Novel syndromic neurodevelopmental disorder caused by de novo deletion of CHASERR, a long noncoding RNA.

Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.

De novo missense variants in ZBTB47 are associated with developmental delays, hypotonia, seizures, gait abnormalities, and variable movement abnormalities.

The collection of known genetic etiologies of neurodevelopmental disorders continues to increase, including several syndromes associated with defects in zinc finger protein transcription factors (ZNFs) that vary in clinical severity from mild learning disabilities and developmental delay to refractory seizures and severe autism spectrum disorder. Here we describe a new neurodevelopmental disorder associated with variants in ZBTB47 (also known as ZNF651), which encodes zinc finger and BTB domain-containing protein 47. Exome sequencing (ES) was performed for five unrelated patients with neurodevelopmental disorders. All five patients are heterozygous for a de novo missense variant in ZBTB47, with p.(Glu680Gly) (c.2039A>G) detected in one patient and p.(Glu477Lys) (c.1429G>A) identified in the other four patients. Both variants impact conserved amino acid residues. Bioinformatic analysis of each variant is consistent with pathogenicity. We present five unrelated patients with de novo missense variants in ZBTB47 and a phenotype characterized by developmental delay with intellectual disability, seizures, hypotonia, gait abnormalities, and variable movement abnormalities. We propose that these variants in ZBTB47 are the basis of a new neurodevelopmental disorder.

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Variant STAT4 and Response to Ruxolitinib in an Autoinflammatory Syndrome.

BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).

An FBN1 deep intronic variant is associated with pseudoexon formation and a variable Marfan phenotype in a five generation family.

Exome sequencing of genes associated with heritable thoracic aortic disease (HTAD) failed to identify a pathogenic variant in a large family with Marfan syndrome (MFS). A genome-wide linkage analysis for thoracic aortic disease identified a peak at 15q21.1, and genome sequencing identified a novel deep intronic FBN1 variant that segregated with thoracic aortic disease in the family (LOD score 2.7) and was predicted to alter splicing. RT-PCR and bulk RNA sequencing of RNA harvested from fibroblasts explanted from the affected proband revealed an insertion of a pseudoexon between exons 13 and 14 of the FBN1 transcript, predicted to lead to nonsense mediated decay (NMD). Treating the fibroblasts with an NMD inhibitor, cycloheximide, greatly improved the detection of the pseudoexon-containing transcript. Family members with the FBN1 variant had later onset aortic events and fewer MFS systemic features than typical for individuals with haploinsufficiency of FBN1. Variable penetrance of the phenotype and negative genetic testing in MFS families should raise the possibility of deep intronic FBN1 variants and the need for additional molecular studies.

Further delineation of the CWC27-associated spliceosomeopathy: Case report and review of the literature.

Pre-mRNA splicing factors are crucial in regulating transcript diversity, by removing introns from eukaryotic transcripts, an essential step in gene expression. Splicing of pre-mRNA is catalyzed by spliceosomes. CWC27 is a cyclophilin associated with spliceosome, in which genetic defects of its components have been linked to spliceosomopathies with clinical phenotypes including skeletal developmental defects, retinitis pigmentosa (RP), short stature, skeletal anomalies, and neurological disorders. We report two siblings (male and female) of Mexican descent with a novel homozygous frameshift variant in CWC27 and aim to highlight the cardinal features among the previously described 12 cases as well as expand the currently recognized phenotypic spectrum. Both siblings presented with a range of ocular and extraocular manifestations including novel features such as solitary kidney and tarsal coalition in the male sibling, together with gait abnormalities, and Hashimoto's thyroiditis in the female sibling. Finally, we highlight ectodermal involvement including sparse scalp hair, eyebrows and lashes, pigmentary differences, nail dysplasia, and dental anomalies as a core phenotype associated with the CWC27 spliceosomopathy.

Loss-of-function variants in MYCBP2 cause neurobehavioural phenotypes and corpus callosum defects.

The corpus callosum is a bundle of axon fibres that connects the two hemispheres of the brain. Neurodevelopmental disorders that feature dysgenesis of the corpus callosum as a core phenotype offer a valuable window into pathology derived from abnormal axon development. Here, we describe a cohort of eight patients with a neurodevelopmental disorder characterized by a range of deficits including corpus callosum abnormalities, developmental delay, intellectual disability, epilepsy and autistic features. Each patient harboured a distinct de novo variant in MYCBP2, a gene encoding an atypical really interesting new gene (RING) ubiquitin ligase and signalling hub with evolutionarily conserved functions in axon development. We used CRISPR/Cas9 gene editing to introduce disease-associated variants into conserved residues in the Caenorhabditis elegans MYCBP2 orthologue, RPM-1, and evaluated functional outcomes in vivo. Consistent with variable phenotypes in patients with MYCBP2 variants, C. elegans carrying the corresponding human mutations in rpm-1 displayed axonal and behavioural abnormalities including altered habituation. Furthermore, abnormal axonal accumulation of the autophagy marker LGG-1/LC3 occurred in variants that affect RPM-1 ubiquitin ligase activity. Functional genetic outcomes from anatomical, cell biological and behavioural readouts indicate that MYCBP2 variants are likely to result in loss of function. Collectively, our results from multiple human patients and CRISPR gene editing with an in vivo animal model support a direct link between MYCBP2 and a human neurodevelopmental spectrum disorder that we term, MYCBP2-related developmental delay with corpus callosum defects (MDCD).

