene-phenotype relationships and participating in widespread data sharing, the CMGs have created resources, tools, and training for the larger community to foster understanding of genes and genome variation. The CMGs have participated in a wide range of data sharing activities, including deposition of all eligible CMG data into the Analysis, Visualization, and Informatics Lab-space (AnVIL), sharing candidate genes through the Matchmaker Exchange and the CMG website, and sharing variants in Genotypes to Mendelian Phenotypes (Geno2MP) and VariantMatcher. CONCLUSION: The work is far from complete; strengthening communication between research and clinical realms, continued development and sharing of knowledge and tools, and improving access to richly characterized data sets are all required to diagnose the remaining molecularly undiagnosed patients.

Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7 Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis.

Despite release of the GRCh38 human reference genome more than seven years ago, GRCh37 remains more widely used by most research and clinical laboratories. To date, no study has quantified the impact of utilizing different reference assemblies for the identification of variants associated with rare and common diseases from large-scale exome-sequencing data. By calling variants on both the GRCh37 and GRCh38 references, we identified single-nucleotide variants (SNVs) and insertion-deletions (indels) in 1,572 exomes from participants with Mendelian diseases and their family members. We found that a total of 1.5% of SNVs and 2.0% of indels were discordant when different references were used. Notably, 76.6% of the discordant variants were clustered within discrete discordant reference patches (DISCREPs) comprising only 0.9% of loci targeted by exome sequencing. These DISCREPs were enriched for genomic elements including segmental duplications, fix patch sequences, and loci known to contain alternate haplotypes. We identified 206 genes significantly enriched for discordant variants, most of which were in DISCREPs and caused by multi-mapped reads on the reference assembly that lacked the variant call. Among these 206 genes, eight are implicated in known Mendelian diseases and 53 are associated with common phenotypes from genome-wide association studies. In addition, variant interpretations could also be influenced by the reference after lifting-over variant loci to another assembly. Overall, we identified genes and genomic loci affected by reference assembly choice, including genes associated with Mendelian disorders and complex human diseases that require careful evaluation in both research and clinical applications.

Xia-Gibbs syndrome is a rare mendelian disorder characterized by Development Delay (DD), intellectual disability (ID), and hypotonia. Individuals with XGS typically harbor de novo protein-truncating mutations in the AT-Hook DNA binding motif containing 1 (AHDC1) gene, although some missense mutations can also cause XGS. Large de novo heterozygous deletions that encompass the AHDC1 gene have also been ascribed as diagnostic for the disorder, without substantial evidence to support their pathogenicity. We analyzed 19 individuals with large contiguous deletions involving AHDC1, along with other genes. One individual bore the smallest known contiguous AHDC1 deletion (∼350 Kb), encompassing eight other genes within chr1p36.11 (Feline Gardner-Rasheed, IFI6, FAM76A, STX12, PPP1R8, THEMIS2, RPA2, SMPDL3B) and terminating within the first intron of AHDC1. The breakpoint junctions and phase of the deletion were identified using both short and long read sequencing (Oxford Nanopore). Quantification of RNA expression patterns in whole blood revealed that AHDC1 exhibited a mono-allelic expression pattern with no deficiency in overall AHDC1 expression levels, in contrast to the other deleted genes, which exhibited a 50% reduction in mRNA expression. These results suggest that AHDC1 expression in this individual is compensated by a novel regulatory mechanism and advances understanding of mutational and regulatory mechanisms in neurodevelopmental disorders.

Genomic sequencing and clinical genomics have demonstrated that substantial subsets of atypical and/or severe disease presentations result from multilocus pathogenic variation (MPV) causing blended phenotypes. In an infant with a severe neurodevelopmental disorder, four distinct molecular diagnoses were found by exome sequencing (ES). The blended phenotype that includes brain malformation, dysmorphism, and hypotonia was dissected using the Human Phenotype Ontology (HPO). ES revealed variants in CAPN3 (c.259C > G:p.L87V), MUSK (c.1781C > T:p.A594V), NAV2 (c.1996G > A:p.G666R), and ZC4H2 (c.595A > C:p.N199H). CAPN3, MUSK, and ZC4H2 are established disease genes linked to limb-girdle muscular dystrophy (OMIM# 253600), congenital myasthenia (OMIM# 616325), and Wieacker-Wolff syndrome (WWS; OMIM# 314580), respectively. NAV2 is a retinoic-acid responsive novel disease gene candidate with biological roles in neurite outgrowth and cerebellar dysgenesis in mouse models. Using semantic similarity, we show that no gene identified by ES individually explains the proband phenotype, but rather the totality of the clinically observed disease is explained by the combination of disease-contributing effects of the identified genes. These data reveal that multilocus pathogenic variation can result in a blended phenotype with each gene affecting a different part of the nervous system and nervous system-muscle connection. We provide evidence from this n = 1 study that in patients with MPV and complex blended phenotypes resulting from multiple molecular diagnoses, quantitative HPO analysis can allow for dissection of phenotypic contribution of both established disease genes and novel disease gene candidates not yet proven to cause human disease.