A recurrent single-exon deletion in TBCK might be under-recognized in patients with infantile hypotonia and psychomotor delay.

Advanced bioinformatics algorithms allow detection of multiple-exon copy-number variations (CNVs) from exome sequencing (ES) data, while detection of single-exon CNVs remains challenging. A retrospective review of Baylor Genetics' clinical ES patient cohort identified four individuals with homozygous single-exon deletions of TBCK (exon 23, NM_001163435.2), a gene associated with an autosomal recessive neurodevelopmental phenotype. To evaluate the prevalence of this deletion and its contribution to disease, we retrospectively analyzed single nucleotide polymorphism (SNP) array data for 8194 individuals undergoing ES, followed by PCR confirmation and RT-PCR on individuals carrying homozygous or heterozygous exon 23 TBCK deletions. A fifth individual was diagnosed with the TBCK-related disorder due to a heterozygous exon 23 deletion in trans with a c.1860+1G>A (NM_001163435.2) pathogenic variant, and three additional heterozygous carriers were identified. Affected individuals and carriers were from diverse ethnicities including European Caucasian, South Asian, Middle Eastern, Hispanic American and African American, with only one family reporting consanguinity. RT-PCR revealed two out-of-frame transcripts related to the exon 23 deletion. Our results highlight the importance of identifying single-exon deletions in clinical ES, especially for genes carrying recurrent deletions. For patients with early-onset hypotonia and psychomotor delay, this single-exon TBCK deletion might be under-recognized due to technical limitations of ES.

Patient and Clinician Perceptions of Precision Cardiology Care: Findings From the HeartCare Study.

BACKGROUND: Routine genome-wide screening for cardiovascular disease risk may inform clinical decision-making. However, little is known about whether clinicians and patients would find such testing useful or acceptable within the context of a genomics-enabled learning health system. METHODS: We conducted surveys with patients and their clinicians who were participating in the HeartCare Study, a precision cardiology care project that returned results from a next-generation sequencing panel of 158 genes associated with cardiovascular disease risk. Six weeks after return of results, we assessed patients' and clinicians' perceived utility and disutility of HeartCare, the effect of the test on clinical recommendations, and patients' attitudes toward integration of research and clinical care. RESULTS: Among 666 HeartCare patients with a result returned during the survey study period, 42.0% completed a full or partial survey. Patient-participants who completed a full survey (n=224) generally had positive perceptions of HeartCare independent of whether they received a positive or negative result. Most patient-participants considered genetic testing for cardiovascular disease risk to have more benefit than risk (88.3%) and agreed that it provided information that they wanted to know (81.2%), while most disagreed that the test caused them to feel confused (77.7%) or overwhelmed (78.0%). For 122 of their patients with positive results, clinicians (n=13) reported making changes in clinical care for 66.4% of patients, recommending changes in health behaviors for 36.9% of patients, and recommending to 33.6% of patients that their family members have clinical testing. CONCLUSIONS: Both patients and clinicians thought the HeartCare panel screen for cardiovascular disease risk provided information that was useful in terms of personal or health benefits to the patient and that informed clinical care without causing patients to be confused or overwhelmed. Further research is needed to assess perceptions of genome-wide screening among the US cardiology clinic population.

Long read sequencing and expression studies of AHDC1 deletions in Xia-Gibbs syndrome reveal a novel genetic regulatory mechanism.

Xia-Gibbs syndrome (XGS; MIM# 615829) 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.

Best practices for the interpretation and reporting of clinical whole genome sequencing.

Whole genome sequencing (WGS) shows promise as a first-tier diagnostic test for patients with rare genetic disorders. However, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading health care and research organizations in the US and Canada, was formed to expand access to high quality clinical WGS by convening experts and publishing best practices. Here, we present best practice recommendations for the interpretation and reporting of clinical diagnostic WGS, including discussion of challenges and emerging approaches that will be critical to harness the full potential of this comprehensive test.

A novel, de novo intronic variant in POGZ causes White-Sutton syndrome.