In October 2020, 62 scientists from nine nations worked together remotely in the Second Baylor College of Medicine & DNAnexus hackathon, focusing on different related topics on Structural Variation, Pan-genomes, and SARS-CoV-2 related research. The overarching focus was to assess the current status of the field and identify the remaining challenges. Furthermore, how to combine the strengths of the different interests to drive research and method development forward. Over the four days, eight groups each designed and developed new open-source methods to improve the identification and analysis of variations among species, including humans and SARS-CoV-2. These included improvements in SV calling, genotyping, annotations and filtering. Together with advancements in benchmarking existing methods. Furthermore, groups focused on the diversity of SARS-CoV-2. Daily discussion summary and methods are available publicly at https://github.com/collaborativebioinformatics provides valuable insights for both participants and the research community.

Summary Robinow syndrome (RS) is a genetically heterogeneous disorder with six genes that converge on the WNT/planar cell polarity (PCP) signaling pathway implicated (DVL1, DVL3, FZD2, NXN, ROR2, and WNT5A). RS is characterized by skeletal dysplasia and distinctive facial and physical characteristics. To further explore the genetic heterogeneity, paralog contribution, and phenotypic variability of RS, we investigated a cohort of 22 individuals clinically diagnosed with RS from 18 unrelated families. Pathogenic or likely pathogenic variants in genes associated with RS or RS phenocopies were identified in all 22 individuals, including the first variant to be reported in DVL2. We retrospectively collected medical records of 16 individuals from this cohort and extracted clinical descriptions from 52 previously published cases. We performed Human Phenotype Ontology (HPO) based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. Individuals with FZD2 variants clustered into two groups with demonstrable phenotypic differences between those with missense and truncating alleles. Probands with biallelic NXN variants clustered together with the majority of probands carrying DVL1, DVL2, and DVL3 variants, demonstrating no phenotypic distinction between the NXN-autosomal recessive and dominant forms of RS. While phenotypically similar diseases on the RS differential matched through HPO analysis, clustering using phenotype similarity score placed RS-associated phenotypes in a unique cluster containing WNT5A, FZD2, and ROR2 apart from non-RS-associated paralogs. Through human phenotype analyses of this RS cohort and OMIM clinical synopses of Mendelian disease, this study begins to tease apart specific biologic roles for non-canonical WNT-pathway proteins., Robinow syndrome (RS) is a genetically heterogeneous disorder caused by WNT/PCP-signaling dysfunction. We identified pathogenic variants in RS individuals (n = 22), including the first DVL2 variant. Quantitative HPO-term-based analyses unveiled gene/allele-specific correlations to disease traits, providing a model for delineating the phenotypic consequences of variants affecting paralogs in the same pathway.

High-throughput mRNA sequencing (RNA-Seq) provides both qualitative and quantitative evaluation of the transcriptome. This method uses complementary DNA (cDNA) to generate several millions of short sequence reads that are aligned to a reference genome allowing the comprehensive characterization of the transcripts in a cell. RNA-Seq has a wide variety of applications which lead to a pervasive adoption of this method well beyond the genomics community and a deployment of this technique as a standard part of the toolkit applied in life sciences. This chapter describes a protocol to perform mRNA sequencing using the Illumina NextSeq or MiSeq platforms, presents sequencing data quality metrics, and outlines a bioinformatic pipeline for sequence alignment, digital gene expression, identification of gene fusions, detection of transcript isoforms, description and annotation of genetic variants, and de novo immunoglobulin gene assembly.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one VH4-34(+) ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of VH4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) (2). Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor (5,9). The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Å resolution (9,14). This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