White-Sutton syndrome (WHSUS), which is caused by heterozygous pathogenic variants in POGZ, is characterized by a spectrum of intellectual disabilities and global developmental delay with or without features of autism spectrum disorder. Additional features may include hypotonia, behavioral abnormalities, ophthalmic abnormalities, hearing loss, sleep apnea, microcephaly, dysmorphic facial features, and rarely, congenital diaphragmatic hernia (CDH). We present a 6-year-old female with features of WHSUS, including CDH, but with nondiagnostic clinical trio exome sequencing. Exome sequencing reanalysis revealed a heterozygous, de novo, intronic variant in POGZ (NM_015100.3:c.2546-20T>A). RNA sequencing revealed that this intronic variant leads to skipping of exon 18. This exon skipping event results in a frameshift with a predicted premature stop codon in the last exon and escape from nonsense-mediated mRNA decay (NMD). To our knowledge, this case is the first case of WHSUS caused by a de novo, intronic variant that is not near a canonical splice site within POGZ. These findings emphasize the limitations of standard clinical exome filtering algorithms and the importance of research reanalysis of exome data together with RNA sequencing to confirm a suspected diagnosis of WHSUS. As the sixth reported case of CDH with heterozygous pathogenic variants in POGZ and features consistent with WHSUS, this report supports the conclusion that WHSUS should be considered in the differential diagnosis for patients with syndromic CDH.

PRUNE1 c.933G>A synonymous variant induces exon 7 skipping, disrupts the DHHA2 domain, and leads to an atypical NMIHBA syndrome presentation: Case report and review of the literature.

Prune exopolyphosphatase-1 (PRUNE1) encodes a member of the aspartic acid-histidine-histidine (DHH) phosphodiesterase superfamily that regulates cell migration and proliferation during brain development. In 2015, biallelic PRUNE1 loss-of-function variants were identified to cause the neurodevelopmental disorder with microcephaly, hypotonia, and variable brain abnormalities (NMIHBA, OMIM#617481). NMIHBA is characterized by the namesake features and structural brain anomalies including thinning of the corpus callosum, cerebral and cerebellar atrophy, and delayed myelination. To date, 47 individuals have been reported in the literature, but the phenotypic spectrum of PRUNE1-related disorders and their causative variants remains to be characterized fully. Here, we report a novel homozygous PRUNE1 NM_021222.2:c.933G>A synonymous variant identified in a 6-year-old boy with intellectual and developmental disabilities, hypotonia, and spastic diplegia, but with the absence of microcephaly, brain anomalies, or seizures. Fibroblast RNA sequencing revealed that the PRUNE1 NM_021222.1:c.933G>A variant resulted in an in-frame skipping of the penultimate exon 7, removing 53 amino acids from an important protein domain. This case represents the first synonymous variant and the third pathogenic variant known to date affecting the DHH-associated domain (DHHA2 domain). These findings extend the genotypic and phenotypic spectrums in PRUNE1-related disorders and highlight the importance of considering synonymous splice site variants in atypical presentations.

What Has the Undiagnosed Diseases Network Taught Us About the Clinical Applications of Genomic Testing?

Genetic testing has undergone a revolution in the last decade, particularly with the advent of next-generation sequencing and its associated reductions in costs and increases in efficiencies. The Undiagnosed Diseases Network (UDN) has been a leader in the application of such genomic testing for rare disease diagnosis. This review discusses the current state of genomic testing performed within the UDN, with a focus on the strengths and limitations of whole-exome and whole-genome sequencing in clinical diagnostics and the importance of ongoing data reanalysis. The role of emerging technologies such as RNA and long-read sequencing to further improve diagnostic rates in the UDN is also described. This review concludes with a discussion of the challenges faced in insurance coverage of comprehensive genomic testing as well as the opportunities for a larger role of testing in clinical medicine.

Genetic testing in ambulatory cardiology clinics reveals high rate of findings with clinical management implications.

PURPOSE: Cardiovascular disease (CVD) is the leading cause of death in adults in the United States, yet the benefits of genetic testing are not universally accepted. METHODS: We developed the "HeartCare" panel of genes associated with CVD, evaluating high-penetrance Mendelian conditions, coronary artery disease (CAD) polygenic risk, LPA gene polymorphisms, and specific pharmacogenetic (PGx) variants. We enrolled 709 individuals from cardiology clinics at Baylor College of Medicine, and samples were analyzed in a CAP/CLIA-certified laboratory. Results were returned to the ordering physician and uploaded to the electronic medical record. RESULTS: Notably, 32% of patients had a genetic finding with clinical management implications, even after excluding PGx results, including 9% who were molecularly diagnosed with a Mendelian condition. Among surveyed physicians, 84% reported medical management changes based on these results, including specialist referrals, cardiac tests, and medication changes. LPA polymorphisms and high polygenic risk of CAD were found in 20% and 9% of patients, respectively, leading to diet, lifestyle, and other changes. Warfarin and simvastatin pharmacogenetic variants were present in roughly half of the cohort. CONCLUSION: Our results support the use of genetic information in routine cardiovascular health management and provide a roadmap for accompanying research.

COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

Exome sequencing in children with clinically suspected maturity-onset diabetes of the young.

OBJECTIVE: Commercial gene panels identify pathogenic variants in as low as 27% of patients suspected to have MODY, suggesting the role of yet unidentified pathogenic variants. We sought to identify novel gene variants associated with MODY. RESEARCH DESIGN AND METHODS: We recruited 10 children with a clinical suspicion of MODY but non-diagnostic commercial MODY gene panels. We performed exome sequencing (ES) in them and their parents. RESULTS: Mean age at diabetes diagnosis was 10 (± 3.8) years. Six were females; 4 were non-Hispanic white, 5 Hispanic, and 1 Asian. Our variant prioritization analysis identified a pathogenic, de novo variant in INS (c.94G > A, p.Gly32Ser), confirmed by Sanger sequencing, in a proband who was previously diagnosed with "autoantibody-negative type 1 diabetes (T1D)" at 3 y/o. This rare variant, absent in the general population (gnomAD database), has been reported previously in neonatal diabetes. We also identified a frameshift deletion (c.2650delC, p.Gln884AsnfsTer57) in RFX6 in a child with a previous diagnosis of "autoantibody-negative T1D" at 12 y/o. The variant was inherited from the mother, who was diagnosed with "thin type 2 diabetes" at 25 y/o. Heterozygous protein-truncating variants in RFX6 gene have been recently reported in individuals with MODY. CONCLUSIONS: We diagnosed two patients with MODY using ES in children initially classified as "T1D". One has a likely pathogenic novel gene variant not previously associated with MODY. We demonstrate the clinical utility of ES in patients with clinical suspicion of MODY.

Phenotypic and protein localization heterogeneity associated with AHDC1 pathogenic protein-truncating alleles in Xia-Gibbs syndrome.

Xia-Gibbs syndrome (XGS) is a rare Mendelian disease typically caused by de novo stop-gain or frameshift mutations in the AT-hook DNA binding motif containing 1 (AHDC1) gene. Patients usually present in early infancy with hypotonia and developmental delay and later exhibit intellectual disability (ID). The overall presentation is variable, however, and the emerging clinical picture is still evolving. A detailed phenotypic analysis of 34 XGS individuals revealed five core phenotypes (delayed motor milestones, speech delay, low muscle tone, ID, and hypotonia) in more than 80% of individuals and an additional 12 features that occurred more variably. Seizures and scoliosis were more frequently associated with truncations that arise before the midpoint of the protein although the occurrence of most features could not be predicted by the mutation position. Transient expression of wild type and different patient truncated AHDC1 protein forms in human cell lines revealed abnormal patterns of nuclear localization including a diffuse distribution of a short truncated form and nucleolar aggregation in mid-protein truncated forms. Overall, both the occurrence of variable phenotypes and the different distribution of the expressed protein reflect the heterogeneity of this syndrome.

Commonalities across computational workflows for uncovering explanatory variants in undiagnosed cases.

PURPOSE: Genomic sequencing has become an increasingly powerful and relevant tool to be leveraged for the discovery of genetic aberrations underlying rare, Mendelian conditions. Although the computational tools incorporated into diagnostic workflows for this task are continually evolving and improving, we nevertheless sought to investigate commonalities across sequencing processing workflows to reveal consensus and standard practice tools and highlight exploratory analyses where technical and theoretical method improvements would be most impactful. METHODS: We collected details regarding the computational approaches used by a genetic testing laboratory and 11 clinical research sites in the United States participating in the Undiagnosed Diseases Network via meetings with bioinformaticians, online survey forms, and analyses of internal protocols. RESULTS: We found that tools for processing genomic sequencing data can be grouped into four distinct categories. Whereas well-established practices exist for initial variant calling and quality control steps, there is substantial divergence across sites in later stages for variant prioritization and multimodal data integration, demonstrating a diversity of approaches for solving the most mysterious undiagnosed cases. CONCLUSION: The largest differences across diagnostic workflows suggest that advances in structural variant detection, noncoding variant interpretation, and integration of additional biomedical data may be especially promising for solving chronically undiagnosed cases.

Transcriptome-directed analysis for Mendelian disease diagnosis overcomes limitations of conventional genomic testing.

BACKGROUNDTranscriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq-guided method to diagnose individuals across a wide range of ages and clinical phenotypes.METHODSOne hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and skin fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network clinical site from 2014 to 2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis.RESULTSThe transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Skin fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts.CONCLUSIONSFor our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.TRIAL REGISTRATIONNot applicable.FUNDINGNIH Common Fund, BCM Intellectual and Developmental Disabilities Research Center, Eunice Kennedy Shriver National Institute of Child Health & Human Development.