Metabolic stress, such as an insufficient supply of nutrients or oxygen owing to inadequate tumor neovascularization, metastasis, or therapy, is a microenvironmental condition frequently encountered by cancer cells [1]. The ability to survive metabolic stress is thus a necessity for tumor initiation and progression. The serine/threonine kinase AKT (also known as protein kinase B) is a primary mediator of survival of both normal and malignant cells [2]. Our recent studies revealed that AKT could be differentially activated by site-specific phosphorylation according to the severity and duration of metabolic stress, enabling cells to shift gears to survive [3]. During canonical AKT activation, growth factors or other stimuli activate transmembrane receptor tyrosine kinases, which in turn activate phosphoinositide 3-kinase to phosphorylate phosphatidylinositol 4, 5-bisphosphate to form phosphatidylinositol 3, 4, 5-trisphosphate on the inner cell membrane. AKT and its upstream kinase, phosphoinositide-dependent kinase-1 (PDK1), are recruited to the cell membrane, which initiates AKT phosphorylation at Thr308 by PDK1 [4]. Mammalian target of rapamycin complex 2 and other potential PDK2 kinases phosphorylate AKT at Ser473, resulting in optimal AKT activation [5]. The active phosphorylated AKT then translocates from the cell membrane to other cell compartments to phosphorylate multiple downstream substrates to fulfill its versatile functions. In contrast with canonical AKT activation with coordinate phosphorylation on both Thr308 and Ser473, we identified a novel AKT activation mechanism induced by glucose deprivation by which AKT is selectively phosphorylated on Thr308 but not Ser473, resulting in targeting AKT to a specific group of substrates [3]. In HeLa cells, short-term glucose deprivation (for up to 6 h) induced a modest increase in AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation (16 h) induced a marked increase in AKT phosphorylation at Thr308 (up to 30-fold) but only a modest increase at Ser473 (twofold to threefold). Phosphorylation at Thr308 continued to increase over a 16-h glucose deprivation period, whereas Ser473 phosphorylation peaked at about 6 h and subsequently declined (Figure). Apparently, at least two independent processes are responsible for AKT phosphorylation during 16-h glucose deprivation, with a switch occurring between 6 and 8 h. Our data indicated that the first process takes place primarily via the release of feedback inhibition from p70S6K that results in coordinated phosphorylation of AKT at both Thr308 and Ser473. The second process likely occurs via the formation of a complex including GRP78, PDK1, and AKT that promotes selective AKT phosphorylation at Thr308. Figure AKT phosphorylation on Thr308 and Ser473 induced by glucose deprivation and a proposed in vivo metabolic stress model AKT is functionally activated by selective Thr308 phosphorylation during prolonged glucose deprivation as indicated by increased phosphorylation of several known AKT substrates, including glycogen synthase kinase 3, mammalian target of rapamycin, and Y-box binding protein 1. Strikingly, several well-established AKT substrates, including PRAS40 and BAD, were not targeted by AKT under glucose deprivation conditions. Thus, with selective Thr308 phosphorylation, AKT may have altered substrate selectivity. We also observed coordinate phosphorylation of multiple residues other than Thr308 and Ser473 on single AKT molecules in an isoform-specific manner [6], which may also contribute to substrate selectivity. Therefore, AKT could be shrewdly activated for the right substrate spectrum under different cellular contexts, enabling cells to specifically survive energy crisis during metabolic stress. Shrewd AKT activation may occur in vivo. When a tumor grows, some tumor cells located away from blood vessels may be subjected to glucose deprivation (Figure). We propose that these cells employ a layered defense against cell death according to the severity and duration of metabolic stress. Mild or transient metabolic stress (e.g., transient glucose deprivation) induces modest AKT phosphorylation at both Thr308 and Ser473, which provides the first line of defense against cell death. However, during prolonged glucose deprivation, this line of defense becomes insufficient to protect cells against death and is shut off. The next tier of the survival mechanism is then activated. It selectively and markedly increases AKT phosphorylation at Thr308, providing a second line of defense against cell death caused by severe metabolic stress. Therefore, cells are capable of shifting gears to survive specific harsh conditions. AKT, with its versatile activation mechanisms, provides a set of important survival gears that cells may use under specific conditions. In summary, glucose deprivation induces site- specific phosphorylation and substrate-specific activation of AKT via distinct mechanisms according to the duration of metabolic stress. These mechanisms may explain how cells tolerate metabolic stress in tumors by using AKT as a survival tool. Our findings revealed a novel AKT-mediated survival mechanism under prolonged metabolic stress that is important to the development and implementation of drugs targeting cell metabolism and AKT signaling.

Targeted therapies are designed to inhibit specific pathways driving the malignant phenotype while concurrently decreasing side effects on normal cells. However, very frequently, inhibition of the targeted pathway activates compensatory mechanisms designed to bring the pathway back into equilibrium allowing for cancer cell survival, a mechanism known as adaptive drug resistance. The purpose of this project is to identify and abrogate the adaptive drug resistance of epithelial cancers to rapamycin, an allosteric inhibitor of mTOR (mammalian target of rapamycin), and to rapamycin analogs in the PI3K (phosphoinositide-3-kinase) pathway, the most commonly dysregulated pathway in epithelial cancers. Using 3D- cultures, rapamycin-induced morphology changes and cell death were recorded. Changes in protein levels due to rapamycin-induced adaptive signaling were detected using reverse phase protein arrays (RPPA), verified by western blotting, and analyzed to determine potential adaptive mechanisms. Based on screening results from RPPA, rapamycin was combined with rationally selected drugs designed to thwart adaptive drug resistance to rapamycin. This co-extinction approach reinforced rapamycin into an effective inducer of cell death, ultimately bypassing drug resistance mechanisms and precluding the emergence of future therapeutic resistance. Successful combinations will be forwarded into animal studies to demonstrate in vivo efficacy for the treatment of epithelial cancers. Note: This abstract was not presented at the meeting. Citation Format: Moez Dawood, Yiling Lu, Gordon Mills. Identifying rational combination therapies with rapamycin for the treatment of epithelial cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3766. doi:10.1158/1538-7445.AM2015-3766